Program & Abstracts Second Meeting of Biochemistry and ... · Guadalupe Espín Dimitris Georgellis...

Second Meeting of Biochemistry and Molecular Biology of Bacteria I

Sociedad Mexicana de Bioquímica, A. C.

Program & Abstracts

Second Meeting of Biochemistry and Molecular Biology of Bacteria

Huatusco, Veracruz November 7 – 11, 2011

Second Meeting of Biochemistry and Molecular Biology of Bacteria II

Organizing Committee Guadalupe Espín Dimitris Georgellis Agustino Martínez Christian Sohlenkamp Local Committee Omar Elind Arroyo Helguera Rocío Coutiño Ramírez Rebeca García Román Hilda Montero Ladrón de Guevara Cristina Ortíz León Clara Luz Sampieri Ramírez Roberto Zenteno Cuevas Instituto de Salud Pública Universidad Veracruzana Technical Edition: and Typesetting: Maria Teresa Castillo Technical Support: Lucero López, Omar Chávez Image Cover: Nayeli Quinto SODIO Published in México, 2011

Second Meeting of Biochemistry and Molecular Biology of Bacteria III

B i e n v e n i d a

¡Bienvenidos al Segundo Congreso de Bioquímica y Biología Molecular de Bacterias!.

Pasaron casi 20 meses desde la primera reunión de nuestra rama celebrada en San Miguel Regla, Hidalgo. Un incentivo para dar luz a este congreso, fue el de querer conjuntar un grupo cohesivo en donde se discutieran con intensidad y profundidad los problemas científicos que nos apasionan. Según la percepción de muchos de los asistentes de aquel congreso, la primera reunión fue un gran paso en esta dirección y por ende un gran éxito. Sin embargo, todavía queda un largo camino delante de nosotros como comunidad para acercarnos a las ideas plasmadas hace 20 meses por el Comité organizador del primer congreso. Les invitamos a todos ustedes a que participen con mucho entusiasmo en las diferentes actividades de este congreso para lograr que éste sea una opción preferida para científicos estudiando bacterias. Como bien decía en la bienvenida del congreso pasado: “De esta manera, nuestra mejor retribución será que el entusiasmo por entusiasmar a los demás, entusiasme a las nuevas generaciones de bacteriólogos mexicanos a fin de que mantengan el espíritu de esta tradición por mucho años venideros;…”. El 11 de febrero del 2011 falleció el Dr. Fernando Bastarrachea Aviles quien fue uno de los pioneros en estudiar la genética molecular bacteriana en México, y cuyo trabajo, rigurosidad cientifica y personalidad influyeron de manera decisiva en el desarrollo de esta diciplina en el Pais. Para conmemorar su papel en la el desarrollo de la microbiología molecular en México, le rendimos homenaje en la sesión IX, en la cual algunos de sus ex –alumnos exponen. También en el año 2011 se celebran los 50 años del nacimiento del concepto del operón en la regulación de la expresión génica. No se dedicó una sesión especial a este tema; este concepto se extiende por un gran número de trabajos presentados en este congreso.

¡Bienvenidos! Guadalupe Espín Dimitris Georgellis Agustino Martínez Christian Sohlenkamp

Segundo Congreso Rama de Bioquímica y Biología Molecular de Bacterias

Sociedad Mexicana de Bioquímica, A.C. 7-11 de noviembre de 2011. Huatusco, Veracruz

Second Meeting of Biochemistry and Molecular Biology of Bacteria IV

Prólogo de la primera edición

“…los privilegiados tienen la obligación de regresar algo al país que les ha permitido obtener esa posición. Porque ¿para qué sirve la experiencia, el conocimiento, el talento, si no se usa para hacer de México un lugar más justo? … ¿Para qué sirve la educación si no se ayuda a los demás a obtenerla? … ¿Para qué sirve ser habitante de un país si no se asume la responsabilidad compartida de asegurar vidas dignas allí?”

Denisse Dresser, 2009

Los conceptos de Denisse Dresser dan el colofón a nuestras palabras de bienvenida. Porque para cualquier región del mundo, para cualquier país, nuestra ciencia con todas sus abstracciones, pasiones y recompensas no tiene ningún sentido si no incide en una sociedad más educada y más sensible. El hacer buena ciencia es nuestra responsabilidad primaria, pero no es suficiente. También es indispensable crear las oportunidades para otros, desde aquellos que desean seguir una carrera científica hasta los individuos de la sociedad civil, que aprenderán de la ciencia su rigor intelectual y de los científicos su pasión y creatividad. Esto es lo que nos mueve a añadir a nuestras múltiples ocupaciones la organización de un congreso como éste. Hemos querido conjuntar un grupo cohesivo, en donde se discutan con intensa profundidad los problemas científicos que nos apasionan. Es por ello que no hay conferencias simultáneas, ni los trabajos están ordenados por áreas del conocimiento. Creemos que todos debemos aprender de todo. Creemos que la evolución hacia una ciencia propia –ya que hemos alcanzado los niveles de una ciencia de calidad internacional- no se dará hasta que entre nosotros encontremos nuestros estilos, nuestra originalidad, como en cualquier otro aspecto de la cultura mexicana que ha alcanzado gran repercusión a nivel mundial. También pensamos que dentro de este espíritu nacionalista debe haber una visión global. Hemos querido que nos acompañen bacteriólogos extranjeros del más alto nivel, que no sólo vengan a impartir una conferencia magistral sino que genuinamente interactúen con todos nosotros, especialmente con los estudiantes y particularmente con aquellos que no han tenido muchas oportunidades de salir al extranjero. Así, hemos insistido en que los resúmenes sean en el idioma inglés y que, de preferencia, también lo sean los carteles y las transparencias de las presentaciones orales. Es de importancia estratégica que nuestro congreso se convierta en uno de los más atractivos para los bacteriólogos de cualquier parte del mundo -pues tenemos todo para lograrlo- en donde los visitantes sepan que no sólo vendrán a compartir su ciencia sino que también se llevarán un aprendizaje valioso. Más aún, un congreso como éste deberá preparar a nuestros principiantes en las lides de lo que implica un congreso en el extranjero De esta manera, nuestra mejor retribución será que el entusiasmo por entusiasmar a los demás, entusiasme a las nuevas generaciones de bacteriólogos mexicanos a fin de que mantengan el espíritu de esta tradición por muchos años venideros; no sólo basándose en lo que se logre en esta ocasión sino aportando de su propia visión y creatividad.

Second Meeting of Biochemistry and Molecular Biology of Bacteria V

Y, finalmente, cerrando con Dresser: “La convicción inquebrantable de mejorar a México. De restañar a la República. De volver a México un país de ciudadanos. Un lugar poblado por personas conscientes de sus derechos y dispuestos a contribuir para defenderlos. Dispuestos a llevar a cabo pequeñas acciones que produzcan grandes cambios. Dispuestos a sacrificar su zona de seguridad personal para que otros la compartan.” Esto es, los invitamos a seguirnos saliendo de nuestra zona de seguridad, para estar dispuestos a llevar a cabo pequeñas acciones que produzcan grandes cambios. ¡Bienvenidos! Edmundo Calva Guadalupe Espín Dimitris Georgellis Bertha González Pedrajo José Luis Puente

Second Meeting of Biochemistry and Molecular Biology of Bacteria VI

Funding Institutions & Sponsors

Second Meeting of Biochemistry and Molecular Biology of Bacteria VII

Table of Contents Program of Events Monday .................................................................................................. 3 Tuesday .................................................................................................. 4 Wednesday ...................................................................................................... 8 Thursday .......................................................................................................... 12 Abstracts Plenary Sessions .............................................................................................. 17 Oral Presentations ........................................................................................... 27 Odd Poster Sessions ......................................................................................... 77 Even Poster Sessions ........................................................................................ 129 Author Index .................................................................................................... 181

PROGRAM

Monday November 7, 2011

Second Meeting of Biochemistry and Molecular Biology of Bacteria 3

11:00 – 18:00 Registration ________________________________________________________________________________________________________________________________________________

18:45 – 19:00 Opening Ceremony, and Opening Talk

________________________________________________________________________________________________________________________________________________

19:00 – 20:00

Opening Lecture Valeria Souza Why so many bacterial species? The roles of food, sex and travel in determining the microbial diversity of our planet Instituto de Ecología – UNAM

Chair: Guadalupe Espín

20:00 – 22:00 Dinner and Welcome Cocktail All oral presentations will be held in the Magno Orquídeas Room All poster presentations will be held in the Palapa

Tuesday November 8, 2011


Oral Session I

Chair: José Luis Puente ___________________________________________________________________

9:00 – 9:20 9:20 – 9:40 9:40 – 10:00 10:00 – 10:20 10:20 – 10:40 10:40 – 11:00

César Augusto Aguilar Martínez Characterization of mutations effects in regulatory genes originated through an adaptive evolution process by rapid growth on glucose in an Escherichia coli strain lacking the PTS system Instituto de Biotecnología – UNAM Morena Avitia Cao Romero Population genetics of Bacillus sp nov from Cuatro Cienegas Coahuila, Mexico Instituto de Ecología – UNAM Víctor Antonio Becerra Rivera The over-expression of cytochrome c550 from Bacillus subtilis has effects on the activity of respiratory complexes FES Iztacala – UNAM Susana Brom Klanner The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain Centro de Ciencias Genómicas – UNAM Rosina Cabrera Ruiz Overexpression and Purification of quorum sensing transcriptional regulator NprR of Bacillus thuringiensis Centro de Investigación en Alimentación y Desarrollo Break



Oral Session II

Chair: Luis Eguiarte Fruns ___________________________________________________________________

11:00 – 11:20 11:20 – 11:40 11:40 – 12:00 12:00 – 12:20 12:20 – 12:40 12:40 – 13:00

Martha Irais Camacho Hernández The global regulators Hfq and CsrA are required for proper uvrY translation Instituto de Fisiología Celular – UNAM Miguel Ángel Cevallos Gaos The origin of replication of a repABC plasmid Centro de Ciencias Genómicas – UNAM Mayra Elizeth Cobaxin Cárdenas Effect Of In Vitro Culture Conditions On The Expression Of Virulence Factors In Vibrio cholerae El Tor Facultad de Medicina - UAEM Susana de la Torre Zavala Gene expression of Pht cluster genes and a putative non-ribosomal peptide synthetase required for phaseolotoxin production is regulated by GacS/GacA in Pseudomonas syringae pv. phaseolicola CINVESTAV – IPN. Unidad Irapuato Alma Laura Díaz Pérez Structural, phylogenetic and functional analysis of the isocitrate lyase from Pseudomonas aeruginosa Instituto de Investigaciones Químico-Biológicas – UMSNH Break



Plenary Session I

Chair: Dimitris Georgellis __________________________________________________________________________________________________________________________________________________

13:00 – 14:00

Roberto Kolter Disassembly of Bacillus subtilis biofilms Harvard Medical School

14:00 16:00 Lunch ______________________________________________________________

Oral Session III

Chair: Herminia Loza Tavera ___________________________________________________________________________________________________________________________________________________

16:00 – 16:20 16:20 – 16:40 16:40 – 17:00

Ma. Dolores Rodríguez Torres Metabolic diversity among Bacillus isolates from the sediment of the Churince system in Cuatro Ciénegas, Coahuila CINVESTAV – IPN. Unidad Irapuato Katy Juárez López Integration Host Factor is a global regulator required for electron transference in Geobacter sulfurreducens Instituto de Biotecnología – UNAM Paula Figueroa Arredondo Vibrio cholerae cytolysin VCC probably causes pyroptosis to THP-1 macrophages by activating MAPKs signaling pathway Escuela Nacional de Medicina y Homeopatía – IPN



Plenary Session II

Chair: Agustino Martínez ____________________________________________________________________________________________________________________________________________________

17:00 – 18:00

Jeff F. Miller Diversity-Generating Retroelements University of California

18:00 – 20:00 Poster Session Odd numbers 20:00 – 22:00 Dinner

Wednesday November 9, 2011


Oral Session IV

Chair: Edmundo Calva Mercado ___________________________________________________________________

9:00 – 9:20 9:20 – 9:40 9:40 – 10:00 10:00 – 10:20 10:20 – 10:40 10:40 – 11:00

Rodolfo García Contreras Quorum Quenching Quandary: Resistance to Antivirulence Compounds Instituto Nacional de Cardiología “Ignacio Chávez” Jaime García Mena Study of protein-protein interaction in the RNA Degradosome of Escherichia coli upon conditional mRNA degradation CINVESTAV – IPN Unidad Zacatenco Andrea González González Genetic diversification and genome size diversity of Mexican Escherichia coli Instituto de Ecología – UNAM Bertha González Pedrajo Molecular mechanisms participating in the biogenesis of the type III secretion system of enteropathogenic Escherichia coli Instituto de Fisiología Celular – UNAM Manuel Alejandro González Vera Transcriptional and posttranscriptional controls involved in the synthesis of flagellin in Rhodobacter sphaeroides Instituto de Investigaciones Biomédicas – UNAM Break



Oral Session V

Chair: Gabriela Olmedo Álvarez ___________________________________________________________________

11:00 – 11:20 11:20 – 11:40 11:40 – 12:00 12:00 – 12:20 12:20 – 12:40 12:40 – 13:00

Isabel M. López Lara FadD is required for utilization of endogenous fatty acids released from membrane lipids Centro de Ciencias Genómicas – UNAM Mildred Castellanos Escamilla RpoS regulator of stationary phase, controls transcriptional expressions of phbR gene and phbBAC operon witch are responsible for PHB production in Azotobacter vinelandii Instituto de Biotecnología – UNAM Rafael Jiménez Mejía Analysis of the environmental conditions involved in regulation of chr genes from Burkholderia xenovorans Instituto de Investigaciones Químico-Biológicas – UMSNH Gamaliel López Leal Analysis of the Rhizobium etli stimulons under heat shock and saline stress by RNA-Seq Centro de Ciencias Genómicas – UNAM Edgardo Galán Vásquez The regulatory network of Pseudomonas aeruginosa and its comparison with other bacterial networks CINVESTAV – IPN. Unidad Irapuato Break



Plenary Session III

Chair: Guadalupe Espín ____________________________________________________________________________________________________________________________________________________

13:00 – 14:00

Stephen Trent Remodeling of the Bacterial Cell Surface: Modification of Lipopolysacchardies University of Texas

13:40 16:00 Lunch ______________________________________________________________

Oral Session VI

Chair: Mayra de la Torre Martínez ____________________________________________________________________________________________________________________________________________________

16:00 – 16:20 16:20 – 16:40 16:40 – 17:00

Ana Elisa Martínez del Campo Chemotaxis signaling pathway of the flagellar system 2 from Rhodobacter sphaeroides Instituto de Fisiología – UNAM Eva Martínez Peñafiel Overexpression of 4.3 protein of phage mEp021 induces pleiotropic effects like inhibition of cell division, release of basal haemolysin HlyE and cell death CINVESTAV – IPN. Unidad Zacatenco Martin Peralta Gil Characterization of multiple arrangements of binding sites of transcription factors in Escherichia coli K-12 and their mechanisms of action in gene regulation Centro de Ciencias Genómicas – UNAM



Plenary Session IV

Chair: Christian Sohlenkamp _______________________________________________________________________________________________________________________________________________________

17:00 – 18:00

Peter Greenberg Sociomicrobiology: Quorum sensing in Pseudomonas aeruginosa University of Washington

18:00 – 20:00 Poster Session Even numbers 20:00 – 22:00 Dinner

Thursday November 10, 2011


Oral Session VII

Chair: Jesús Campos García ___________________________________________________________________

9:00 – 9:20 9:20 – 9:40 9:40 – 10:00 10:00 – 10:20 10:20 – 10:40 10:40 – 11:00

Daniel Segura González Effect of phaG gene expression on polyhydroxyalkanoate structure synthesized by a recombinant strain of Azotobacter vinelandii Instituto de Biotecnología – UNAM David Zamorano Sánchez Identification of FikR, the response regulator that completes the fixL-fixK cascade in Rhizobium etli CFN42 Centro de Ciencias Genómicas – UNAM Roberto Zenteno Cuevas Characterization of the molecular mechanisms producers of drug resistance in Mycobacterium tuberculosis Instituto de Salúd Pública – UV César Quiñones Valles Regulatory circuits controlling asymmetrical cell division in bacteria CINVESTAV – IPN. Unidad Irapuato Eria Alaide Rebollar Caudillo Evolutionary history and niche differentiation of Exiguobacterium, a halophilic bacterial genus living at Cuatro Cienegas Basin, Mexico Instituto de Ecología – UNAM Break



Oral Session VIII

Chair: Heliodoro Celis Sandoval ___________________________________________________________________________________

11:00 – 11:20 11:20 – 11:40 11:40 – 12:00 12:00 – 12:20 12:20 – 12:40 12:40 – 13:00

Gloria Alejandra Sarmina Leonel Characterization of a null mutant in the mazG gene of Rhodobacter sphaeroides Instituto de Fisiología Celular – UNAM Omar Alejandro Sepúlveda Robles Genetic diversity of phages of Pseudomonas aeruginosa: isolation, characterization and identification of new species CINVESTAV – IPN. Unidad Zacatenco Hortencia Silva Jiménez Construction of a prototype sensor kinase from the hybrid kinase TodS of Pseudomonas putida DOT-T1E Consejo Superior de Investigaciones Científicas Miguel Ángel Vences Guzmán Function of ornithine lipids in Agrobacterium tumefaciens Centro de Ciencias Genómicas – UNAM Roche Diagnostics Metagenomics and Microbial Diversity Technical Talk Break



Plenary Session V

Chair: Dimitris Georgellis __________________________________________________________________________________________________________________________________________________

13:00 – 14:00

Susan Gottesman Regulating with small RNAs in E. coli

National Cancer Institute

14:00 16:00 Lunch

Oral Session IX

In Memorian FERNANDO BASTARRACHEA

Chair: David Romero __________________________________________________________________________________________________________________________________________________

16:00 – 16:30 16:30 – 17:00 17:00 – 17:30 17:30 – 18:00

Luis Servín González Genetics and genomics coming together to understand the methyl-specific restriction system of Streptomyces coelicolor Instituto de Investigaciones Biomédicas – UNAM Laura Camarena Motility in Rhodobacter sphaeroides is mediated by a complex rotation system Instituto de Investigaciones Biomédicas – UNAM Gloria Soberón Chávez The RhlR regulon in Pseudomonas aeruginosa quorum‐sensing response. Instituto de Investigaciones Biomédicas – UNAM David René Romero Camarena Gene Conversion in Rhizobium etli: role of systems for initiation and migration of the Holliday junction Centro de Ciencias Genómicas – UNAM

ABSTRACTS PLENARY SESSION

Opening Lecture. November 7, 2011


Why so many bacterial species? The roles of food, sex and travel in determining the microbial diversity of our planet

Valeria Souza and Luis E. Eguiarte Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, CU, AP 70-275, Coyoacán 04510 The microorganisms that inhabit modern-day microbial mats coexist in space due to the differences in oxygen regime, permitting the co-occurrence of aerobic microbes, different types of photosynthesis and ancient anoxic methanogens, within the same architectural complex. Although microbial mats and microbialites once had broad geographical distribution, they now are only found in extreme environments, where algae cannot prosper. This is the case of the extremely oligotrophic oasis of Cuatro Ciénegas Basin (CCB) in Coahuila Mexico. By describing the microbial diversity in CCB we detected that bacterial clonal lineages were diversifying locally, generating a large total diversity. Moreover, molecular evolution analyses indicate that they are closely related to marine microorganisms that once shared the common ocean of Panthalassa. Further exploration of this link was done through comparative genomics, finding that CCB represents a “lost world” were ancient lineages persisted despite the fact that the ocean left the region ca. 35 million years ago. In order to explore a possible ecology of the past, we carried out detailed comparative metagenomic analyses of 4 microbialites with different N:P content, observing that each one was unique in its taxonomical composition. However, all of them converged to an amazing array of metabolic pathways that had the potential of performing the majority of the biogeochemical cycles and also of degrading most of the known compounds described in KEGG database. Our inference is that in the Precambriam ocean, microbes survived different environmental catastrophes in part due to their community metabolic cohesion and redundance. Our data and analyses for Cuatro Cienegas and their extrapolation to earlier Earth suggests that it is precisely the low Phosporous (P) content that increase reproductive isolation by limiting all the mechanisms of HGT. Low HGT is followed by local diversification of small population sizes and high metabolic coherence were communities have been competing and coexisting for so long that newcomers are not welcome. On the other hand, in model systems such as E. coli, is precisely the abundance of food that allows ample space for sex and travel, maintaining an enormous cosmopolitan genetic pool that has taken a very long time to diversify and speciate from it sister genes Salmonella.

Plenary Session I. November 8, 2011


Disassembly of Bacillus subtilis biofilms Roberto Kolter Harvard Medical School Most microbes can switch between unicellular and multicellular lifestyles. Depending on environmental conditions, there are times where each of these lifestyles can provide a selective advantage and thus aid in the survival of the species. As a consequence, most microbes have evolved the capacity to respond to environmental cues by switching lifestyles. The most common form of multicellularity among bacteria is the formation of cellular aggregates that are held together by an extracellular matrix. Such aggregates are generally found associated to surfaces and are referred to as biofilms. Depending on the setting where biofilms form, they can have very different consequences for human life. The biofilms that form on the surfaces of the human body are largely mutualistic and most of the time protect the human. However, when normal human defenses are breached, pathogens can colonize surfaces forming biofilms that are the foundation of chronic infections. Because of the importance of biofilms on human health it is therefore important to understand the processes that lead to their disassembly. We are studying these processes in the model bacterium Bacillus subtilis and we have learned much about the molecules involved in biofilm disassembly. What we have learned from these studies can be extended to important pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. We are hopeful that from the basic knowledge we have obtained we will be able to develop strategies to control biofilms that are problematic in clinical settings.

Plenary Session II. November 8, 2011


Diversity-Generating Retroelements Huatao Guo, Mari Gingery, Diego Arambula, Elizabeth Czornyj and Jeff F. Miller Dept. of Microbiology, Immunology and Molecular Genetics UCLA Host-parasite interactions are often driven by mechanisms that promote genetic variability. In the course of our studies on bacterial pathogenesis, we discovered a group of temperate bacteriophages that generate diversity in a gene that specifies tropism for receptor molecules on host Bordetella species, which cause respiratory infections in humans and other mammals. This microevolutionary adaptation is produced by a “diversity-generating retroelement” (DGR) that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats, one serving as an donor template (TR) and the other as a recipient of variable sequence information (VR). Recent work has focused on the genetic basis of diversity-generation. The directionality of information transfer is determined by the initiation of mutagenic homing (IMH) sequence present at the 3’ end of VR. We have demonstrated that DGR function occurs through a TR-containing RNA intermediate by a unique target-primed reverse transcription mechanism that precisely regenerates target sequences. This non-proliferative, “copy and replace” mechanism enables repeated rounds of protein diversification and optimization of ligand-receptor interactions. The potential utility of DGRs is illustrated by the identification of over 150 related elements in bacterial, phage, and plasmid genomes. DGRs are present in human pathogens (Treponema, Legionella spp.), human commensals (Bacteroides, Bifidobacterium spp.), green sulfur bacteria (Chlorobium, Prosthecochloris spp.), cyanobacteria (Trichodesmium, Nostoc spp.), magnetotactic bacteria (Magnetospirillum spp.), and many other diverse species. DGRs comprise a new family of retroelements with the potential to confer powerful selective advantages to their host genomes.

Plenary Session III. November 9, 2011


Remodeling of the Bacterial Cell Surface:

Modification of Lipopolysacchardies

M. Stephen Trent The University of Texas at Austin

Bacteria synthesize remarkable surface structures that are often required for growth and infection of the human host. In response to extracellular stimuli, gram-negative organisms frequently remodel lipopolysaccharide (LPS) structures to promote their survival. Often these modifications occur on lipid A, a saccharolipid that anchors LPS in the outer membrane. Notably, lipid A is the endotoxic portion of LPS that is recognized by the TLR4-MD2 receptor of the mammalian innate immune system. Enzymatic remodeling of the lipid A provides resistance to cationic antimicrobial peptides that bind to the anionic surface of bacteria and promotes immune evasion by modulating TLR4-MD2 recognition. The level of diversity seen in LPS modification systems is quite extraordinary and our laboratory has focused on the characterization of modification machinery from a number of organisms including Escherichia coli, Campylobacter jejuni, Helicobacter pylori, Vibrio cholerae, Salmonella typhimurium, and others. More recently, we have discovered that LPS modification machinery can serve in the assembly of additional bacterial structures such as flagella and peptidoglycan. Understanding how bacteria remodel their cell surface is of fundamental importance and may yield new therapeutic strategies for intervention in bacterial infections.

Plenary Session IV. November 9, 2011


Sociomicrobiology: Quorum sensing in

Pseudomonas aeruginosa E. Peter Greenberg University of Washington, Seattle USA For most of the last century biologists held the belief that except for rare exceptions bacteria were not social organisms. This belief has given way to a new understanding that bacteria are like other creatures. As a result of selection, social activities have evolved in many bacterial species. This has led to the development of an emerging discipline that now counts hundreds of scientists as practitioners; sociomicrobiology. The rationales for studying the social behavior of bacteria are as follows: Studies of bacterial sociality can significantly increase our understanding of sociobiology in general. If one hopes to understand bacterial activities in any comprehensive way it is critical to include social activities in the equation. Finally, we have observed that bacterial social activities are important for successful infections by a variety of pathogenic bacteria. Therefore, it may be possible to develop new medicines as a result of sociomicrobiology investigations. Our studies of bacterial social activity began about thirty years ago with the beginnings of an understanding that bacterial cells produced pheromones that enabled cell-to-cell communication. This form of communication allows individuals of a given species to monitor the abundance of that species, and at sufficiently high population densities the community responds by activating specific genes that result in production of public goods (eg extracellular enzymes, antibiotics, light production in special cases). We thus termed this activity quorum sensing and response. This presentation will cover recent investigations of quorum sensing signal receptors in the opportunistic pathogenic bacterium Pseudomonas aeruginosa. There will be a particular on new information regarding the key receptor LasR

Plenary Session V. November 10, 2011


Regulating with small RNAs in E. coli Susan Gottesman Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD. 20892 E. coli grows in a variety of environments, and thus must adjust its metabolism and regulation as it moves from environment to environment. Transcriptional regulation is at the core of these changing responses, but it is becoming increasingly clear that other layers of regulation exist. I will discuss the small non-coding RNAs (sRNAs) that regulate mRNA stability and translation. The best-studied family of sRNAs binds the RNA chaperone Hfq and pairs with target mRNAs. Expression of the sRNAs are highly regulated, and in many cases the sRNAs act as bridges between different regulatory cascades. For instance, the Arc two-component system negatively regulates ArcZ, which in turn positively regulates the RpoS sigma factor and negatively regulates the regulator of motility, flhDC. We use a combination of microarrays, bioinformatics, and screens of sRNA libraries to identify which sRNAs regulate which mRNAs, with the aim of understanding how the sRNAs modulate regulatory networks. I will describe our recent findings in dissecting these networks.

Plenary Sessions


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Oral Session I. Tuesday


Characterization of mutations effects in regulatory genes originated through an adaptive evolution process by rapid growth on glucose in an

Escherichia coli strain lacking the PTS system César Aguilar*, Adelfo Escalante, Noemí Flores, Ramón de Anda, Guillermo Gosset, Francisco Bolívar Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología (UNAM). Apdo. Postal 510-3 Cuernavaca/Morelos 62250, México. *Corresponding author: Tel. +52 777 329 1648, Fax: 52(777)329-1601, E-mail address: [email protected]

The intracellular availability of phosphoenolpyruvate (PEP) is very controlled; now a days engineering of metabolic pathways has succeeded in increasing this availability by eliminating in a strain of Escherichia coli (JM101) the ptsHlcrr operon, which codes to the phosphotransferase system (PTS), which consumes more than 50% of PEP originated from glycolysis (Flores, N. et. al 2005). From the strain PTS-, and by a process of selective pressure for rapid growth in glucose, we generated the PB12 strain, which partially recovered the ability to assimilate glucose (PTS- Glc+) and the ability to grow efficiently using glucose as the only carbon source. Through metabolic engineering, we were able to redirect part of PEP not used by PTS to the synthesis of aromatic compounds with high yield in PB12 strain (Escalante, et al. 2010; Cortés-Tolalpa et al. 2011). It has been shown that strain PB12 overexpressed most of the genes in the glycolytic pathway, as well as genes involved in the metabolism of ppGpp (Flores, N. et. al. 2005, 2007, 2008). In order to know accurately the changes arising during the evolution process that gave rise to PB12 strain, it was sequenced by two methods: using a comparative sequencing performed by Roche-Niblegen and by a second method performed with the Genome Analyzer system GAIIx company (IBT, UNAM). Several point mutations were found including mutations in genes involved in regulation (arcB, barA, rna, rpoD, rssA, yjjU and ypdA genes). Also the absence of a chromosomal fragment comprising 10.3 Kb's long where 12 genes are located (including relevant ones such as the rppH, mutH and galR genes) was detected. This work try to explain the possible consequences of each of the mutations in the regulatory genes originated in the strain PB12 during the evolution process thorough a performed set of experiments, including complementation, inactivation and RT-qPCR experiments. Results show the relevance of certain mutations on growth recovery in strain PB12, and explain some of the metabolic characteristics of this evolved strain and furthermore the importance of eliminating chromosomal region as important part of the adaptation process, which is speculated to be among the first mutational events occurring during the evolution process of this strain.



Population genetics of Bacillus sp nov from Cuatro Cienegas Coahuila, Mexico

Avitia Cao Romero Morena, Souza Saldívar Valeria, Eguiarte Fruns Luis Instituto de Ecología, Universidad Nacional Autónoma de México Correo electrónico: [email protected] Dirección postal: Apartado Postal 70-275 Ciudad Universitaria, UNAM 04510 México, D.F. Circuito exterior s/n anexo al Jardín Botánico Exterior

Population genetics studies of bacterial species have focused mainly on pathogenic and symbiotic organisms. However there are only a few works exploring the population structure of free-living bacteria. Cuatro Cienegas Basin (CCB) is located in Coahuila, Mexico, and it is a place with great diversity of aquatic environments. These environments are characterized by the presence of complex bacterial communities called microbial mats, as well as diverse planktonic communities with high beta diversity. Within that ample microbial diversity, few genera are abundant and diverse in many sites. One of these groups with broad distribution is Bacillus. Previous studies of Bacillus from Cuatro Cienegas have allowed us to understand part of the evolutionary history of this group and some aspects of its adaptation to the particular environmental conditions of this basin. The aim of the present work was to explore the population structure of a new particular lineage of free-living bacteria from the Bacillus genus, which is widely distributed throughout the CCB. We described the evolutionary forces that have been shaping such populations in contrast with other lineages with restricted distribution. The multilocus analysis of this group of microorganisms showed that this population has a clonal structure and its distribution does not seem to correlate with environmental factors.



The over-expression of cytochrome c550 from Bacillus subtilis has

effects on the activity of respiratory complexes

Becerra Rivera Victor Antonio, Cabellos Avelar Tecilli y Gutiérrez-Cirlos Madrid Emma Berta Unidad de Biomedicina, FES Iztacala UNAM, [email protected], Av. De los Barrios #1. Los Reyes Iztacala. Tlalnepantla, Edo. de México. c.p. 54090, Tel: 5623-1333, ext 39783. Fax: 5623-1138 Cytochrome c-type is a small mobile protein that transports electrons from complex III to the terminal oxidase in the intermembrane space. It may be present in two forms: as soluble periplasmic proteins or attached to the membrane with a hydrophobic N-terminal extension. The mechanism of action of the later is still unknown. The information available from these proteins is limited in Gram-positive bacteria, in contrast to the amount of information available in Gram-negative bacteria and eukaryotes, therefore studies of this cytochrome in Gram-positive bacteria as Bacillus subtilis are of extreme importance. The c-type cytochrome in B. subtilis is encoded by the gene cccA and is called cytochrome c550. This protein of 13 kDa is composed of a α helix as a transmembrane binding domain and a heme domain. It is located on the outer surface of the plasma membrane, as all bacterial c-type cytochromes. In this work, we cloned and over expressed the membrane-bound cytochrome c550 to evaluate its effect on the respiratory complexes in the bacterium Bacillus subtilis grown in LB medium and MSR (3% succinate) and harvested at three different times (5, 9 and 23 hr of growth). We found that cells grown in MSR medium give a higher yield as well as over-expression of cytochrome c550 demonstrated by spectrophotometric analysis of the isolated membranes. The maximum concentration obtained for cytochrome c550 was similar (36.7 and 40.4μM) for LB and MSR respectively. It is important to note that it is required of smaller volume of cells grown in LB than MSR to obtain this concentracion. Polyacrylamide gels using lithium dodecyl sulfate showed the kinetics of expression of cytochrome c550, which increased according to time. We observed that in LB medium, expression of cytochrome c550 increases from 5 to 9 hr and then decreases at 23 hr. Also the enzymatic activity of the four respiratory complexes was measured spectrophotometrically. We found that the activity of complex I, increases significantly after 9 hr of growth in the case of LB medium but in MSR it increases at 23 hr. Complex II shows a significant increase in both media at the three different times of harvesting. Complexes III and IV also show a significant increase in the two media but only at 9 hr. Finally, complex IV activity increased significantly only between 5 and 9 hr of growth in both media. Thus, this paper introduces the expression of proteins of the respiratory chain of B. subtilis, which varies depending on the carbon source where it grows.



The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the

chromosome of a Sinorhizobium strain Susana Brom, Laura Cervantes, Patricia Bustos, Lourdes Girard, Rosa Isela Santamaría, Guillermo Dávila, Pablo Vinuesa, and David Romero Centro de Ciencias Genómicas. UNAM. [email protected]. Av. Universidad 1001, Cuernavaca, Mor., México. Tel. (777) 329 16 91

Beans and their symbiont Rhizobium etli originated in Mesoamerica, while soybean and its symbiont Sinorhizobium fredii evolved in East Asia. S. fredii strains (e.g. GR64) have been isolated from bean nodules in Europe, suggesting the occurrence of conjugative transfer events among introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pRet42d) requires cointegration with the endogenous self-transmissible plasmid pRet42a. With the purpose of further understanding the generation of diversity among bean nodulating strains, we studied the plasmids of S. fredii GR64 (pSfr64a and pSfr64b).

Plasmid pSfr64b corresponds to the symbiotic plasmid, although it allows bean-nodulation, it differs from typical R. etli symbiotic plasmids, concerning various features, such as the replication functions, and reiteration of nifH genes.

Regarding conjugative transfer, plasmid pSfr64a was found to be self-transmissible, and required for transfer of the symbiotic plasmid (pSfrGR64b). We obtained the complete sequence of pSfr64a, finding 166 ORFs. The plasmid showed three large segments of different evolutionary origins; one of them presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; another one harbored 51 ORFs with highest similarity to genes from pRet42d, including the replication, but not the symbiosis genes; as a result, pSfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, although it did not contribute to symbiosis. The last sector contained 36 ORFs with highest similarity to genes localized on pRet42a, 20 of them involved in conjugative transfer. Nevertheless, pRet42a was unable to substitute pSfr64a for induction of pSym transfer, furthermore pRet42a transfer frequency was significantly diminished in GR64 background. Our results indicate that S. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We suggest that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid cointegrate from R. etli to a S. fredii strain, and at least two recombination events among the R. etli plasmids and the S. fredii genome. As in R. etli CFN42, the S. fredii GR64 transmissible plasmid is required for the conjugative transfer of the symbiotic plasmid. In spite of the similarity in the conjugation related genes, the transfer process of these plasmids shows a host-specific behaviour, exposing their adjustment to diverse host environments. Acknowledgments: To DGAPA for PAPIIT grant IN203109.



Overexpression and Purification of quorum sensing transcriptional

regulator NprR of Bacillus thuringiensis

aRosina Cabrera-Ruiz, bAdriana Guzmán-Soto, aRefugio Robles-Burgueño, aJorge Rocha-Estrada, cGabriel Guarneros y aMayra de la Torre aCentro de Investigación en Alimentación y Desarrollo, A. C., Departamento de Ciencia de los Alimentos, Hermosillo Sonora 83304, bUniversidad de Sonora, Departamento de Medicina, Hermosillo Sonora 83000, cCINVESTAV, Departamento de Genética y Biología Molecular, México DF 07000 [email protected] Bacterial cells use quorum sensing mechanism (QS) to interact with each other and coordinate gene expression in response to cell density. RNPP is a family of QS intracellular-receptors that bind directly to its peptide effector. One of the proteins that integrate this family is the Neutral Protease Regulator (NprR). In Bacillus subtilis and Bacillus stearothermophilus, NprR was described as activator of a neutral protease and it has been recently related to sporulation and expression of cry genes in the entomopathogenic bacterium Bacillus thuringiensis (Bt). However, NprR has not been characterized, this work represent the first step to know the molecular mechanism and function that this protein performs as a QS transcriptional regulator. We overproduced the protein NprR using a heterologous expression system (Escherichia coli BL21), at the same time, to avoid the rapid degradation or aggregation of the recombinant protein we co-expressed two teams of chaperones (DnaK-DnaJ-GrpE y GroES-GroEL) to assist protein folding and this leads to increased production of active proteins. The protein was purified by affinity chromatography using Ni-NTA and its purity was analyzed by SDS-PAGE at 10%. This expression system allowed the overexpression of NprR as a soluble protein.



NOTES

Oral Session II. Tuesday


The global regulators Hfq and CsrA are required for proper uvrY translation

Martha I. Camacho, Adrián F. Álvarez y Dimitris Georgellis Departamento de Genética Molecular, Instituto de Fisiología Celular. Universidad Nacional Autónoma de Mé[email protected] In prokaryotic cells, the sensing and processing of environmental signals depends mainly on two-component signal transduction system (TCS). These systems consist of a membrane-bound histidine kinase (HK) and a cytosolic response regulator (RR). The BarA/UvrY TCS of Escherichia coli consists of BarA as the HK, and UvrY as its cognate RR. BarA has been shown to autophosphorylate in response to extracellular acetate or other aliphatic carboxylic acids, and transphosphorylate UvrY. UvrY-P, in turn, acts as a transcriptional regulator that directly activates the expression of the noncoding RNAs (sRNAs) CsrB and CsrC. These sRNAs act as antagonists of CsrA, an RNA binding protein with profound effects on many physiological processes, such as central carbon metabolism, virulence, motility and biofilm formation. Curiously, csrB transcription, which directly depends upon UvrY, is not activated in a csrA mutant strain, suggesting that CsrA may affect directly or indirectly UvrY expression. Here, we present the results of experiments aiming at elucidating the regulatory interactions of BarA/UvrY and CsrA/CsrB. A simple model for this regulatory circuitry is presented and discussed.



The origin of replication of a repABC plasmid

Miguel A. Cevallos, Ramón Cervantes Rivera, Francisco Pedraza López, Gabriela Pérez Segura Programa de Genómica Evolutiva; Centro de Ciencias Genómicas–UNAM. Avenida Universidad s/n, col. Chamilpa, CP: 62210. Cuernavaca, Morelos, [email protected], (777)3114663

repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 -proteobacterial genera and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not enter an R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC SymA was able to replicate in the presence of p42d. Conclusions: RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The oriV of this plasmid resides within the repC gene and is located close to or inside of a large A+T region. RepC can act as an incompatibility factor, and the last 39 amino acid residues of the carboxy-terminal region of this protein are involved in this phenotype.



Effect Of In Vitro Culture Conditions On The Expression Of Virulence Factors In Vibrio cholerae El Tor

Cobaxin Cárdenas Mayra Elizeth1, 2 and Sánchez Castillo Joaquín2 Facultad de Ciencias1 and Facultad de Medicina2, UAEM. Cuernavaca Morelos, Mexico. Phone: (777) 3 29 70 00 Ext. 6030 [email protected],

Cholera, which is characterized by voluminous watery diarrhea, is produced when the gram-negative curved bacillus Vibrio cholerae colonizes the upper small intestine of its human host. Two major virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP), play major roles in the pathogenesis of this infection. The two major biotypes of V. cholerae, classical and El Tor each require ToxR and ToxT for activation of virulence factors, and potential regulatory sequences are largely conserved between the two biotypes. Nevertheless, the mechanism controlling expression of toxT appears to be biotype specific, because classical strains express CT and TCP under a wide range of experimental conditions, while El Tor strains require specific growth conditions, termed AKI, for detectable expression of the ToxR regulon. Such conditions comprise a biphasic culture where vibrios are first statistically grown for a 4-h period and then shifted to shaking. However, previous work indicated that CT synthesis is induced in single phase still cultures if the surface area of the exposed culture is increased and shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype by enhancing ToxT activity. On the other hand, proteomic studies indicated that the ambient oxygen concentration plays a role in V. cholerae virulence gene expression and at the molecular level CT expression is controlled by AphB, which induces expression of TcpP, TcpP activates synthesis of ToxT and ToxT directly enhances transcription of cistrons encoding CT. It has recently been shown that the activity of AphB is increased under low oxygen concentrations and the activity of ToxT is induced by exogenous bicarbonate. Both of these responses are compatible with stimulation of CT synthesis by AKI conditions. In an attempt to more precisely define how growth conditions may function to induce expression of CT, in this work we followed the kinetics of consumption of dissolved oxygen during bacterial growth; results suggest a working model that may account for induction of CT expression under both AKI and non-AKI conditions.



Gene expression of Pht cluster genes and a putative non-ribosomal peptide synthetase required for phaseolotoxin production is regulated

by GacS/GacAin Pseudomonas syringaepv. phaseolicola. De la Torre-Zavala, Susana1, Hernández-Flores, José Luis1, Aguilera Selene1, Hernández-Morales, Alejandro, Alvarez-Morales, Ariel1 1 Departamento de Ingeniería Genética de Plantas. CINVESTAV-Irapuato. Km 9.6 Libramiento Norte, carretera Irapuato-León. Irapuato, Gto. México. C. P. 36821. Tel.: +52 (462) 623 96 00 ext. 439, email: [email protected] Pseudomonas syringaepv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This toxin inhibits the enzyme ornithine carbamoyltransferase involved in the arginine biosynthesis pathway produced at low temperatures (18-20°C). Phaseolotoxin [N´-(N´-sulphodiaminophosphinyl)-ornithyl-alanyl-homoarginine] biosynthesis and regulation have not been determined, although it has been demonstrated that genes withing the Pht cluster are required for phaseolotoxin production. To achieve full pathogenicity and virulence, the bacterium expresses numerous pathogenicity and virulence factors that lead to a successful invasion and infection. Signal transduction pathways coupling the sensing of an environmental signal to modulation of the expression of the relevant target genes frequently involve the use of two-component systems, consisting of sensor histidine kinases coupled to their respective response regulators to modulate de expression of transcriptional factors that activate functionally-related genes. Among these, the GacS/GacA two-component system, which is widespread in Pseudomonas spp. and other Gram-negative bacteria, plays a major role in the regulation of important pathogenicity and virulence factors. However, it was previously reported that GacS did not exert control over phaseolotoxin production. In the present study we hybridized a genomic microarray of P. syringaepv. phaseolicola NPS3121 to compare transcriptional profiles from wild type strain and a gacA-null mutant with a Tox- phenotype. Our results show that GacA controls expression of genes within the Pht cluster as well as another group of clustered genes located in a different region in the bacterial chromosome and it contains at least one gene that we unambiguously showed to be directly involved in phaseolotoxin biosynthesis. Our results suggest that this cluster is a new pathogenicity island containing genes whose regulation is also under the GacA regulatory cascade.



Structural, phylogenetic and functional analysis of the isocitrate lyase from Pseudomonas aeruginosa

Díaz-Pérez1, A. L., C. Díaz-Pérez1, C. R. Sosa-Aguirre1, and J. Campos-García1* 1Instituto de Investigaciones Químico-Biológicas, UMSNH. Edificio B-3, Ciudad Universitaria, CP 58030, Morelia, Mich., México. *[email protected] Isocitrate lyase (ICL) is an enzyme that plays an essential role in Pseudomonas aeruginosa when grows on fatty acids, acetate, acyclic terpenes and amino acids, such as leucine, channelizing their final metabolites to the glyoxylate pathway [1]. In P. aeruginosa, ICL is encoded by the aceA gene (ORF 2634) [1]. In the present work we carried out the molecular modeling, phylogeny and functional analysis by site directed mutagenesis of P. aeruginosa ICL. The phylogenetic analysis suggests that ICL superfamily is divided in two families, the AceA (constituted for 5 subfamilies) and the PrpB ICLs. The ICL from P. aeruginosa belongs to subfamily 3, showing the characteristic QIENQVXDEKQCGHQDGK fingerprint. Molecular modeling of ICL shows two particular domains, named D3 and D4, which are missing in the subfamily 1, subfamily 2 and PrpB´s. Alignment of fingerprint and active site showed that Q211, N214 and Q221 were unique for subfamily 3. ICL from P. aeruginosa was expressed as His-tag recombinant protein showing a molecular mass of 68 kDa. The recombinant ICL at 30oC and pH 7.5 as optimal conditions shows a Michaelis-Menten behavior with kinetic constants of Vmax of 29 mmolmin-1mg-1 and Km of 0.82 mM for isocitrate. Site directed mutagenesis for residues Q211H, N214D, E219A, and Q221K, conserved in the ICL subfamily fingerprint, indicated that N214 is essential for enzymatic activity at least in P. aeruginosa. E219A not shows effect, while the change Q221K caused an increment in ICL substrate affinity. Mutations in Q211H and Q221K caused shift in optimal temperatures toward the thermofilic region, which was implicated with a reduced thermostability of ICL at 45oC, suggesting that these residues are implicated in the thermodynamic properties of ICL.



NOTES

Oral Session III. Tuesday


Metabolic diversity among Bacillus isolates from the sediment of the Churince system in Cuatro Ciénegas, Coahuila

Rodríguez-Torres, María Dolores1, Islas, Africa1, Souza, Valeria2 and Olmedo Álvarez, Gabriela1 1 Departamento de Ingeniería Genética de Plantas. CINVESTAV-Irapuato. Km 9.6 Libramiento Norte, carretera Irapuato-León. Irapuato, Gto. México. C. P. 36821. Tel.: +52 (462) 623 96 00 ext. 439, email: [email protected] 2Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, CU, AP 70-275 Coyoacán 04510 México DF. Although there is more evidence of the great genetic diversity of microorganisms, the ecological interpretation of such diversity is not understood. Classical microbiological studies rely on the analysis of single isolates to infer the function of bacterial populations. Recent studies have revealed that there is diversity even among taxonomic groups considered to be the same species, and this has made researchers question some ecological theories. There are mainly two ecological theories to try to explain why organisms assemble in a particular place and why trophically similar species can co-occur. The theory of the classical niche indicates that species within a community differ in their niches, while the neutral theory proposes that similarity can explain their great diversity in the natural communities. Within the sediment of the Churince water system, in the valley of Cuatro Ciénegas, Coahuila, a large number of bacteria of the Bacillus genus have been found. This is hard to explain according to the theory of competitive exclusion, in which two species in biological competence for a resource cannot coexist. We analyzed 158 Bacillus from this system, representing 9 taxonomic groups. We undertook different analysis to determine the metabolic diversity, from sugar utilization, growth response to different glucose concentrations, growth rate, and ability to produce biofilm. Mothur was used to evaluate the metabolic properties relative to the genetic distance of the different Bacillus species in the communities. Our results revealed that there is metabolic diversity even within taxonomic groups, thus suggesting that these isolates may occupy a different ecological niche.



Integration Host Factor is a global regulator required for electron

transference in Geobacter sulfurreducens Katy Juárez, Ana Lilia Tirado, Sonia Dávila, Alfredo Mendoza, Enrique Morett, Ángel Andrade Instituto de Biotecnología. UNAM. Av. Universidad #2001, Colonia Chamilpa. 62210 Cuernavaca, Morelos, México. (+5255)5622 7641. [email protected] Geobacter species are the predominant Fe(III)-reducing microorganisms in a diversity of subsurface environments (1). The insoluble nature of Fe(III) oxides requires that Geobacter species transfer electrons outside the cell via a long-range electron transfer mechanism in order to reduce Fe(III) (2). In addition, most Geobacteraceae, can also use properly poised electrodes as electron acceptors and produce electricity (3). The 54 seems to play an essential role in G. sulfurreducens physiology (4). The expression of 54-dependent promoters requires efficient communication between enhancer proteins (EBP) and bound promoter RNA polymerase, which are usually separated by 100–140 bp. It is generally believed that the heterodimeric protein, IHF (integration host factor) binds and induce DNA bending between the promoter enhancer region lowering the energetic cost of DNA looping therefore facilitating the RNA polymerase-EBP communication (5). On account of the relevance and number of 54 promoters in G. sulfurreducens (4) and one of the organisms sequenced to date that presents more EBPs, we hypothesized that IHF plays a relevant role in G. sulfurreducens physiology and electron transfer. For this reason, in this work we decided to carry out a genetic characterization of IHF of G. sulfurreducens. Firstly, we noticed duplicity in G. sulfurreducens ihf subunits (denominated as ihfA-1, ihfA-2, ihfB-1 and ihfB-2), which are codified in different operons. In order to analyze ihf genes are transcribed, we carried out a semi-quantitative RT-PCR assay, the results showed a higher level of ihfA-1 and ihfB-2 over the two other ihf copies. The functionality of ihfA-1 and ihfB-2 was confirmed through the generation of G. sulfurreducens null mutant strains. The phenotype observed was impaired to reduce fumarate or Fe(III) citrate in both mutants, in contrast with the ihfB-1 mutant that showed no phenotype. Finally, as multiple cytochromes are involved in electron transfer in G. sulfurreducens we decided to evaluate the cytochrome profile in ihf strains; and consistently with our previous results, we observed a remarkable deficit in ihfA-1 and ihfB-2 strains on the subcellular compartments analyzed. However, the cytochrome profile in ihfB-1 strain was similar to the wild-type strain. Overall these results demonstrate a relevant role of IHF in G. sulfurreducens physiology and electron transference. References 1. D. E. Holmes, K. T. Finneran, R. A. O'Neil, D. R. Lovley, Appl Environ Microbiol 68, 2300 (May, 2002). 2. S. Srikanth, E. Marsili, M. C. Flickinger, D. R. Bond, Biotechnol Bioeng 99, 1065 (Apr 1, 2008). 3. D. R. Bond, D. R. Lovley, Appl Environ Microbiol 69, 1548 (Mar, 2003). 4. C. Leang et al., BMC Genomics 10, 331 (2009).

5. K. K. Swinger, P. A. Rice, Curr Opin Struct Biol 14, 28 (Feb, 2004)..



Vibrio cholerae cytolysin VCC probably causes pyroptosis to THP-1

macrophages by activating MAPKs signaling pathway

Figueroa-Arredondo P1, Pérez-Franco V1, Enríquez-Rincón F2, Luna-Arias JP2 and Pedraza-Alva G3 1 Programa Institucional de Biomedicina Molecular de la ENMH-IPN Avenida Guillermo Massieu Helguera 239 Fracc. La Esacalera, Ticomán D.F. CP 07320. México D.F., 2Departamento de Biología Celular and Laboratorios Centrales del Cinvestav Campus Zacatenco and 3 Instituto de Biotecnología IBT-UNAM Cuernavaca Morelos Méx. Corresponding author: 1 [email protected]

In this work we would like to find out how the pore forming toxin of Vibrio cholerae VCC, is interacting with macrophages by establishing the activation of molecules along the way of the MAPKs signaling pathway and pyroptotic cell death.

Pore forming toxins are bacterial products that usually self assemble as oligomers that eukaryotic cells uptake by endocytosis, destabilizing the normal vesicular traffic showing striking vacuolation of the cytoplasm. Interactions of pore forming microbial toxins, with innate immune system cells such as macrophages and dendritic cells, today has not being fully understood. Therefore, eukaryotic cell interactions with the toxin may be well described using in vitro models as useful tools to study the signaling activated with this toxin.

The innate immune system evolved to detect pattern recognizing receptors (PRR). They in turn, identify molecular motifs from pathogens named PAMPs (pathogen associated molecular patterns). Toll like receptors also called TLRs, are an important branch of PAMPs and they are capable to detect damage associated molecular patterns (DAMPs) such as signals for cellular damage or even thr presence of bacterial toxins. TLRs also may induce the cell to pyroptosis, a lytic programmed cell death undergone by cells that have initiated activation of the inflammasome. The inflammasome represents tha molecular platform that early senses danger to the cell and initiates non specific defense mechanisms of the cell.

Objective. The goal of this study is to gather in vitro experimental evidence of molecular interaction of the Vibrio cholerae cytotoxin (VCC) with innate immune cells such as macropahages.

In vitro assays were performed using purified VCC. Treatments with the toxin were applied to the cells and then incubated overnight (aprox.18 h); challenged cells and supernatants were collected separately and tests were performed. Cytotoxic in vitro assays were performed as follows: THP-1 macrophages were chalenged with 20 ng per ml of the toxin in 24 well plates until showing 80-100% vacuolization, microscopically at 40X in a Nikon TS100, then photodocumented using the NIS (Nikon image software). We chose to first identify elements of the MAPKs signaling pathways by conventional Western blot, we found out that ERK is activated by treatments with the toxin. Cell lysis suggesting pyroptosis, was also observed with VCC intoxication, then estimated by an enzymatic assay that showed how lactic dehidrogenase (LDH) was released to the medium. We were capable to reproduce in macrophages the vacuolating effect elicited by VCC, then determined that toxin treated cells activate ERK, one of the kinases from MAPKs signaling pathway, and subsequently cells undergo pyroptosis, presumably by releasing LDH. Therefore, from above results may we suggest that the VCC initiates assembly of the inflammasome through the MAPKs signaling pathway activating ERK and then pyroptosis is induced. Whether this toxin interaction with macrophages is started by TLRs, still remain to be studied.



NOTES

Oral Session IV. Wednesday


Quorum Quenching Quandary: Resistance to Antivirulence Compounds Rodolfo García-Contreras4,5, Toshinari Maeda1,3, Wendy Rangel4 , Mingming Pu1, Lili Sheng1, L. Rene Garcia2, Maria Tomás6, and Thomas K. Wood1,2 1Department of Chemical Engineering and 2Department of Biology, Texas A&M University, C. S., Texas, USA 3Department of Biological Functions and Engineering, Kyushu Institute of Technology,

Kitakyushu, JAPAN 4Department of Biochemistry, National Institute of Cardiology, México DF, MEXICO 5Department of Molecular Cell Physiology, VU University, Amsterdam, THE

NETHERLANDS 6Unidad Investigación-Microbiología, C. H. U. A Coruña INIBIC, La Coruña, SPAIN [email protected], phone: 55732911/1517

Background: Quorum sensing (QS) is the regulation of gene expression in response to the concentration of signal molecules, and its inactivation (quorum quenching) has been suggested to have great potential to attenuate microbial virulence since QS activates the expression of multiple virulence factors and it is assumed that there should be low selection pressure for resistance against QS inhibitors. However, this assumption is suspect because it is based in growth in rich medium. Methods: Using Pseudomonas aeruginosa, and inhibitors of the N-acyl homoserine lactone QS systems (C-30) and the quinoline dependant QS system (AA analogues), it was shown that resistance to QS inhibitors can arise easily. To generate conditions in which QS inhibition could affect growth, growth on adenosine as the sole carbon source to select strains resistant to C-30 was used, and growth with low iron to select resistance to AA analogues. Adenosine was used since its utilization requires QS and is related to disease in the GI tract, and low iron since the expression of siderophores requires QS. Multiple QS related phenotypes were studied, and a C. elegans virulence model. Results: Growth on adenosine was inhibited with C-30, and within 15 generations, both transposon and spontaneous resistant mutants were found. Mutations were identified in mexR and nalC, which are repressors of the mexAB-oprM multi-drug resistance operon; inactivation of MexR produces efflux of C-30. The mexR mutant was pathogenic to C. elegans in the presence of C-30 whereas virulence of the wt strain was decreased. Critically, P. aeruginosa strains found in cystic fibrosis infections have the same mutations and also show resistance to C-30. In the presence of AA analogues, growth in low iron was inhibited, and resistant mutants were obtained. The mutations are being identified. Conclusion: Single mutations in key loci give rise to resistance against QQ compounds. Therefore, bacteria may readily have resistance to many new pharmaceuticals that are under development under the rationale they are impervious to resistance.



Study of protein-protein interaction in the RNA Degradosome of Escherichia coli upon conditional mRNA degradation

Jaime García-Mena, Candelaria Merino-Jiménez, Lilianha Domínguez-Malfavón Departamento de Genética y Biología Molecular. Cinvestav-IPN Unidad Zacatenco. Av IPN #2508, Col. Zacatenco. México DF 07360. +52(55) 5747-3800 X5328. [email protected]

The RNA Degradosome (RNAD) of Escherichia coli is a macromolecular complex involved in regulated mRNA degradation composed of the endoribonuclease E (RNase E), whose carboxy-terminal domain serves as a molecular scaffold for the organized assembling of the phosphopyruvate dehydratase enzyme (enolase), besides to an ATP-dependent RNA helicase (RhlB), and an Pi-dependent exoribonuclease called polynucleotide phosphorylase (PNPase). Although it has been extensively documented the importance of the functional role of additional resident proteins of the RNAD in vivo, it has been shown that in vitro, this complex is capable of conducting RNA degradation, with a minimal assembly including only RNase E, RhlB, Enolase and PNPase. In our work we have been studying the RNAD of E. coli, using functional fusions to fluorescent proteins of the minimal resident enzymes and following fluorescent signals and performing fluorescence resonance energy transfer assays (FRET). Using this approach we have been able to show that this complex indeed assembles in vivo in bacteria in defined foci. In the present work and by using a combination of FRET assay and qPCR analysis, we present data illustrating that important rearrangements occur in the interaction among the resident components of the RNAD, upon conditional degradation of the ptsG mRNA. This transcript encodes the major glucose transporter and is rapidly degraded in an RNase E−dependent manner in response to the accumulation of glucose 6-P (or fructose 6-P) when the glycolytic pathway is blocked. Our data strongly suggest an involvement of the RNAD complex in vivo in bacteria in ptsG mRNA degradation in addition to just the endoribonuclease E. We believe this could be a general mechanism of degradation for additional transcripts in the cell. This work was partially supported by CONACyT and Cinvestav-IPN.



Genetic diversification and genome size diversity of Mexican Escherichia coli

Andrea González1, Luna Sánchez1, Gabriela Delgado2, Luis E. Eguiarte1, Valeria Souza1 1 Departamento de Ecología Evolutiva, Instituto de Ecología, UNAM, México. 2 Departamento de Genómica Bacteriana, Facultad de Medicina, UNAM, México.

Bacterial genome evolution is driven by different genetic processes such as mutation, homologous and non-homologous recombination, genomic rearrangements, deletions and duplications. These mechanisms are responsible for genetic diversity which explains how evolutionary forces shape and extinguish bacterial lineages, the process of adaptation to new niches and the mechanisms of population differentiation. In this context, patterns of genetic variation will depend on the degree of population clonality. It is therefore necessary to consider the extent of mutation and recombination in the genetic diversification of a species. Escherichia coli exist as both a free-living organism or as pathogen or commensal within the colons of mammals and birds. The objective of this study was to determine the evolutionary mechanisms that generate population diversity and structure of commensal and pathogenic E. coli strains from different ecological niches in Mexico. This was investigated by using Pulsed Field Gel Electrophoresis and Multilocus Sequence Typing to screen a collection of 128 isolates. Specifically, this collection was derived from wild mammalian and avian hosts, as well as environmental isolates from mud, water and from both healthy and diseased humans. We found intermediate levels of genetic diversity under stabilizing selection. Overall, this appeared to be predominantly linked to recombination and to a lesser extent, point mutations during genetic diversification. Comparing the Mexican sample with E. coli MLST database, we found that Mexican isolates do not represent a restricted subset of E. coli strains and also comprise new allelic forms. Great variability in fingerprints, chromosome size and megaplasmid number were identified throughout the sample. No remarkable differences, however, were detected between either commensal or pathogenic strains. Consequently, this data suggests that levels of genetic diversity facilitate the adaptation of Mexican strains of E.coli to different ecological niches. Furthermore, the commensal strains may serve as a possible reservoir, harboring potentially adaptive factors in this case.



Molecular mechanisms participating in the biogenesis of the type III secretion system of enteropathogenic Escherichia coli

Mariana Romo-Castillo, Ángel Andrade, Elizabeth García-Gómez, Julia Monjarás, Norma Espinosa and Bertha González-Pedrajo Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ap. Postal 70-243, México, D.F., 04510. [email protected] Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. It colonizes the small intestine producing a characteristic histopathology referred to as the attaching and effacing (A/E) lesion. All the factors needed for the development of the A/E phenotype are contained within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). The LEE encodes a type III secretion system (T3SS or injectisome) used to deliver virulence effector proteins directly into enterocytes. Translocated effectors subvert diverse host cellular processes leading to the intestinal disease. The T3SS is composed of more than 20 different proteins that assemble into a syringe-like structure consisting of a multi-ring base that spans both bacterial membranes, and a needle-like projection and filamentous extension that protrudes out of the cell from the bacterial surface, and serves as a conduit for the translocation of effectors. A fundamental component of all T3SSs is a highly conserved ATPase that is thought to energize the secretion process. It provides a docking interface for chaperone-effector complexes and it is proposed to induce chaperone release and unfolding of the secreted protein in an ATP-dependent manner. EscN is the ATPase associated with the T3SS in EPEC and it is essential for the virulence of this bacterial pathogen. In order to have a better understanding of how ATPase activity is regulated, we have isolated EscN non-functional mutants and characterized the protein interactions in which this enzyme is engaged. Here we show that EscO, an essential component of the T3SS, interacts with EscN and regulates its enzymatic activity. A molecular dissection of this interaction will be presented. Our results show that the ATPase activity of EscN needs to be up-regulated in order to achieve protein secretion.



Transcriptional and posttranscriptional controls involved in the

synthesis of flagellin in Rhodobacter sphaeroides

Manuel González-Vera, Clelia Domenzain-Reyna, Aurora Osorio, Sebastián Poggio, Georges Dreyfus and Laura Camarena*

Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City,Mexico *Correspondingauthor: Dept. de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, U.N.A.M. México, D. F., 04510 México It has been shown that two different flagella are assembled in Rhodobactersphaeroides. One single polar flagellum encoded by a cluster of flagellar genes known as fla-1; and several polar flagella that are encoded by other flagellar cluster named fla-2. Under the growth conditions commonly used in the laboratory, the flagellar genes (fla2) are silenced. However, mutants that allow fla2 expression can be isolated in a fla1- genetic background. Phylogenetic analysis indicated that the fla1 genes were acquired through an event of horizontal transfer probably from a gamma proteobacteria; whereas the fla1 are the endogenous genes of R. sphaeroides. The regulatory mechanisms underlying flagellum 1 (Fla1) biogenesis have been characterized. In contrast, there is no information regarding the mechanisms that control flagellar formation of Fla2. Flagellin is the most abundant flagellar protein,there are about 30000 flagellin subunits forming the filament. Given that an important amount of energy is required to synthesize flagellin, its expression and synthesis are tightly regulated. To determine the transcriptional activity of the flaA promoter, the upstream region of flaA was fused to the reporter gene ‘lacZ. This transcriptional fusion gave -galactosidase activity in the Fla2+ strain. However, a reduction in the amount of the enzyme was observed if the genes encoding for proteins of the early flagellar structure were affected. A reduction in the amount of flagellin detected by inmmunoblotwas also observed by western blot. Mapping of the transcription start site of flaA revealed a UTR region of 120 bases, suggesting post-transcriptional regulation. In Caulobactercrescentus and Brucellamelitensis, which are also a-proteobacteria, this post-transcriptional regulation is mediated by the FlbT protein. A copy of the flbT geneis present in R. sphaeroides. In order to determine if it is involved in the regulation of flagellin expression, mutant strains with an insertion in flbT were isolated. The mutants were non-motile, and an important reduction in the amount of flagellin was detected byimmunobloting but the -galactosidase activity from the flaA-‘lacZ fusion was not affected. Based on these results we propose that flagellin is controlled by two different mechanisms; one of them controls the transcription level of flaA, and a post transcriptional mechanism that modulate flagellin synthesis.



NOTES

Oral Session V. Wednesday


FadD is required for utilization of endogenous fatty acids released

from membrane lipids Isabel M. López-Lara*1, Ángel Pech-Canul1, Joaquina Nogales2, Alfonso Miranda-Molina3, Laura Álvarez3, Otto Geiger1, and María José Soto2

1Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México. Cuernavaca, Morelos,Mexico. 2Estación Experimental del Zaidín, CSIC,Granada, Spain 3Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico. *Programa de Ecología Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México. Apdo. Postal 565-A, Cuernavaca, Morelos. C.P. 62210. Mexico. E-mail: [email protected] FadD is an acyl-coenzyme A (CoA) synthetase responsible for the activation of exogenous long chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobiummeliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. melilotifadD mutants accumulate a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool together with the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase are in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulate free fatty acids released from membrane lipids in the stationary phase. This phenomenon does not occur in a mutant of E. coli in the fatty acid transporter FadL, suggesting that the accumulation of fatty acids is not the result of cellular lysis. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed in S. meliloti revealed that a functional FadD is required for the up-regulation of genes involved in fatty acid degradation and suggest that in the wild type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth .



RpoS regulator of stationary phase, controls transcriptional expressions of phbR gene and phbBAC operon witch are responsible for PHB production in Azotobacter vinelandii

Castellanos, M., Hernández Eligio, J. A., Moreno León, S., and Espín, G. Instituto de Biotecnología UNAM. Av. Universidad No. 2001 Col. Chamilpa, Cuernavaca Morelos, Mex. [email protected] The sigma factor RpoS is a central regulator during stationary phase in bacteria. In A. vinelandii RpoS is involved in the formation of mature cysts and is also required for survival under oxidative stress conditions. Poly-hydroxybutyrate (PHB) is a polymer synthesized by a wide range of microorganisms and is used to synthesize plastics that are biodegradable, biocompatible and do not cause toxicity. Synthesis of PHB in A. vinelandii occurs entering the stationary phase from three enzymatic reactions, which are encoded in the operon phbBAC. Upstream and the opposite direction to phbBAC operon is the phbR gene, which encodes for a protein AraC/XylS family member. Recently it was found that mutations in rpoS and phbR gene affect negatively the PHB production. By S1 nuclease protection experiments, was observed that the rpoS and phbR mutations affect the transcripts of the phbBAC operon and phbR gene in exponential and stationary phase. Additionally, in the phbBAC-phbR intergenic region were found 6 possible binding sites for PhbR protein. Therefore, the objective of this work was study the transcriptional regulation of the phbBAC operon and phbR gene mediated by PhbR and RpoS regulators. To exclude that the negative effect on PHB in the mutant strains is due to a polar effect, first we carried out complementation of the phbR and rpoS mutant strains and confirming that the PHB defective phenotype was due to the phbR and rpoS mutations. By RT-qPCR and transcriptional fusions we observed a strong decrease of the expression of phbB and phbR genes in the mutant strains in exponential and stationary phase. By EMSA experiments, we observe in vitro interaction of PhbR with 4 of the 6 proposed sites in the intergenic region phbBAC-phbR and by footprinting assay we delineate the binding sequence of PhbR 18 nt (TGTCACCAA N-4 CANTA). With these results, we propose that PhbR and RpoS activate the expression of phbBAC and phbR; RpoS directly activating one of two promoters phbBAC and indirectly to phbR. PhbR directly activates the first promoter of phhBAC and itself indirectly (phbR).



Analysis of the environmental conditions involved in regulation of

chr genes from Burkholderia xenovorans

Rafael Jiménez, Karina Salinas, Martha I. Ramírez and Carlos Cervantes [email protected]. Instituto de Investigaciones Químico-Biológicas, UMSNH, Morelia Michoacán.

The CHR superfamily comprises transporters that confer chromate resistance by extruding toxic chromate ions from cytoplasm. The relatively large genomes of species of the β-Proteobacteria Burkholderia genus commonly encode multiple CHR homologs of different subfamilies. The B. xenovorans LB400T genome (9.7 Mpb) encodes six CHR homologs, including four long-chain proteins (ChrA1a, ChrA1b, ChrA2, and ChrA6) and two short-chain proteins (Chr1NCa and Chr1NCb) (Díaz-Pérez et al., 2007). Previously was found that the six CHR homologs from B. xenovorans LB400T confer chromate resistance to Escherichia coli, although in a differential fashion (León-Márquez, 2009). It was also found that expression of most CHR homologs in B. xenovorans is repressed by high concentrations of sulfate and induced by chromate exposure (Luna-Luna, 2010). With the objective to analyze whether the six chr systems from B. xenovorans were expressed differentially in response to environmental growth conditions, in the present work we made transcriptional fusions of chr regulatory regions to the promoterless lacZ gene and its expression was monitored in the heterologous host Pseudomonas aeruginosa PAO1. The expression of each fusion was analyzed in different culture media, temperatures and in the presence or absence of chromate. We found that three transcriptional fusions (chrA1a-lacZ, chr1NCa-lacZ and chrA1b-lacZ) were expressed at very low levels or not expressed in any growth condition, whereas the other three (chrA6-lacZ, chr1NCb-lacZ and chrA2-lacZ) were expressed at considerably higher levels than the others. The highest expression was observed in the late exponential phase of growth and the major level of expression was observed for the chrA6 fusion whereas the lower level was for the chr1NC fusion. From the analysis of expression at two different growth temperatures, 30°C and 37°C, the chr genes were affected in response to this change, chrA6 was expressed about 20 times higher when the cultures were grown at 37°C with respect to 30°C; chrA2 was expressed two times higher at 37°C, whereas the opposite was observed for the expression of chr1NCb which was 2 times higher at 30°C. When bacteria were grown in different culture media as LB broth, nutrient broth (NB) and minimal medium (MM), transcriptional fusions were expressed at different levels, although the differences were more discrete than the changes observed by the effect of temperature; the most notable effect was observed for the chrA6 gene, which was expressed 2.5 times higher in LB and NB with respect to MM. Even more, we observed that in the presence of chromate the expression of the gene fusions was modified, indicating that it could act as modulator of chr expression. Our results indicate that the chr systems from B. xenovorans are expressed differentially in response to environmental conditions, suggesting that the redundancy of these systems ensures a functional chrA system in any condition.



Analysis of the Rhizobium etli stimulons under heat shock and saline stress by RNA-Seq

López-Leal Gamaliel*1; Mendez-Jimenez Cyntia1; Santillan-Godinez Orlando1; Miguel A. Ramírez Romero1 1Programa de Genómica Evolutiva, Centro de Ciencias Genómicas-UNAM/ Av. Universidad s/n Col. Chamilpa. Fax: 01(777) 3175581. correo electrónico: [email protected]

The more distinguishing characteristic of bacterial growth is the ability to adapt in different stress condition. Investigation on the control stress response revels that transcriptional control mechanism are of paramount importance. In Escherichia coli (Szalewska-Palasz 2007. et al), It has been shown that the alternative sigma factor RpoH plays a crucial role in the response of heat shock (Nonaka 2006. et al), as well as in oxidative stress, DNA damage, phage infection, among others stress (Nover 1991.). Rhizobium etli is a nitrogen fixation α-proteobacteria which inhabit the soil and has 22 alternative sigma factors. An important aspect of Rhizobium etli is the presence of redundant alternative sigma factors (González 2006. et al), which in that it, appear to be involved in particular stress condition, for example we have observed that the gene rpoH1 and rpoH2, which encode the alternative sigma factors RpoH1 and RpoH2 respectively, are essential in different stress conditions, because a deletion in rpoH1 results in cell-viability loss during heat shock, furthermore bean plants nodulated by this strain are unable to fix nitrogen. On the other hand it has been shown that a deletion in the rpoH2 gene is lethal in NaCl2 80mM growth conditions (Martínez-Salazar 2009. et al). With this observation it can be assumed that the participation of these sigma factors is condition-dependent. In order to analyze the RpoH-dependent sigmulon, we studied RNA-seq data under heat shock (42°C) and salin (NaCl280mM) stress conditions. Our results suggest that the response to these stresses can be mediated by plasmids and a small fraction of the chromosome. We observed that the plasmids that contribute with more expressed genes in the heat shock stress are plasmid A and plasmid F; and the plasmids A, D and F contribute in the salin stress. This peculiarity was also observed in the growth kinetics of strains without plasmids. References Szalewska-Palasz A, Wegrzyn G, Wegrzyn A. Mechanisms of physiological regulation of RNA synthesis in bacteria: new discoveries breaking old schemes. J Appl Genet. 2007;48(3):281-94. Review. Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA. Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress. Genes Dev. 2006. 20(13):1776-89. Nover L. Inducers of HSP synthesis: heat shock and chemical stressors. In L. Nover (Ed). Heat Shock Response. CRC Press.1991. 5-40. González V, Santamaría RI, Bustos P, Hernández-González I, Medrano-Soto A, Moreno-Hagelsieb G, Janga SC, Ramírez MA, Jiménez-Jacinto V, Collado-Vides J, Dávila G. The partitioned Rhizobium etli genome: genetic and metabolic redundancy in seven interacting replicons. Proc. Natl. Acad. Sci. 2006 103(10):383. Martínez-Salazar JM, Sandoval-Calderón M, Guo X, Castillo-Ramírez S, Reyes A, Loza MG, Rivera J, Alvarado-Affantranger X, Sánchez F, González V, Dávila G, Ramírez-Romero MA. The Rhizobium etli RpoH1 and RpoH2 sigma factors are involved in different stress responses. Microbiology. 2009.155(Pt 2):386-97.



The regulatory network of Pseudomonas aeruginosa and its

comparison with other bacterial networks Edgardo Galán-Vásquez1, Beatriz C. Luna1 y Agustino Martínez-Antonio1

1Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Campus Guanajuato, Irapuato 36500, México. [email protected]

Pseudomonas aeruginosa is a Gram-negative bacterium and one of the most important opportunistic pathogens, in humans it is able of cause mortal infections in patients with immunodeficient systems, cystic fibrosis, and severe burns.1 This makes of P. aeruginosa the bacterium most studied about pathogenic and virulence determinants and the third bacterial model in its molecular biochemistry only after of E. coli and B. subtilis. The regulation of genes is an important process as determine its metabolism, adaptive and pathogenic capacities. The regulatory interactions among transcription factors, sigma factors, anti-sigma factors and target genes can be represented in the form of transcriptional regulatory networks as we made in this work.2

Methodology We search and cure regulatory interactions from original published papers on P. aeruginosa. Following, we reconstruct and determine the functional structure of the network from P. aeruginosa and compare it with those from B. subtilis and E. coli 3, 4. Results Until May of 2010 we got from literature a network of 1020 regulatory interactions with 690 genes; this includes 76 transcription factors, 14 sigma factors (9 of them with extracytoplasmic function -ECF- ), 7 anti-sigma factors and 593 non-regulatory genes5. The network obtained represents the 12% of predicted genes in P. aeruginosa; it follows a scale-free network. Most of regulatory interactions in the network are positive and 38% of the transcription factors self-regulate, 55% of them in a positive way. We also identified the main functional modules in the network: for alginate biosynthesis, iron metabolism, motility, biofilm, virulence factors and antibiotic resistance all them coordinated by the quorum sensing. Finally, we use the software CONSENSUS to identified possible consensus sites for 38 regulators in the network of P. aeruginosa and identified orthologous genes among the networks of P. aeruginosa, E. coli y B. subtilis . References 1) Yeung A et al. J Bact. 2009, 5592-5602. 2) Björn H. & Falk S. Wiley Interscience. 2008. 3) Martínez-Antonio A & Collado-vides J. Curr Opin Microbiol. 2003, 482-489. 4) Martinez-Antonio A et al. J Mol Biol. 2008, 381(1):238-247. 5) Galán-Vásquez E. et al. Microb. Inform. & Exp. 2011, 1:3



NOTES

Oral Session VI. Wednesday


Chemotaxis signaling pathway of the flagellar system 2

from Rhodobacter sphaeroides.

Ana Martínez del Campo1, Teresa Ballado1, Javier de la Mora1, Laura Camarena2 and Georges Dreyfus1 1Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. Apdo. Postal 70-243, Cd. Universitaria 04510.México, D.F. Tel. 56225618. E-mail: [email protected] 2Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Rhodobacter sphaeroides is a purple non-sulfur bacterium that belongs to the alpha subgroup of proteobacteria. It is considered a metabolically diverse bacterium, since it can grow aerobically and anaerobically, and can also photosynthesize and fix nitrogen. R. sphaeroides genome encodes two flagellar gene clusters. The genes that belong to the first cluster are expressed constitutively under laboratory conditions and are required for the synthesis of the well-characterized single subpolar flagellum (Fla1); which allows the swimming of the WS8 wild-type strain. The genes of the second flagellar cluster are not expressed in the wild-type strain, however strains that express it can be isolated. In contrast with the case for other dual-flagellum systems, the Fla2 genes of R. sphaeroides produce polar flagella that are required for swimming. In addition to having two flagellar systems, R. sphaeroides posses several reiterated chemotactic genes: 4 cheW (adaptor protein), 2 cheB (methylesterase), 3 cheR (methyltransferase), 4 cheA (kinase) and 6 cheY (response regulator), which are encoded in three operons: cheOp1, cheOp2 and cheOp3. Only cheOp2 and cheOp3 are essential when the cell is swimming with the fla1 flagellum. By contrast CheY1, CheY2 and CheY5, encoded incheOp1, are required for the chemotactic control of the Fla2 flagellum. This evidence suggests that cheOp1 could be involved in the tactic control of the flagellar system 2. To test this hypothesis we proceeded to individually mutate each gene encoded in this operon (RSP2444, mcpB, tlpS, mcpA, cheD, cheX, cheA1, cheW1, cheR1 and RSP2432) in two different genetic backgrounds: Fla1+Fla2− and Fla1−Fla2+. We analyzed the swimming behavior of all the mutants by soft agar motility assays and computer cell tracking. Mutants in the Fla1+Fla2− background have the same phenotype as the wild-type strain, while the same mutants in a Fla1−Fla2+ background show a non-chemotactic phenotype. The experimental evidence suggests that the chemotatic signalling pathway of the Fla2 flagella is similar to the pathway found in Escherichia coli, with the exception of the methylation system. In this work, we demonstrate that when R. sphaeroides swims with Fla2 flagella the cheOp1 controls its chemotactic behavior; and constitutes its signaling pathway.

This work was supported by CONACyT (106081) and DGAPA (IN213408).



Overexpression of 4.3 protein of phage mEp021 induces pleiotropic effects like inhibition of cell division, release of basal haemolysin

HlyE and cell death

Martínez-Peñafiel Eva1, Ishida Cecilia2, Fernández-Ramírez Fernando3, Reyes-Cortés Ruth2, Sepúlveda-Robles Omar1, Guarneros-Peña Gabriel1, Bermúdez-Cruz Rosa Ma.1 and Kameyama Luis1* 1Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN. 2Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN; Av. Instituto Politécnico Nacional No. 2508, C.P. 07360, México D.F., México. 3Departamento de Genética, Hospital General de México, SSA. Dr. Balmis No. 148, Col. Doctores. C.P. 06726, México, D.F. Mexico Phone: 57 47 38 00 Ext: 5365 E-mail: [email protected] mEp021 is a temperate coliphage isolated from human feces. Its main feature is the induction of a haemolytic phenotype in its lysogen. Haemolysis depends on host HlyE hemolysin and it has been detected when a 77 aa protein (i.e. ORF 4.3) of mEp021 is overexpressed. This protein is encoded in ORF 4, which has the following features: 4 putative AUG start codons (ORF 4.3 protein starts at the third one) and no consensus Shine-Dalgarno sequence (SD). We suggest that in vivo translation of ORF 4 depends on upstream ORF 3 translation, since the latter harbors a typical SD and both ORFs are overlapped. In addition to the haemolytic phenotype, ORF 4.3 expression induces the arrest of cell division, observed since the second hour of induction, leading to the formation of different bacterial structures such as filaments, bacterial ghosts, minicells and vesicles. Released vesicles carry the basal haemolysin, producing the lysis of erythrocytes. None of the other proteins translated from ORF 4 (ORF 4.1, -4.2 and -4.4) induced these activities. Microarray data are consistent with the phenotypic observations, showing altered expression of genes related to cell shape regulation, among others. In addition, we present evidence that ORF 4 peptides display toxic effects, leading to reduced bacterial viability.



Characterization of multiple arrangements of binding sites of

transcription factors in Escherichia coli K-12 and their mechanisms of action in gene regulation

*Peralta-Gil Martin1, Muñiz-Rascado L. 1, Guzmán J. 2 and Julio Collado-Vides1. 1Program on Computational Genomics, Center for Genomic Science UNAM, Cuernavaca, Mor., México, 2Institut of Biotechnology UNAM, Cuernavaca Mor. Méx. *Corresponding author: *[email protected] RegulonDB is one of the most important databases worldwide, and it is focused primarily on the transcriptional regulation of Escherichia coli K-12. This database contains approximately 300 transcription factors (TFs), and only 179 have been characterized experimentally. To better understand the mechanisms of action of the TFs, we have analyzed 1,988 binding sites for 152 different TFs. In E. coli, binding sites of the TFs (TFBSs) are represented by small regions of DNA, from 7 to 24 bp on average, and their function depends on the central position with respect to the transcription start (+1). Our analysis has identified, for the first time, the length, symmetry, orientation, and consensus sequence binding sites for 129 TFs of E. coli. In some cases the lengths of the binding sites are variable and can have different orientations. Other important features of the binding sites are specific variations in the sequences and therefore in their affinities, their distribution in tandem or overrepresentation of motifs, presence of false sites, overlapping and cooperativity of binding sites, and combinatorial mechanisms. The symmetries of the TFBSs show that regulators in this microorganism tend to form dimers as a regulatory strategy, to expand the region of contact with the DNA and increasing specificity. Twelve percent of the TFs bind as monomers to asymmetric regions, while 4% of the TFs can bind as monomers or dimers, and the highest percentage (82%) corresponds to TFs that function as homodimers that bind to symmetric regions. A smaller percentage, 2%, binds as heterodimers to asymmetric regions. The role of TFs is linked with the central position of the binding site, in relation to the initiation of transcription and its interaction with the promoter. The central positions of the binding sites are closely linked with the turns of DNA and the RNA polymerase phase; for this reason, we propose a restructuring of the classification proposed by Aiba in 1989, this classification includes only the CRP-dependent promoters. In conclusion, all these biological characteristics of TFBSs are important for deciphering the mechanisms of action of the different regulators and understanding both the simple and the complex regulatory rules. This knowledge will allow us to identify the frames of regulation, as well as build more accurate computer models for both E. coli K-12 and for related microorganisms.



NOTES

Oral Session VII. Thursday


Effect of phaG gene expression on polyhydroxyalkanoate structure synthesized by a recombinant strain of Azotobacter vinelandii.

Miguel Mejía, Carlos Peña, Guadalupe Espín y Daniel Segura Departamento de Microbiología Molecular, Instituto de Biotecnología, UNAM Apdo. Post. 510-3, Cuernavaca, 62210 Morelos, MÉXICO Tel: (777) 3 29 16 29, e-mail: [email protected] Azotobacter vinelandii produces polyesters known as polyhydroxyalkanoates (PHAs) that are of industrial interest, because it can be used as biodegradable plastics. This bacterium produces polyhydroxybutyrate when it is grown on carbohydrates, and a copolymer of hydroxybutyrate and hydroxyvalerate on pentanoate and heptanoate. However, Pseudomonas putida is able to synthesize PHAs with different hydroxyalkanoates from carbohydrates. In this case the formation of precursors for the synthesis of PHAs occurs through the synthesis of fatty acids. In P. putida, the link between the fatty acids synthesis pathway and the synthesis of PHAs, is provided by a β-hydroxyacyl ACP:CoA acyltransferase. The aim of this study was to construct a recombinant strain of A. vinelandii capable of expressing the β-hydroxyacyl-ACP:CoA acyltrasferase in order to produce different types of bioplastics from carbohydrates with controlled monomer composition. To express the enzyme ACP:CoA acyltransferase a system was implemented that allows the control of the expression of phaG (the gene coding for this enzyme) from P. putida in A. vinelandii. phaG was fused with the promoter of scrX (pscrX), which is inducible by sucrose, and it was integrated in the chromosome. The recombinant strain produced a polymer composed of hydroxybutyrate units under non-inducing condition. When phaG was induced, there was a notorious effect on the monomer composition of the polymers produced, twelve hours after phaG gene induction, the PHA monomer composition was changed, obtaining a copolymer composed of hydroxybutyrate-co-hydroxyhexanoate units (~ 90 and 10% respectively). To assess the possibility to manipulate the type of PHA synthesized by the recombinant strain, we evaluate different concentrations of the inducer to control the level of phaG gene expression. This control allowed to change the percentage of hydroxyhexanoates of the co-polymer in a range of 1 to 10 % only by modifying the concentration of sucrose added. However, phaG gene expression had a negative effect on the growth of the strain, causing a decrease in the biomass. These results indicate that the ACP:CoA acyltransferase from P. putida is functionally active in A. vinelandii and is sufficient to confer the ability to use intermediaries of the pathway for fatty acids synthesis and produce novel PHAs in this bacterium, allowing the generation of new bioplastics.



Identification of FikR, the response regulator that completes the fixL-

fixK cascade in Rhizobium etli CFN42

David Zamorano-Sánchez1 Alma Reyes-González1,2, Nicolás Gómez-Hernández1, Patricia Rivera1 and Lourdes Girard1 1Centro de Ciencias Genómicas, UNAM. 2Facultad de Ciencias, UAEM. Ap. Postal 565-A, Cuernavaca Mor, 62271, México. Telephone: 777 3291896. E-mail: [email protected] Rhizobium etli CFN42, the Phaseolus vulgaris symbiont, employs a complex regulatory model for fix genes with several novel characteristics when compared to the conventional systems, Sinorhizobium meliloti and Bradyrhizobium japonicum. Some important regulatory elements described in otherrhizobia, such as fixL and fixJ, are not found on the pRet42d (symbiotic plasmid) in R. etli CFN42. fixL encodes an orphan histidine kinase located on a pRet42f region containing copies of the fixK, fixNOQP and fixGHIS genes. fixNOQP reiterations are controlled by a novel fixL-fixKf cascade, where the transcriptional activator of fixKf remains to be described (Girard et al., 1996; Girard et al., 2000; Gonzálezet al., 2003; Gonzálezet al., 2006). A similar distribution of fix genes is shared by R. leguminosarum bv. viciae VF39 (Patschkowskiet al., 1996). Previous efforts in our lab to identify a response regulator related to FixJ in R. etli by cross hybridization and transposon mutagenesis were unsuccessful. In this work expression analysis, coupled with mutation of different response regulators located on pRet42f, let us identify a new response regulator indispensable for fixKf activation in microaerobiosis. We present genetic evidence that supports that the R. etli CFN42 ORF RHE_PF00530 encodes a previously uncharacterized fixKf regulator (FikR). We also identified a FikR-like protein in R. leguminosarumbv. viciae that was able to substitute for FikR transcriptional control of fixKf in a R. etlifikR mutant strain. We propose that in addition to FixL, FikR is a regulatory element indispensable for the activation of fix genes in R. etli in response to oxygen. We thank M.P. Elizabeth Salas and Rosa Ma. Ocampo for their excellent technical assistance. D. Zamorano-Sánchez, and A. Reyes-González received a doctoral scholarship from CONACyT. Partial financial support was provided by PAPIIT-UNAM (grant IN202109)



Characterization of the molecular mechanisms producers of drug resistance in Mycobacterium tuberculosis

Cuevas-Córdoba B.1, Gonzalez O.1, Rios A.2, Cuellar A.2, Zenteno-Cuevas R.1*. 1Instituto de Salud Pública, Universidad Veracruzana. Xalapa, Veracruz, México. 2Laboratorio Estatal de Salud Pública de Veracruz. *[email protected]. T. (01) 228-8418934 (13321) Introduction: The drug resistant phenomenon in tuberculosis (TB-DR) explains by the presence of mutations or changes in some genes. In such a way in rifampin (R) is involved the gene rpoB; the genes katG and inhA to isoniazid (I), embB to etambuthol (E) , rrS to Streptomycin (S) and pncA to pyrazinamide (P) . The frequencies of these changes are different according to the geographical locations. Have this information is important for the development and evaluation of existing genotypic diagnostic tools. For that reason the goal of this work was to characterize the changes presented in the genes associated to the TB-DR, in clinical isolates of tuberculosis from Veracruz, México. Methods: From patients with a suspicious of been affected by a TB-FR, the mycobacteria was isolated and the drug resistant profile was determined by BACTEC or MGIT. DNA was purified, and the specific locus of rpoB, katG e inhA, embB, rrS y pncA, were sequenced by capillary electrophoresis (AG 3500, Applied Biosystems 3500), allowing a maximum error in the assignation of the nucleotide base inferior to the 1%. Results: From 2007-2011, 167 isolates were collected. 67% showed resistance to S, 83% to I, 53% to E and 38% to P. 66% were MDR and 24 SIREP. 387 fragments were sequenced. 71% of the 56 fragments from rrS (S), not showed mutations, and the most frequent mutation was at codon 513 (8%). 30% of the 80 fragments of katG (i) had mutations at codon 315. 17% of the 77 fragments of inhA showed mutations in six codons. 67% of the 71 fragments of rpoB (R) had mutations at codon 531. 37% of the 61 fragments showed mutations at codon 306 of embB. Finally, 81% of the 42 fragments of pncA (P) showed deletions (4), insertions (4) and mutations (18). Conclusions: 20% of the mutations found have not been referred in the literature. Also there was a low percent of mutations in the key codons; considered like the typical marker for drug resistance. This information evidence a low sensibility in the implementation of the traditional molecular diagnostic tools. Finally shows an important diversity of drug resistant isolates circulating in the state, conforming a complex epidemiological landscape.

Funded by CONACyT-COVECyT No. 95819. C-CB was a CONACyT fellow No. 171183...



Regulatory circuits controlling asymmetrical cell division in bacteria César Quiñones-Valles2 and Agustino Martínez-Antonio1 CINVESTAV Unidad Irapuato, Departamento de Ingeniería Genética, Km. 9.6 Libramiento Norte Carr. Irapuato-León 36821, Irapuato Gto. México, Tel. 52(462)6239673, [email protected], [email protected]

The cell division is a natural process that allows bacteria to increase or maintain their population and persist on time. This process normally generates two identical cells from a mother cell. Some species of bacteria, however, developed a mechanism of asymmetric cell division. This phenomenon is characterized by the generation of two daughter cells with morphological and functional differences between them even when originated from the same mother cell. One example of this is observed in Bacillus subtilis, which under certain environmental conditions, like nutritional stress, this bacterium divides asymmetrically giving place to the formation of an endospore. The spore is a cellular form that is extremely resistant to adverse environmental conditions. The molecular mechanism that regulates this process includes several sigma and transcription factors acting in a hierarchical cascade triggered by a master regulator. Other organism that presents asymmetric cell division is Caulobacter crescentus. This bacteria can divides in two morphologically different cells: the motile flagellated cell, where DNA replication is silenced; and the other one non motile, this last type remain attached to some solid surface and is the only one that can divides and generates the two cell types. The two cell types differ in the form they execute the regulatory programs for cell division. The systems of control are globally mediated by some transcriptional factors like: DnaA, GcrA y CtrA. Here we shall present our advances on the compilation and analyzes of the regulatory networks controlling asymmetric cell division in B. subtilis and C. crescentus, and compare it to E. coli as an organism which not presents asymmetric cell division but where the most is known about the regulation of cell division. Key references: Holtzendorff, J., Hung, D., Brende, P., Reisenauer, A., et al. (2004). Oscillating global regulators control the genetic circuit driving a bacterial cell cycle. Science, 304, 983-987. Li, S., Brazhnik, P., Sobral, B., & Tyson, J. J. (2009). Temporal Controls of the Asymmetric Cell Division Cycle in Caulobacter crescentus. PLoS Computational Biology, 5(8), e1000463. Quiñones-Valles, C., Espíndola-Serna, L., and Martínez-Antonio, A. (2011). Mechanisms and Controls of DNA replication in bacteria. Chapter 7: DNA replication/Book1. InTech, In press Acknowledgements: CQ-V thanks CINVESTAV-IPN and UAA for a PhD scholarship. This work is partially supported by the CONACYT grant 102854 give to AM-A.



Evolutionary history and niche differentiation of Exiguobacterium, a halophilic bacterial genus living at Cuatro Cienegas Basin, Mexico

Rebollar Eria A.1, Eguiarte Luis E1, Mora Lucy2 and Souza Valeria1

1Instituto de Ecología, Universidad Nacional Autónoma de México, 2Instituto de Geología, Universidad Nacional Autónoma de México. Correo Postal: Circuito exterior s/n anexo al Jardín Botánico Exterior, Apartado Postal 70-275 Ciudad Universitaria, UNAM 04510 México, D.F. Correo electrónico: [email protected] Tel:(5255)56229006 Prokaryotes are both the most diverse and abundant organisms in the planet. The microorganisms have played essential biological and geochemical functions since the origin of life on earth. One of the central subjects in evolutionary microbiology is to understand the reasons for this enormous genetic, metabolic and ecologic diversity. The Cuatro Cienegas Basin (CCB) is part of the chihuahuan desert and it comprises several oligothrophic aquatic systems with contrasting environmental conditions. The heterogeneity of this place make it an excellent model to explore the role of ecological and geographical factors in the diversification and adaptation of prokaryotes to different environments and the molecular mechanisms that allow population divergence and differentiation. We studied the molecular evolution of 183 isolates of the Exiguobacterium genus. This genus is comprised by halophilic and alcaliphilic non-sporulating bacteria and is extremely abundant in the sediments and water from the CCB aquatic systems. The complete sequence of the 16srRNA gene and four partial housekeeping genes (citC, rpoB, recA and hsp70) were obtained. Based on the concatenated phylogeny of the four coding genes we defined three main phylogroups with contrasting genetic diversity values. These phylogroups are not specific to an aquatic system or a determined salinity. However the phylogenetic reconstruction indicates the existence of several small clusters composed by isolates from sediment or water only. In addition, Unifrac and AdaptML analyses revealed a clear differentiation pattern associated to sediment and water habitats. This work supports previous observations of niche differentiation in other bacterial lineages and represents the first work showing ecological divergence associated to water and sediment habitats.



NOTES

Oral Session VIII. Thursday


Characterization of a null mutant in the mazG gene of Rhodobacter sphaeroides

Sarmina Leonel, Gloria Alejandra; Celis Sandoval, Heliodoro y Suaste Olmos, Fernando Instituto de Fisiología Celular, UNAM. [email protected], Circuito Exterior S/No. Ciudad Universitaria. Coyoacán PO Box 70-243, Zip Code 04510, México D. F. Tel. 56225667

Over the last two decades, programmed cell death (PCD) has been observed on unicellular organisms including bacteria (1). In prokaryotes, this event is related to a toxin-antitoxin module, composed of two major genes; mazE and mazF. A third ORF called mazG has been recently discovered; it is located downstream the mazEF genes, and encode for a protein whit nucleotide pyrophosphohydrolase activity (2). This gene, is transcribed in the same polycistronic mRNA as mazEF, suggesting relation with PCD.

The mazG enzyme catalyzes the hydrolysis of guanosine 3', 5’-bispyrophosphate (ppGpp), synthesized by relA under amino acid starvation conditions. This process is involved in the regulation of the stringent response (3). The phototrophic bacteria, Rhodobacter sphaeroides, does not produce ppGpp under amino acid starvation, however, this compound is synthesized under other stimulus like light intensity downshift.

In order to understand the biological role of mazG in Rhodobacter Sphaeroides under stress conditions, a strain of this bacterium in which mazG gene has been mutated by deletion, was characterized.

1. Aizenman, E., Engelberg-Kulka, H. and Glaser, G. 1996, PNAS, Vol. 93, pp. 9059-

9063. 2. Gross, M., Marianovsky, I. and Glaser, G. 2006, Mol Microbiol, Vol. 59, pp. 590 601. 3. Boutte, C. and Crosson, S. 2011, Mol Microbiol, Vol. 80, pp. 695-714.



Genetic diversity of phages of Pseudomonas aeruginosa: isolation,

characterization and identification of new species Sepúlveda-Robles O., Kameyama-Kawabe L. y Guarneros-Peña G. Genetics and Molecular Biology Department.CINVESTAV-IPN.Av. Instituto Politécnico Nacional 2508. Col. San Pedro Zacatenco. C.P. 07360. México, D.F. 14-740, 07000. Phone: 5747-3800 Ext. 5352. E-mail: [email protected] Pseudomonas aeruginosa is an opportunistic pathogen and a major nosocomial problem due to itsmultiple-resistance to antibiotics. We aimed atusing phagesin search of a quick way to identify P. aeruginosa variants and, eventually, for treatment of infected patients. We isolated and characterized 68 Mexican bacteriophages that infected clinical isolates of P. aeruginosa. Electron microscopy studies showed that most of the phages have the typical tail and head morphology of DNA phages, belonging to the order Caudovirales and to the families Siphoviridae, Podoviridae, and Myoviridae. Only four were small tail-less isometric head RNA phages belonging to the Leviviridae family. Through the analysis of the phages host range against 142 P. aeruginosa clinical isolates, we identified ten phages capable of lysing the 79.5% of the strains. Subsequent strain typification could be based on the susceptibility to infection with other phages in our collection. The analysis of the tailed-phage DNAs by RFLP, cross hybridization, electron microscopy and host range allowed to classify them in ten species. The comparative analysis of the genomic sequences of phages, representing each of these species, against the sequences of the P. aeruginosa phages deposited in GenBank, defined five new species. With the discovery of these new species we expanded by 50% P. aeruginosa known phage diversity. We plan to use our phage collection to quickly identify strains of P. aeruginosa prevalent in México City hospitals hoping to abbreviate the time to apply the appropriate patient treatment. Acknowledgments: We specially thank to Ma. de Lourdes Rojas Morales and Sirenia González Pozos for technical assistance in electron microscopy. This work was supported by Instituto de Ciencia y Tecnología del Distrito Federal (ICyTDF) and CONACyT grants.



Construction of a prototype sensor kinase from the hybrid kinase TodS

of Pseudomonas putida DOT-T1E Hortencia Silva-Jiménez*, Juan Luis Ramos and Tino Krell Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain. Phone: +34958181600, ext. 118. *e-mail: [email protected] Two-component systems (TCS) play key roles in the adaptation of bacteria to environmental changes. In prototype TCSs a single phosphoryl transfer between the sensor kinase and response regulator occurs, whereas phosphorelay TCSs are characterized by a His1-Asp1-His2-Asp2 phosphorylation cascade. It remains unclear why some processes are regulated by prototype systems whereas others by phosphorelay systems. The phosphorelay TodS/TodT TCS controls the expression of a toluene degradation pathway. TodS is composed of 7 domains and its activity involves an internal phosphorelay prior to TodT phosphorylation. Based on the phosphorelay TodS/TodT TCS we report the construction of a minimal form of TodS (Min-TodS) composed of only 3 domains. We report a comparative analysis of the phosphorelay TCS with its prototypal derivative. Using microcalorimetry we demonstrate that Min-TodS binds TodS effectors tightly. Min-TodS forms a TCS with TodT and increases the amount of TodT-P in response to toluene. Toluene does not stimulate Min-TodS autophosphorylation but increases TodT phosphorylation. The half-life of Min-TodS-P was significantly increased as compared to TodS. Analysis of TodSD500A revealed that the hydrolysis of the acylphosphate of the receiver domain is responsible for the reduced half-life of TodS. The regulation of PtodX expression by Min-TodS/TodT and TodS/TodT in response to different effectors are compared. The Min-TodS/TodT system was characterised by a higher basal activity but a lower magnitude of response. The TodS agonist ortho-chlorotoluene had an agonistic action on Min-TodS. To our knowledge this is the first study of its type and reveals mechanistic details between both types of TCSs.



Function of ornithine lipids in Agrobacterium tumefaciens

Miguel Á. Vences-Guzmán,1 Ziqiang Guan,2 Otto Geiger1 and Christian Sohlenkamp1 1 Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Apdo. Postal 565-A, Cuernavaca, Morelos, CP62210, Mexico. 2 Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

Ornithine lipids (OLs) are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α -amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. It has been shown that OLs can be hydroxylated within the secondary fatty acyl moiety and within the ornithine moiety. These modifications have been related to increased stress tolerance and symbiotic proficiency in Rhizobium tropici. Analyzing the membrane lipid composition of the plant pathogen Agrobacterium tumefaciens we noticed that it forms two different OLs, corresponding to the unmodified OL S1 and the hydroxylated OL S2. In the present work we wanted to study if OLs play a role in stress tolerance and pathogenicity in A. tumefaciens. Mutants deficient in the OLs biosynthesis genes olsB and olsE were constructed and characterized. They either completely lack OLs (olsB-) or only form the unmodified OL (olsE-). Here we present a characterization of both OL mutants under stress conditions and in a plant transformation assay using potato tuber discs.



Metagenomics and Microbial Diversity

Roche Diagnostics Applied Science Av. Santa Fe 485-4 piso Col. Cruz Manca Santa Fe 05349 México D.F. Tel. +52(0155) 50-81-58-22 Microorganisms have experienced around 3.8 billion years of evolution on earth and comprise the vast majority of biological diversity. Genetic characterization of these life forms helps us understand the physiological diversity, community, ecology and biogeochemistry. All these knowledge led researchers to the development of novel compounds and processes for biotechnology, pharmaceutical and other applications in industries. On the healthcare area for instance, the Human Microbiome Project was initiated to examine the microorganisms that human hosts with the objective to address the relationship between diseases and changes in the human microbiome. Advances in next-generation DNA sequencing technologies have created a new era for metagenomics, allowing comprehensive examination of microbial communities, even those comprised of uncultivable organisms. The high-throughput 454 pyrosequencing technology introduced possibilities for deeply sequenced data collections and strategies aimed at microbial identification via single genetic targets or whole-genome methodologies. Pyrosequencing has been successfully applied in a variety of applications, including genotyping and SNP analysis, microorganism identification, and point mutations detection in antimicrobial resistance genes to explore the presence of drug-resistant microbes. Long read lengths are the key to accurate assemblies and unambiguous pathogen detection when speed to result matters most. Proven applications include detection of novel organisms, discovery of unknown variants without prior knowledge of the organism, metagenomic pathogen surveys, and many more. 454 Life Science, a Roche company, has developed two long read systems with different throughput in order to suit accordingly costumers need. The GS Junior System can process in parallel around 100.000 DNA fragments with 400 bp read lengths, and the GS FLX+ System produces approximately 1.000.000 fragments up to 1,000 bp lengths.



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Oral Session IX. Thursday


Genetics and genomics coming together to understand the methyl-

specific restriction system of Streptomyces coelicolor Luis Servín-González Instituto de Investigaciones Biomédicas, UNAM. Apartado Postal 70228. Cd. Universitaria, D.F. TF: 5622-8928. E-mail:[email protected]. Streptomyces coelicolor is a Gram positive filamentous actinomycete that has been the subject of intense studies in the past few decades, making it the best known streptomycete at the genetic and molecular biology levels. Its manipulation can be complicated by the fact that it harbors a potent methyl-specific restriction system (MSRS) that impedes entry of methylated DNA. This is in contrast to Streptomyces lividans, a closely related species that is very similar at the genomic level to S. coelicolor, but that does not exhibit methyl-specific restriction. Analysis of the S. coelicolor genome sequence revealed two genes that appeared to encode enzymes involved in restriction of methylated DNA that were absent from S. lividans, and that show similarity to Escherichia coli Mrr and McrA. Null mutations in these genes, however, showed little effect on the restriction of methylated DNA. We therefore used a positive selection method to isolate mutants in the MSRS, by introducing genes encoding DNA methylases into S. coelicolor. The sphM gene of Streptomyces phaeochromogenes, which encodes the M.SphI methylase, could be easily introduced into S. lividans, and resulted in methylation of SphI sites; expression of this gene in S. coelicolor, however, proved to be lethal due to antagonism with the MSRS. This lethality provided a positive selection method to isolate mutants affected in the MSRS. Classical genetic mapping of the mutation allowing survival in the presence of M.SphI methylation revealed that it was localized to a region of the chromosome where a large plasmid-like element is integrated into a tRNA gene. Further analysis revealed that the strain surviving the lethal expression of sphM had in fact lost the integrated element. This element carries several genes encoding putative nucleases, which were cloned individually in S. lividans. One of these nucleases was shown to be responsible for antagonism with the M.SphI methylase, whereas the other was shown to be involved in restriction of methylcytosine-containing DNA. Analysis of the S. coelicolor genome allowed the identification and deletion of additional genes encoding putative type IV nucleases, showing that the MSRS of S. coelicolor is complex. All these genes are localized in laterally acquired regions of the chromosome, implying that S. coelicolor, unlike S. lividans, evolved under selective pressure to restrict methylated DNA or to keep out methylation systems



Motility in Rhodobacter sphaeroides is mediated by a complex rotation system

Laura Camarena Instituto de Investigaciones Biomédicas, UNAM. Apartado Postal 70228. Cd. Universitaria, D.F. E-mail:[email protected] Bacterial flagellar motility is dependent on a motor that is embedded in the inner membrane. This motor can be divided in two functionally distinctive units: the rotor and the stator. In E. coli and other bacteria the stator is a proton channel formed by MotA(4)-MotB(2) that uses the proton motive force to provide the energy for flagellar rotation. MotB has a short cytoplasmic N-terminus, a single TM helix, and a large periplasmic domain. It has been proposed that the single TM domain of MotB together with TM3 and TM4 of MotA form a functional proton channel. The rotor can be divided in three different regions: the basal body, the hook and the filament. In the cytoplasmic region of the basal body, the FliG protein is required to generatetorque through its interaction with MotA. We recently found that a fliL strain of R. sphaeroides shows Mot- phenotype (the cells have a flagellum but are unable to rotate it). From the fliL strain we isolated eight independent pseudorevertants. In each of these strains a single nucleotide change in motB was identified affecting only three residues located in the periplasmic side of MotB. It has been suggested that this region acts as a plug of the proton channel. We envision that FliL could control the activity of the proton channel. However, double hybrid assays showed that the periplasmic regions of MotB and FliL do not interact, indicating that FliL may exert its function indirectly. Recent results suggest that other flagellar components could be involved in controlling the opening of the MotA/MotB proton channel. These components are: the membrane protein MotF, and the periplasmic protein MotP. motF is unique to R. sphaeroides strains, whereas motP is conserved in several gamma-proteobacteria. Mutants in any of these genes yield Mot- phenotype. Two pseudorevertants of motF, were located in motB; additionally the motB alleles isolated as suppressors of fliL also suppressed the Mot- phenotype of the motF strain. In contrast, the Mot- phenotype of motP was not fully suppressed by these alleles even though, in vitro,MotP and MotB interact. Together these results suggest that besides the MotA/MotB complex, FliL, MotF and MotP also promote flagellar rotation. If these proteins truly control the opening of the proton channel they could do it only once during flagellar biogenesis or constantly during swimming in order to modulate the rate of stop-start events that modulate swimming of this bacterium.



The RhlR regulon in Pseudomonas aeruginosa

quorum-sensing response Victoria Grosso-Becerra1, Gerardo Croda-García1, Enrique Merino-Perez2, Luis Servín-González1, Abigail González Valdez1, Marisela Aguirre-Ramírez3, Valentín Tejeda1 and Gloria Soberón-Chávez1* 1Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas Universidad Nacional Autónoma de México. Apdo. Postal 70228, 04510. México, D. F. Tel: 0155-56229201, 56229202. Fax: 0155-56229212.; 2 Departamento de Microbiología Molecular, Instituto de Biotecnología, UNAM; 3Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Chihuahua, México. *E-mail: [email protected]

Pseudomonas aeruginosa is a free-living -proteobacteria, but is also one of the most important opportunistic pathogens; it produces acute infections in immune-compromised individuals and is the first cause of death among Cystic Fibrosis patients. The production of many virulence factors by P. aeruginosa is regulated by the quorum sensing response (QSR). In this regulatory network LasR and RhlR bound to their corresponding autoinducers play a central role and operate through a hierarchical organization: LasR/3O-C12-HSL activates the transcription of rhlR and RhlR/C4-HSL activates the transcription of several genes, including the rhlAB operon encoding for enzymes involved in the production of the biosurfactant rhamnolipids. The rhlAB operon is located immediately upstream of rhlR; this gene presents four transcription start sites, two of which are located in the rhlB coding region. In this work we will present different aspects of the RhlR regulon, including the unique mechanism of RhlR transcriptional regulation and specificity for DNA sequences, as well as the role of different proteins and environmental factors on rhlR transcription



Gene Conversion in Rhizobiumetli: role of systems for initiation and migration of the Holliday junction

David Romero1, Mildred Castellanos2and Fares Osam Yáñez1

Centro de Ciencias Genómicas1 and Instituto de Biotecnología2, UNAM. Ave. Universidad 1001, Col. Chamilpa.62210, Cuernavaca, Mor. México. Tel. +52 (777) 3175867, e-mail [email protected] Gene conversion, the localized nonreciprocal transfer of information that occurs during homologous recombination, is recognized as the main process that maintains identity between members of multigene families. Genetic requirements of this process have been studied mainly in eukaryotes. However, prokaryotic genome sequences have revealed an astonishing wealth of sequence repeats (mean abundance 6.5%). This abundance, and the well-studied genetics of recombination in prokaryotes, makes these interesting models for the study of gene conversion.

To gain insight into the mechanism of gene conversion in Rhizobiumetli, we have studied the anatomies of tracts undergoing gene conversion in this organism. Our results revealed that

(i) Crossover events were almost invariably accompanied by a gene conversion event occurring nearby.

(ii) Gene conversion tractlengths ranged in size from 150 bp up to 800 bp. (iii) Geneconversion events displayed a strong bias, favoring the preservationof

incoming sequences. (iv) The MutS mismatchrepair system plays an important role in determining the

lengthof gene conversion segments. (v) The interplay between the systems in charge of migrating the Holliday

junction is one of the main factors that determine the length of gene conversion segments.

More recently, we have focused on the reasons for the strong bias, favoring

the preservation of incoming sequences, observed in gene conversion events. We have found that initiation mediated by double-strand breaks is the main reason for the observed bias.

Santoyo, G. and D. Romero. (2005). Gene conversion and concerted evolution in bacterial genomes.FEMS Microbiol. Revs. 29:169-183. Santoyo, G., J. Martínez-Salazar, C. Rodríguez and D. Romero. (2005). Gene conversiontractsassociatedwithcrossovers in Rhizobiumetli.J. Bacteriol., 187:4116-4126. Castellanos, M., and D. Romero. (2009). The extent of migration of the Holliday junction is a crucial factor for gene conversion in Rhizobiumetli. J. Bacteriol. 191:4987-4995.

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1

In vivo analysis of the interactions between SecY, YidC and FtsY in Escherichia coli

José Luis Aguilar•, Eli O. van der Sluis, Fabiola Jaimes Miranda•, Roland Beckmann and Soledad Funes• •Departamento de Genética Molecular, Instituto de Fisiología Celular, Circuito Exterior s/n Ciudad Universitaria 04510 UNAM (México) and Gene Center, University of Munich (Alemania) Tel. 5556225679 [email protected]

In gram negative bacteria the main pathway for protein insertion into the cytoplasmic membrane is mediated by the SEC system, a heterotrimeric membrane protein complex that consists of three proteins termed SecY, SecE and SecG. The essential function of this complex is to translocate proteins across the membrane in an unfolded state and to integrate the transmembrane segments into the lipid phase.

One of the mechanisms which delivers proteins to the SEC complex identifies the protein synthesis machinery at a very early stage of translation in order to guide them to the membrane where the translocation machinery is located. This mechanism ensures that the synthesis of the protein and its insertion can occur in a coupled manner. Cotranslational protein translocation in Escherichia coli begins with a targeting phase where the N-terminal of a nascent polypeptide emerging from the ribosomal exit tunnel is recognized by the signal recognition particle (SRP). Then, the ribosome-nascent chain-SRP complex is targeted to the membrane where SRP interacts with its receptor (FtsY). In a following step, the translation machinery is delivered to the SEC complex in order to translocate, fold or further assemble membrane proteins.

YidC is an essential membrane protein that facilitates the insertion of a subset of inner membrane proteins in E. coli. YidC works in a cooperative manner with the SEC complex stabilizing hydrophobic transmembrane alpha helixes after they are inserted into the membrane in order to release them as a properly folded protein into the lipid bilayer. YidC seems to function primarily in the insertion of small membrane proteins that need to assemble into larger membrane protein complexes.

Little is known about the dynamics of the physical interactions between the SEC translocon and YidC or the FtsY receptor. In order to address this functional issue, we have fused SecY to either YidC or FtsY and chose an in vivo approach which allows us to evaluate whether the interactions between these components are stable or not.



3

Study of HexR2, a transcriptional regulator of the Entner-Doudoroff pathway and alginate production in Azotobacter vinelandii E strain

Alejandrez Martínez Norma, Castañeda Lucio Miguel, Núñez López Cinthia Centro de Investigaciones en Ciencias Microbiológicas, BUAP, Edif. 103J, Ciudad Universitaria, Puebla, Pue., CP 72570. Tel: (222)2295500 Ext.2527. [email protected] Azotobacter vinelandii is a Gram-negative soil bacterium that is capable of fixing nitrogen under aerobic conditions. This bacterium produces a family of extracellular polysaccharides called alginates. A. vinelandii metabolizes the hexoses exclusively through the Entner-Doudoroff pathway (ED). Thus, when this bacterium grows in the presence of glucose the ED pathway provides the precursors for alginate biosynthesis.1 However, in A. vinelandii the molecular genetics and the regulation of the ED pathway is poorly understood. Previously, a mutant, named GG88, exhibiting higher specific alginate production was generated by random Tn5 mutagenesis. GG8 strain carried a Tn5 insertion within a gene (called hexR1) homologous to hexR of Pseudomonas aeruginosa, which encodes a repressor of the ED pathway.2 The predicted HexR1 protein of A.vinelandii showed 87% identity with its homologue in P. aeruginosa. There is evidence indicating that HexR1 directly controls the transcription of ED genes and indirectly regulates alginate production. Interestingly, in A. vinelandii the genes of the ED pathway are reiterated. We found a hexR paralogue, called hexR2, its deduced amino-acid sequence showed 82% identity to HexR1. The hexR2 locus also contains others ED genes and its genetic arrangement is highly conserved with respect to the hexR loci of P. aeruginosa and Pseudomonas putida.3 We generated a hexR2 mutant of A. vinelandii and found significant differences in growth, with respect to the GG88 and the wild type strains. The production of alginate appears to be also regulated by HexR2. Current work is focused on establishing the role of HexR2 in the regulation of the ED pathway; this will be conducted by determining the activities of the key enzymes in this pathway and by quantifying their corresponding mRNA by qRT-PCR. 1. Beale J., M. Jr., and Foster J. L. (1996) Biochemistry.35:4492-501. 2. Daddaoua A., Krell T., Ramos JL. (2009) Journal of Biological Chemistry. 32:21360-21368 3. del Castillo T., Duque E., Ramos JL. (2008) Journal of Bacteriology 190: 2331-2339.



5

Cloning, expression and glycosylation of the antigenic MPT83

lipoprotein of Mycobacterium tuberculosis in Streptomyces lividans.

Isaí Arista Carrera, Laura E. Córdova-Dávalos, G. González-Cerón & Luis Servín González Departamento de Biología Molecular y Biotecnología. Instituto de Investigaciones Biomédicas. UNAM. Apartado Postal 70228, Ciudad Universitaria, D.F. TF: (55)5622-8929. Correo electrónico: [email protected] MPT83 is one of the major antigens of Mycobacterium tuberculosis, and, together with MPT70, seems to be associated with tropism of this bacterium towards bone tissue. MPT83 is a glycosylated lipoprotein anchored to the cell membrane of M. tuberculosis, and is expressed at low levels compared to MPB83, its homolog from Mycobacterium bovis. Members of the genus Streptomyces are non-pathogenic actinobacteria that have the ability to glycosylate proteins using a mechanism that is homologous to that of M. tuberculosis; we therefore explored the use of Streptomyces lividans as a host for the high level expression of glycosylated MPT83. The gene for this protein (Rv2873) was amplified from M tuberculosis H37Rv DNA and cloned in the high copy number expression vector pIJ6021, either keeping its own signal peptide, which contains an anchoring signal or “lipobox” for signal peptidase II, or replacing it with the secretory signal peptide of a Streptomyces extracellular lipase. Immunoblot analysis with anti-MPT83 antibodies demonstrated the presence of the secreted protein in culture supernatants of S. lividans carrying either construct. In addition, the protein was present in membrane fractions from cultures carrying the construct in which the protein retains its own signal peptide and “lipobox”. We also showed by reaction with the mannose-specific lectin concanavalin A that the MPT83 protein obtained from Streptomyces cultures is indeed glycosylated.



7 Nitric oxide and chemotactic motility regulate biofilm formation in

Azospirillum brasilense. Arruebarrena Di Palma A 1,2, Moreno Ramírez L 1,2, Pereyra C2. Xiqui Ma. L.1 Creus CM2. and Baca BE1 1 Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla. Cdad Universitaria, Av. San Claudio S/N. Puebla Pue. México. 2 Facultad de Ciencias Agrarias. Universidad Nacional de Mar del Plata INTA Balcarce CC 276 (7620) Balcarce, Argentina. [email protected] The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. Bis-(39-59)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger controlling diverse bacterial responses such as the transition from free-living motile cells to biofilm lifestyle in bacteria. This molecule is synthesized by diguanylate cyclases (DGCs) [1], proteins that contain the GGDEF domain, and can be degraded by specific phosphodiesterases (PDEs) containing the EAL domain [2]. Nitric oxide (NO) is a pluripotent signaling and effector molecule that is implicated in a variety of biological roles [3]. We constructed A. brasilense Sp245 WT and its isogenic periplasmic nitrate redutase (Nap) minus FAJ0164 strain [4] harboring plasmid coding for the phosphodiesterase ChsA [2]. It has been described that FAJ0164 produces very little quantity of NO, when the bacterium was grown in a minimal medium with nitrate as N source [3]. To test for their ability to form a biofilm on an abiotic surface, the cells were allowed to grow on two minimal media supplemented with either nitrate or ammonium as N sources. The nap mutant had lower biofilm formation compared to that of the wild type, indicating that Nap positively regulates biofilm formation, probably through the NO production. Genetic interaction analysis revealed that a chsA, gene which control chemotactic motility in A. brasilense [2], showed higher biofilm formation in the genetic FAJ0164 background. It has been reported that motility is necessary for both biofilm formation on biotic and abiotic surfaces: Therefore our results suggested that chemotactic motility and NO production both are required for biofilm production. Acknowledgements: The authors are grateful for the financial support of the CONACYT- México and MINCyT-Argentina. References: 1.Paul R, Weiser S, Amiot NC, Chan C, Schirmer T, et al. (2004) Cell cycledependent dynamic localization of a bacterial response regulator with a novel diguanylate cyclase output domain. Genes Dev 18: 715–727. 2.Carreño-López C., Sánchez A., Camargo N., Elmerich C., Baca, B.E. ( 2009) Characterization of chsA, a new gene controlling the chemotactic response, in Azospirillum brasilense Sp7. Arch. Microbiol. 191:501-507. 3. Molina Favero C, Creus C M, Lanteri M L, Simontacchi M, Puntarulo S, Lamattina L. (2008) Aerobic nitric oxide producción by Azospirillum brasilense Sp245 and its influence on root architecture in tomato. Molecular Plant-Microbe Interactions, 7:1001-1009. 4. Steenhoudt O, Keijers V, Okon Y, and Vanderleyden. (2001). Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245. Arch. Microbiol. 175:344-352.



9

Characterization of Staphylococcus aureus isolates from bovine mastitis in backyard farms: evaluation of culture media, antibiotic

resistance and selection of polimorphic virulence genes

Gerardo Uriel Bautista Trujillo1, Irma Rentería Solórzano2, Saúl Carranza-Germán3, José Luis Solorio-Rivera3, Víctor Manuel Baizabal-Aguirre1, Marcos Cajero-Juárez1, Alejandro Bravo-Patiño1, Juan José Valdez-Alarcón1 1 Centro Multidisciplinario de Estudios en Biotecnología; 2 Unidad de Servicios de Apoyo al Diagnóstico; 3 Facultad de Medicina Veterinaria de la Universidad Michoacana de San Nicolás de Hidalgo. Email: [email protected]; Tel/Fax: (443) 295-8029. Centro Multidisciplinario de Estudios en Biotecnología, UMSNH

Palabras clave: Staphylococcus aureus, selective medium, antibiotic resistance, virulence genes

The cell envelope of Staphylococcus aureus contains surface virulence proteins. Because of its importance in the infection, efforts to identify genetic polymorphism in virulence genes between different hosts and the identification of clonal relationship using molecular biology and epidemiological parameters, have allowed to establish the molecular basis to search for control methods. Milk samples were collected from bovine mastitis and human of dairy farm backyard systems of Michoacán. Epidemiological parameters were determined and a phenotypic characterization of isolates by analysis of antibiotic resistance was performed. Final identification was made by partial sequencing the 16S rRNA gene. The prevalence of mastitis in 18 backyard farms of the central region of the state of Michoacán was 41.3%. We evaluated the appropriateness of four selective media to isolate bovine-related S. aureus strains. Salt-Manitol Agar (86 %) provided better sensitivity (P <0.001, Kappa 0.08) for S. aureus over Agar S-110 and CHROMagar StaphAureus® and Blood Agar (75%, 69% y 75% respectively). Most of the strains were resistant to penicillin, ampicillin and/or lincomycin. The MIC for oxacillin was 2 µg/ml thus leading to designation of isolates as methicillin sensitive. The presence of the mecA gene associated to methicillin resistance was not detected in our isolates. No resistance to vancomycin was detected. We aim to identify specific alleles of virulence genes encoding cell surface proteins of S. aureus that may be associated with specific hosts (humans or animals). fnbA, fnbB, clfA, clfB, sdrC, sdrE, sbi, efb, ebps and map, were adhesins selected by a bioinformatic analysis of in which nucleotidic diversity index (π) values were higher than 0.1 and dN/dS values were higher than 0.2. The polymorphism of these genes will be used to correlate with host specificity. .



11

Glycolipid synthesis in Agrobacterium tumefaciens under phosphorus-limiting conditions

José Roberto Bermúdez Barrientos, Miguel Angel Vences Guzmán, Christian Sohlenkamp Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apdo. Postal 565-A, Cuernavaca, Morelos CP62210, Mexico. Cellphone: 7771751175 Lab: (777) 3131697 Mail contact: [email protected], [email protected] Under conditions of phosphate limitation some bacteria are able to modify their membrane lipid composition: phospholipids such as phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine are replaced by phosphorus-free membrane lipids such as ornithine lipids, sulpholipids, diacyglyceryl- N,N,N-trimethyl) homoserine (DGTS), and less frequently glycolipids. Glycolipids are common and well studied in plants, animals and gram-positive bacteria but are rare in gram-negative bacteria where there is far less information. The plant pathogen Agrobacterium tumefaciens forms in addition to the phospholipids phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and cardiolipin two different ornithine lipids when grown under phosphate-replete conditions. Under phosphate-limiting conditions we observed that the amount of OLs does not change, whereas the phospholipids in general decrease and DGTS and several glycolipids are newly formed. As no genes involved in glycolipid biosynthesis under phosphate limitation had been described in gram-negative bacteria we chose a bioinformatic approach to identify genes possibly under the regulation of the transcriptional regulator PhoB. Among those PhoB-regulated genes were two putative glycosyltransferases. Both genes encoding glycosyltransferases were expressed in Escherichia coli. Mutants deficient in one or both glycosyltransferases were constructed in A. tumefaciens. Currently we are characterizing the mutants and we are trying to purify the glycolipids for a structure determination.



13

Analysis of the binding of ferredoxin NADP+ reductase (FNR) in the phycobilisomes of Tolypothrix PCC 7601 and

Fremyella diplosiphon UTEX B590 Tecilli Cabellos-Avelar1, Lourdes Elizabeth Leyva-Castillo2, Carlos Gómez-Lojero2, and Emma Berta Gutiérrez-Cirlos Madrid1 1Unidad de Biomedicina, F.E.S. Iztacala. U.N.A.M. Av. de los Barrios No.1. Col. Los Reyes Iztacala. Tlalnepantla, Edo. de México. C.P. 54090. Tel. 56231333 ext. 39783. Mail: [email protected]. 2Departamento de Bioquímica, CINVESTAV, Zacatenco, IPN. Av. Instituto Politécnico Nacional No. 2508. Col. San Pedro Zacatenco. México, D. F. C.P. 07360. Tel: 57473948. Mail: [email protected].

Cyanobacteria Tolypothrix PCC 7601 and Fremyella diplosiphon UTEX B590 can produce two alternative phycobilisome (PBS) rods depending on the light quality they are grown. PE-PBSs with one phycocyanin (PC) disk and phycoerythrin (PE) disks were found in cells grown under green light (GL). The PC-PBSs from cells grown under red light (RL) have only PC disks. In most cyanobacteria, the ferredoxin NADP+ reductase (FNR-3D) is attached to PBS. We are interested in studying how FNR binds to PBS adapted to different light intensities. The petH gene encoding (FNR) from F. diplosiphon was cloned in two different plasmids: pQE30UA that contains 6xHis tag at the N-terminus of the recombinant protein and pET3a to express the native protein.

In this study, we found FNR-3D attached to the PBS in the two cyanobacteria F. diplosiphon and Tolypothrix grown in RL or GL. A protein band of 46 kDa was identified by western blotting using PBSs from both species and probed with antiserum directed against A. maxima FNR. The FNR was associated in both types of PBSs from the two cyanobacteria. The PC PBS of both cyanobacteria have a cap in the rods that limits the amount of rFNR-3D that can bind the PBS. PE-PBS without a rod caps can receive up to six rFNR-3D per PBS. With these data we are able to propose a detailed model of how FNR binds to PE-PBS and PC-PBS.

Present data showed that the difference of binding of rFNR-3D to PC-PBS and to PE-PBS is consistent with the proposal that binding of FNR to the PBS is at the distal PBP with respect to the core. No more than two sites were available in the PC-PBS for FNR-3D, which, correspondingly, must have at least four sites blocked by a polypeptide linker. However, six sites are available for FNR-3D binding in the PE-PBS lacking the linker. Titration experiments using r-FNR-3D with a Tag of 6x- His (in the N-terminal side) were performed. This enzyme failed to bind to the PBSs of Tolypothrix or F. diplosiphon.



15

Role of ribosomal protein S1 in the binding of adenine-rich

mRNAs to ribosomes

Castañeda Montes María Azucena, Jacinto Loaeza Eva, Castillo Méndez Manuel, Guarneros Peña Gabriel y Hernández Sánchez Javier Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional. [email protected] Av. Instituto Politécnico Nacional 2508 Col. San Pedro Zacatenco, C.P. 07360 México, D.F. Apartado postal 14-740, 07000 México, D.F. Teléfono: 5747 3800 Ext: 5352 Adenines close to translational start codon promote protein synthesis. S1 ribosomal protein has also been associated with an increase in the translation of messengers, especially those containing adenines close the Shine-Dalgarno sequence. Therefore, it is important to analyze whether the high affinity of adenine-rich mRNAs for 30S subunit is promoted by the interaction with S1. To this aim, binding specificity was tested with synthetic messengers labeled with 32P, harboring adenines or guanines at codon positions 2 and 3 and at ORF´s +1, +2 or +3 triplets. Homopolymers of As or Gs were also analyzed. We found that adenine-rich messengers showed higher binding affinity to 30S subunit compared with the guanine containing messengers. Both, A and G homopolymers also presented a poor binding to 30S subunit. These results suggest that adenines close to start codon allow messengers to bind strongly to the ribosome in an independent codon context. S1 coding gene (rpsA) will be cloned and the expressed protein will be purified by affinity columns. Competition binding assays will be used to analyze whether S1 inhibits the binding of mRNAs with 30S subunits.



17

Prevalence of virulence genes in Escherichia coli strains isolated from

piglets in the suckling and weaning period in México

Ana María Castro, Andrea Toledo Rojas, Daniela Gómez, Celene Cruz, Rosalba Carreón, Silvia Giono Depto Salud Pública, Facultad de Medicina, Universidad Nacional Autónoma de México. e-mail: [email protected]. Tel: 56232149. Faecal samples from suckling (n= 503) and weaning piglets (n=450) with and without diarrhoea from 10 farms in México were examined for identification and prevalence of virulence genes of Escherichia coli. E. coli isolates were tested further for enterotoxin (LT, STa, STb STx1, STx2 and EAST1), fimbrial (F4, F5, F6, F17, F18 and F41), and eae adhesin genes by Multiplex PCR. Of the 953 isolates of E. coli examined by mPCR, 650 (68.2%) isolates were positive for at least one adhesin gene. Among the adhesin isolates, F41 adhesin (54%) was the most prevalent followed by eae adhesin (18.3%), F6 (8.6%), F5 (6.2%), F17 (6.0%), F18 (4.6%), and F4 (2.8%). Enterotoxin genes were detected in 424 (44.5%) of the isolates; from which STa (37.5%) and EAST1 (36.1%) were the most common, followed by STb (15.6%), Stx2 (6.0%) and LT (4.7%). Stx1 was found only in three of the isolates of E. coli (0.6%). The virotypes most prevalent were F41:STa, F41:EAST1, EAST1:eae, F41:F6, F41:STa:STb, STa: EaST1, STb:EaST1 and F41:eae. The present study examined for the first time the prevalence of 13 virulence genes among E. coli strains isolated from piglets with and without diarrhoea, in Mexico. The results suggest that there is a wide variety of virulence genes associated with diarrhoea in piglets. This study provides baseline information of the significance of specific virotypes associated with suckling and weaning period in piglets in Mexico.



19

Identification and characterization of the transfer region of plasmid PGR64a of Sinorhizobium fredii GR64

Laura Cervantes, Yaxal Ponce and Susana Brom Programa de Ingeniería Genómica. Centro de Ciencias Genómicas, UNAM. Av. Universidad 1001, col Chamilpa, Cuernavaca, Mor., México. Tel. (777) 329 16 91. email: [email protected] Rhizobium strains are able to develop symbiotic relationships with the roots of leguminous plants, through the formation of nodules where differentiated bacteria fix atmospheric nitrogen. Bacterial genetic information required for this process is usually localized on plasmids. We are interested in understanding the conjugative transfer mechanisms that participate in the generation of Rhizobium diversity. In Rhizobium etli CFN42 we have described a transfer mechanism for the symbiotic plasmid (pSym), through its cointegration with the self-transmissible endogenous plasmid pRet42a. Based on 16SrDNA analysis, bean-nodulating strain GR64, was classified as Sinorhizobium fredii, which usually nodulates soybean. The genome of S. fredii GR64 is constituted by a chromosome and 2 plasmids: pSfr64a (183 kb) and pSfr64b (~ 400 kb). Plasmid p64b is the pSym; pSfr64a is self-transmissible and required for conjugative transfer of pSfr64b (pSym). The sequence of pSfr64a shows approximately 40 kb shared with the R. etli CFN42 self-transmissible plasmid pRet42a, including the transfer region. Both plasmids contain traR and traM genes, and traCDG, traAFBH, trbEJKLFGHI, and traItrbBCD operons. The plasmids differ in three ORF’s encoding hypothetical conserved proteins, located between traH and traM. Also, pSfr64a lacks a cinR gene present in pRet42a. Conjugative transfer of plasmids from Rhizobium and Agrobacterium strains has been shown to be regulated by quorum sensing through traI and traR genes. Analysis of a GR64 derivative carrying a mutation in traR, showed a significant decrease in transfer frequency, suggesting that pSfr64a is also subject to quorum sensing regulation. In contrast, the transfer frequency of the traI mutant was not altered, implying the presence of additional functional traI genes located elsewhere in the genome. Interestingly, a mutation in the ORF147 encoding a hypothetical protein, completely abolished transfer of p64a, revealing a new gene involved in positive regulation of conjugative transfer. Plasmid pSfr64a was able to promote transfer of a construct carrying the oriT of pRet42a and viceversa, indicating that the oriT sites of pRet42a and pSfr64a may be processed by the relaxases of either plasmid. The data obtained indicate a similarity in the overall conjugative transfer regulation scheme of the R. etli and S. fredii transmissible plasmids, but also highlight notable differences, which may be related to their adaptation for functional expression in diverse hosts. Acknowledgments: To DGAPA for PAPIIT grant IN203109.



21

Comparison of secreted proteins and actin polymerization among diarrhea and control Enteropathogenic E. coli (EPEC)

strains isolated from children

Contreras C.A.1, Bustamante V.H.1, Ochoa T.J.2,3, Cleary T.G. 3 and Puente J.L. 1 1Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Av. Universidad 2001, Col. Chamilpa. Cuernavaca, Mor., 62210. Tel. (52) (777) 3291627; Fax. (52)(777) 3138673. [email protected] 2Universidad Peruana Cayetano Heredia, Instituto de Medicina Tropical Alexander von Humboldt, Lima, Perú. 3University of Texas School of Public Health, Houston, USA.

Background and aims: EPEC pathogenesis includes the production and translocation of bacterial proteins through a “needle complex” via a type III secretion system (TTSS), and actin polymerization-associated intimate attachment and pedestal formation. The aim of this study was to compare the TTSS-dependent secretion of E. coli secreted proteins EspA and EspB/D and of the EspC auto transporter and EPEC-induced actin polymerization evaluated by FAS (Fluorescent-actin staining) among diarrhea and control EPEC strains. Methods: 69 representative EPEC strains (46 diarrhea-associated and 23 controls) isolated from children under 1 year of age living in Lima, Perú, were analyzed for: Protein secretion (EspA, EspB/D and EspC) by SDS-PAGE and actin polymerization by FAS test in HEp-2 cells. Typical EPEC (tEPEC) strains were distinguished from atypical EPEC (aEPEC) by the presence of the bfpA gene as determined by PCR. Results: Secretion of the EspB/D proteins varied between the different isolates grown under standard secretion inducing conditions (e.g. DME medium at 37ºC): diarrhea-associated (DA) (25/46, 54%) and control (17/23, 74%) strains, as well as between tEPEC (10/13, 77%) and aEPEC (26/56, 46%). EspA was observed with similar frequency in DA and controls (22/46, 48% and 10/23, 43% respectively), but with higher frequency in tEPEC than in aEPEC (9/13, 69% vs 23/46, 50%). EspC was found with similar frequency among strains isolated from diarrhea cases and controls. Secretion of all translocator proteins EspA and EspB/D was more common in strains isolated from diarrhea cases than from controls (19/46, 39% vs. 2/23, 9%, p<0.05). FAS was positive for 16/46 (35%) DA strains vs. 4/23 (17%) for strains from control subjects, and for 9/13 tEPEC (69%) vs. 11/56 (20%) (p<0.05). Conclusions: Our findings indicate that there is high heterogeneity among EPEC strains isolated from Peruvian children. Few EPEC strains secrete all 3 type III translocator proteins (EspA and EspB/D) under the conditions tested, which are known to favor type III secretion in prototype EPEC strains; however, it correlates with diarrhea cases. The small proportion of strains that induce actin polymerization in HEp-2 cells correlate with the small frequency of tEPEC in our population.



23

Exploring the phenotypic diversity in the ability to sporulate of different groups of Bacillusfrom CuatroCiénegas, Coahuila

De la Torre-Zavala, Susana1, Rodríguez-Torres, Ma. Dolores1, Islas, Africa1, Souza, Valeria2 and Olmedo Álvarez, Gabriela1. 1 Departamento de Ingeniería Genética de Plantas. CINVESTAV-Irapuato. Km 9.6 Libramiento Norte, carretera Irapuato-León. Irapuato, Gto. México. C. P. 36821. Tel.: +52 (462) 623 96 00 ext. 439, email: [email protected] de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, CU, AP 70-275 Coyoacán 04510 México DF. TheBacillusare best known for their ability to sporulate, that gives them great resistance to environmental stresses such as nutrition scarcity, dessication, and high temperature. However, understanding the ecological role of the Bacillus is challenging, as the spores can be found anywhere and this can be a confounding factor when trying to infer what their actual niche is. Comparison of more than 20 complete genome sequences revealed that althoughthere is a core of conserved sporulation genes, greatvariability is found in the signal transduction machinery required to sense environmental cues, both to sporulate and to germinate. Bacillus subtilisis the most extensively studied sporulatingBacillus.Sporulation in B. subtilisis known to be repressed by glucose and triggered by nutritional stress such as limiting phosphorous.In B. subtilisit has been shown that a drop of GTP triggers sporulation and that the CodY regulator is involved in this response. A GTP drop can be artificially obtained using decoyinine, a GMP synthetase inhibitor. We have been studying Bacillus isolates from the sediment of an oligotrophic water system located in CuatroCienegas, Coahuila. Among 400 isolates we can recognize seven dominant Bacillus groups. Since very few studies have been carried out with wild isolates we had several questions regarding the phenotypic diversity we would observe among a representative group from thisBacillus collection: Are theseisolatessubject to catabolite repression? Do they respond to decoyinine? What fraction of a population can differentiate into spores?These results will be discussed.



25

Characterization of MotP, a new essential component for the flagellar rotation inRhodobactersphaeroides

Salvador Fabela, Clelia Domenzain, Sebastian Poggio, Aurora Osorio, Georges Dreyfus y Laura Camarena Instituto de Investigaciones Biomédicas, UNAM. [email protected]; 56229222

The bacterial flagellum is a widely conserved structure among the eubacteria. In E. coli and Salmonella, more than forty genes govern the assembly of this structure. The regulation of the flagellar genes in several species may be different, however, the structural components show a strong conservation degree. In all the hitherto studied flagellated-species of bacteria, have been identified orthologous proteins forming the filament, the hook, and the basal body complex. Recently, a couple of exceptions were recognized: the proteins MotX and MotY form an additional structure inside the basal body complexes of the polar flagellum in Vibrioparahaemolyticus.Also, specific components allowing the rotation of flagella through a Na+ gradient -instead of protons- were found. The proteins that form the flagellum in Rhodobactersphaeroides have a 50-70% similarity with those ofE. coli and Salmonella, nevertheless, the genomic sequence revealed several open reading frames (ORFs) of unknown function embedded within the flagellar locus. One of these ORFs, named RSP_6086, predicts a periplasmic protein without homology in other bacterial species. The mutant strain in RSP_6086 shows a Mot- phenotype, which means the cells are able to assemble the flagellum, but unable to rotate it. The protein encoded by RSP_6086 was fused to a hexahistidine-tag and purified. This protein, termed MotP, was used to establish possible interactions with other flagellar components involved in the control of flagellar rotation, such as MotB and FliL. Co-precipitation assays demonstrate that MotP interact with both proteins. These interactions were confirmed by two-hybrid assays in yeast. The connection between MotP and MotB was reinforced by the isolation of a pseudo-revertant strain in the allele motP::aadA, which recovered the swimming ability in a similar level to the wild type. The suppressor mutation was mapped to the aminoacid 62 in the motB gene, changing from serine to proline. The region affected by the mutation in MotB is located in the periplasmic region of the protein, and is functionally associated to the proton flux, which is indispensable for the rotation of the flagellum.Furthermore, we observed that an incompleteperiplasmic region of the membrane-associated proteinMotF, involved in the flagellar rotation of R. sphaeroides, interacts with MotP. Nonetheless, this interaction does not occur when the complete periplasmic region of MotF is present. This suggests that conformational changes in the flagellarperiplasmic components modify the interaction among these proteins and they possibly modulate the proton flux across the channel formed by MotB.



27 PCR-Multiplex based on K. variicola Sequencing Genome to distinguish

K. variicola of K. pneumoniae Clinical Isolates and its Prevalence Garza-Ramos Ulises1, Silva-Sánchez Jesús1, Martínez-Barnetche Jesús1, Tinoco Perla1, Gómez-Barreto Rosa1, Tellez Juan1, Garcia-Lopez David1, Martínez-Romero Esperanza2, Reyes-Prieto Mariana2 1Instituto Nacional de Salud Pública. Av. Universidad 655. Col. Sta. Ma. Ahucatitlan. 62100. Cuernavaca, Morelos. 2Centro de Ciencias Genómica, UNAM, Cuernavaca, Morelos. México. [email protected] Background: The genus Klebsiella includes the species Klebsiella pneumoniae, Klebsiella oxytoca and Klebsiella variicola. K. variicola has been found in plants and as opportunistic human pathogen in nosocomial infections and recently was found as symbiont in ants. K. variicola is capable of fixing nitrogen however is biochemically indistinguishable of K. pneumoniae. Objectives: Sequencing the K. variicola 801 genome and compare it with K. pneumoniae genomes for identify unique genes and develop a PCR-multiplex that differentiate both species, as well as determine the prevalence of K. variicola between ESBL-producing clinical isolates, previously identified as K. pneumoniae. Materials y Methods: K. variicola 801 genome was sequenced by pyrosecuencing in a Sequencer FLX 454 and the genome was ensemble mediated the Sanger Institute service. The unique genes of each bacteria specie were identified mediated the K. variicola 801, K. variicola At-22, K. pneumoniae 342, K. pneumoniae MGH78578 y K. pneumoniae NTUH-K2044 genomes comparison, using the BLASTp algorithm. The functional categories were determined by BLASp and subsequently analyzed by discontiguous megablast (BLASTn) in GenBank data base. The PCR-multiplex was validated using ATCC bacterial control (K. pneumoniae, K. oxytoca, R. terrígena, and R. planticola). The PCR-multiplex was used for determinate the prevalence of K. variicola among a collection of ESBL-producing K. pneumoniae clinical isolates, obtained from 1990 to 2009. Resultados: The genome possesses a 5.7 MB (5,705,981 pb), based 368,985 sequences and 5,663 ORFs were identified. Initially, the genome comparison identified 114 and 54 unique genes of K. variicola and K. pneumoniae, respectively. Subsequently, considering the metabolic and structural genes by discontiguous megablast, were identified four (3 metabolic and 1 structural genes) and two (metabolic genes) unique genes (probes) of K. variicola and K. pneumoniae, respectively. The PCR-Multiplex was developed according the specific oligonucleotides of each probe and the mtnC gene was selected by as a probe of Klebsiella genus. The validation of PCR-Multiplex using the ATCC bacterial control showed a specificity of 100% and the prevalence the K. variicola was of 2.6% between 190 ESBL-producing K. pneumoniae clinical isolates. Conclusions: The genome of K. variicola and its comparison with the others Klebsiela genomes, allowed to develop a PCR-multiplex based in unique genes of K. variicola. This bacterial specie is endophyte of plants and symbiont in ants and was identified as multirresistant bacteria in several Mexican hospitals causing nosocomial infections in humans. This work highlight that K. variicola could to be emergent bacteria in nosocomial infections.



29

A putative histidine kinase is required for chemotactic motility in Azospirirllum brasilense Sp245 strain

Villarreal Astorga C, Cosme Herrera YI, Aguilar Piedras, JJ, and Baca BE Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla. Cdad Universitaria, Av. San Claudio S/N. Puebla Pue. México. [email protected]

Bacteria can colonize very different niches because they can adapt to changing environmental conditions by altering their metabolism, physiology and behaviour. This capacity is the result of the coordinated action of regulatory systems that are triggered in response to environmental and/or physiological stimuli or signals to control the metabolic, physiological and behavioural traits that allow the cell to thrive in the new conditions. Histidine kinase (HKs) are sensory proteins that participate in two component signal transduction systems that convert environmental stimuli into a signal that elicits a cellular response through alterations in gene expression [3] Vegetative A. brasilense cells grow as short, curved rods that have a polar flagellum for swimming through aqueous environments, or they can transform into elongated, hyperflagellated swarmer cells for motility over a solid surface [2]. The present study reports the identification of a gene which deduced translation product contains a periplasmic sensor domain and a cytosolic autokinase domain linked by a transmembrane region and a HAMP domain. The structure of the cytoplasmic fraction contains the histidine phosphotransfer (DHp) domain that harbors the phosphor accepting histidine and the globular catalytic ATP binding (CA) domain. In addition the sequence translation analyses indicated that an internal receiver (Rec), and subsequently an HPT domains occurred; suggesting that the HK identified consist of more sophisticated hybrid system that involve multiple phosphotransfer reactions. In order to determine if the identified gene was functional a mutant was constructed. Chemotaxis assay were conducted using a procedure that has been described previously [1]. Size of the chemotactic rings and the rates at which hskA::tc R mutant formed were greatly affected compared to the wild type, indicating that swimming motility was significantly delayed. It is interesting to note that the putative HK contribute to chemotaxis in Azospirillum. Acknowledgements: The authors are indebted for the financial support of the Vicerrectoría de Investigación y Estudios de Postgrado (VIEP) Grant. References: 1.Carreño-López C., Sánchez A., Camargo N., Elmerich C., Baca, B.E. ( 2009) Characterization of chsA, a new gene controlling the chemotactic response, in Azospirillum brasilense Sp7. Arch. Microbiol. 191:501-507 2.Hall PG, Krieg NR (1983) Swarming of Azospirillum brasilense on solid media. Can J Microbiol 29:1592–1594 3.Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69:183–215



31

Blue Native/High Resolution Clear Native/SDS-PAGE and Mass Spectrometry Analysis of the Respiratory Chain Super complexes in

Gram-Positive Bacterium Bacillus subtilis Led Yered García Montes de Oca, González de la Vara Luis Eugenio, Chagoya López Alicia, Cabellos Avelar Tecilli y Gutiérrez-Cirlos Madrid, Emma Berta Unidad de Biomedicina, F.E.S. Iztacala. U.N.A.M. [email protected] Av. de los Barrios #1. Los Reyes Iztacala. Tlalnepantla, Edo. de México. c.p. 54090. Tel. 5623-1333 ext.39783. CINVESTAV Unidad Irapuato. Apdo. Postal 629, México 36503. [email protected] The protein complexes responsible for oxidative phosphorylation in both the inner membranes of mitochondria and the cytoplasmic membranes in prokaryotes are arranged in ordered structures so-called supercomplexes. Super complexes have been revealed by blue native electrophoresis (BN-PAGE), and have been the most popular strategy in the structural analysis of the respiratory chain in different organisms. Based on this procedure, the existence of super complexes has been found in respiratory chains, such as bacteria, yeasts, higher plants and mammals. In the other hand, high-resolution clear native electrophoresis (hrCNE) has also been used to detect super complexes by their activity.

In this paper, using BN-hrCNE/SDS-PAGE combined with mass spectrometry, we study the associations of different supramolecular complexes comprising the respiratory chain of the Gram-positive bacterium Bacillus subtilis 168. We detected four different super complexes: III+IV+cit c550+cit c551, III+IV+cit c550, IV+cit c550+cit c551 y cit c550+cit c551. In the 2D gels analysis by staining with TMBZ we were able to identify two of the subunits of the complex b6c (III) (cytochrome b6 and cytochrome c) the cytochrome c of cytochrome oxidase (complex IV) and the small cytochromes c550 and cytochrome c551. On the other hand, the histochemical staining of respiratory complexes showed that the five respiratory complexes of B. subtilis are obtained in active form after detergent solubilization with digitonin at 4%. It was also shown that neither complex I nor complex II or complex V are associated with any of the respiratory chain complexes.



33 The absence of any of the terminal oxidases in the respiratory chain of

Bacillus subtilis increases the b6c complex activity García Hernández Diana Edisa1; Cabellos Avelar Tecilli2; Gutiérrez-Cirlos Madrid Emma Berta2 1Escuela Nacional de Ciencias Biológicas. IPN. C.P. 11340. México D.F. 2Laboratorio 2. Unidad de Biomedicina (UBIMED). FES Iztacala. UNAM. Tlalnepantla, Edo. de Méx. C.P. 54090. Tel: 5623-1333, ext 39783. Fax: 5623-1138. [email protected].

Bacillus subtilis is a Gram-positive aerobic bacterium that can grow anaerobically under some conditions. Aerobically, B. subtilis presents a branched respiratory chain, with several quinol oxidases that can take electron from menaquinol the natural substrate of the bc-type complex. Expression of the oxidases depends on environmental and developmental conditions. There are at least three terminal oxidases in B. subtilis: cytochrome aa3 present in exponential growth phase and well aereated conditions, and pumps protons. Cytochrome bd is found in stationary phase and in low oxygen conditions, and does not pump protons. The caa3-type (COX) is expressed during growth on non-fermentable substrates such as succinate and also pumps protons. Another possible oxidase is coded by genes ythAB, and has homology to other Bacillus quinol oxidases. We are interested in isolating the b6c complex because it has photosynthetic characteristics in a non-photosynthetic bacterium. Due to the presence of quinol oxidases, isolation of this complex has proven difficult. By using three mutants lacking some oxidases to increase the electron flux to the b6c complex in the final exponential phase, we are trying to obtain the b6c complex in higher amounts. We selected three mutants: LUW46 defective in oxidase aa3; LUW138 defective in oxidase aa3 and Yth; LUW196 defective in oxidase bd, in COX and Yth. From these mutants the isolation of membranes was performed along side with the wild type strain 168. We determined the protein concentration and calculated the quantity of each cytochrome by absorbance spectra. For the activity experiments, we used substrates and inhibitors of each respiratory complex in spectrophotometric assays. Also the quinol oxidase activity was determined with a Clark-type oxymeter. Finally, a comparison of the protein composition between the mutants and the wild type by SDS-PAGE geles was done. We conclude that LUW46 will be the best mutant to isolate the b6c complex.



35

Studies on the role of SepL in the hierarchical secretion of proteins through the type three secretion system of enteropathogenic

Escherichia coli

Meztlli Gaytán Enríquez*, Miguel Díaz Guerrero*, Norma Espinosa and Bertha González-Pedrajo Departamento de Genética Molecular, Instituto de Fisiología Celular, UNAM. Apartado postal 70-243, México D.F., 04510. Tel. (5255) 56225965. e-mail: [email protected] * These authors contributed equally to this work. Enteropathogenic Escherichia coli (EPEC) is one of the main etiological agents of infantile diarrhea in developing countries. It uses a Type Three Secretion System (T3SS or injectisome) to translocate virulence effector proteins directly into enterocytes. Type III secretion is a coordinated process with a hierarchy of export during the assembly of the injectisome. Secreted proteins are targeted to the secretion apparatus in an ordered fashion: first, the early substrates (rod and needle subunits); then the intermediate substrates (translocators); and finally the late substrates (effectors). The substrate specificity switching from intermediate to late substrates is controlled by a protein complex composed of SepL and SepD. The deletion of either sepL or sepD abolishes secretion of translocators and leads to hypersecretion of effector proteins; however, the precise mechanism by which this differential secretion is regulated is still unknown. In this work we purified a recombinant version of SepL (His-SepL) that carries an N-terminal His-Tag, and generated polyclonal antibodies against this protein. We performed complementation and multicopy assays to verify the functionality of His-SepL. Our results suggest that the different functions associated with SepL i.e., to facilitate the secretion of translocators and inhibit an early secretion of effectors, can reside in different domains of the protein. In addition, we show for the first time that SepL interacts with EscN, the ATPase associated with the T3SS of EPEC. We generated different truncated versions of SepL and analyzed their functionality and interactions with EscN. Recently, it was proposed that SepL is an aberrant effector, i.e., that although it has a secretion signal it is not secreted by the T3SS. Here, we propose a model in which SepL is recognized by EscN as an effector, and that this interaction blocks the recognition site of effector-chaperone complexes, regulating in this way, the secretion of this kind of substrates. Finally, it has been reported that SepL not only interacts with SepD but also with CesL and that both of these proteins could act as molecular chaperones. Using the truncated versions of SepL, we are currently mapping the interacting regions between these proteins.



37

The permeable form of short-chain carboxylic acids act as signaling

molecules to the BarA sensor kinase González-Chávez, Ricardo and Georgellis, Dimitris Departamento de Genética Molecular, Instituto de Fisiología Celular, UNAM, México, D.F. 04510.Tel. 56-22-57-38. [email protected] The BarA/UvrY two-component system (TCS) of Escherichia coli comprises the BarA protein as the histidine sensor kinase (SK) and UvrY as the cognate response regulator (RR). This system, which its homologues in other gram-negative bacteria, such as BarA/SirA of Salmonella, ExpS/ExpA of Erwinia, VarS/VarA of Vibrio, and GacS/GacA of Pseudomonas species, have been shown to positively control expression of the noncoding CsrB (RsmY) and CsrC (RsmZ) RNAs. These small regulatory RNAs together with the CsrA protein constitute the Csr (carbon storage regulation) system, a post-transcriptional regulatory system that has profound effects on central carbon metabolism, motility and biofilms formation.

It was demonstrated that formate, acetate as well as other aliphatic short chain fatty acids act as stimulus that activate the BarA/UvrY two-component system. In addition, it has also been shown that the carboxyl group of weak acids is essential for the activation of this system. In this study, we provide experimental evidence that the uncharged protonated form of the carboxylic acids is the signaling molecule. The results of this analysis and its implication in cell physiology are discussed.



39

Study of the interaction between the RhlR and LasR proteins and their target DNA sequence in Pseudomonas aeruginosa

González-Valdéz, A. A., Leyva-Hernández, E. and Soberón-Chávez, G* Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, UNAM. Apdo. postal 70228. Ciudad Universitaria, 04510, México, D. F. Tel. 0155-56229201. Fax: 56229212. [email protected], [email protected]* Pseudomonas aeruginosa is a γ-proteobacteria widely distributed in nature. It is an opportunistic pathogen of immunocompromised individuals and it is the main cause of morbility and mortality in cystic fibrosis patients. Expression of diverse virulence factors in P. aeruginosa is regulated by the so called quorum sensing response (QS). This response is controlled by two regulons which are thought to function in a hierarchical manner and are regulated by LuxR homologous proteins, which is the QS regulatory protein of Vibrio fisheri. The first regulatory protein in this QS cascade is LasR which, once bound to its autoinducer N-3-oxododecanoyl homoserine lactone (3OC12-HSL), function as a transcriptional activator of genes that encode proteases (elastases A and B), exotoxin A and an acyl-HSL synthase (LasI). LasR also stimulates the transcription of the transcriptional regulator called RhlR, which after binding with the autoindicer N-butanoyl homoserine lactone (C4-HSL) activates the transcription of the genes encoding enzymes involved in rhamnolipid synthesis (rhlAB), as well as other genes encoding virulence factors. Both QS signaling systems, LasR/3OC12-HSL and RhlR/C4-HSL bind to 20-bp specific sequences designated as las boxes. Some of these las boxes are exclusively recognized by LasR/3OC12-HSL, but there are also boxes recognized not only by LasR/3OC12-HSL but also by RhlR/C4-HSL, with different specificities though. The aim of the present work is the use of an in vivo system in the Escherichia coli background to evaluate the contribution to protein binding specificity of different positions in las boxes sequence that are recognized either by RhlR, by LasR, or by both proteins. Some of our results were validated using EMSA assays.

To measure in vivo RhlR and LasR binding to different DNA sequences (las-boxes) we constructed hybrid promoters with consensus -10 and -35 RNA-polymerase recognition sequences separated by 19 base pairs (bp) with the sequence corresponding to either rhlAB, rmlB, phzA1 or lecA las boxes.



41

Study of three flagellar loci in the coral pathogen Vibrio shilonii and

its involvement in cell motility

Yael González Tinoco1, Daniela Venegas1, Guillermo Mendoza-Hernandez3, Laura Camarera2, Georges Dreyfus1 1Instituto de Fisiología Celular, 2Instituto de Investigaciones Biomédicas and 3Facultad de Medicina, Universidad Nacional Autónoma de México. 04510 D.F. Mexico. Tel. 5622-5618. [email protected].

Vibrio shilonii is the best studied pathogenesis model in coral Oculina patagonica. Little is know about its motility system. In a resent study, we explored the swimming behavior of this bacterium under different concentrations of agar; as the density of the media increases, the cells show morphological changes; from one polar sheathed flagellated cells to elongated lateral flagellated cells and round non flagellated cells. The ion preference for torque generation was also determined by the sodium-channel blocker amiloride. The functional study was complemented with the isolation and characterization of the filament-hook-basal body complex of the polar sheathed flagellum. The ultra structure was resolved by transmission electron microscopy and eight internal protein sequences were identified by mass spectrometry. The comparison of these sequences with the protein data base from the complete Vibrio shilonii genome, lead us to the finding of a new third flagellar gene cluster atypical in other members of the Vibrio group. A reverse genetic study could gives light on the function of this new group of flagellar genes, whose involvement in cell motility is not clear. We are currently working on the generation of mutant strains that will allow us to study the three flagellar loci present in this marine bacteria. We thank Javier de la Mora and Teresa Ballado for technical assistance; this work is supported by CONACYT (106081) and DGAPA (IN213408).



43

Molecular characterization of Pet autotransporter protein in Proteus mirabilis RTX334 strain

Gutiérrez-Lucas Luis Raúl*1; Eslava-Campos Carlos2; Mendoza-Hernández Guillermo2; González-Pedrajo Bertha2; Sainz-Espuñes Teresita del Rosario1 1Universidad Autónoma Metropolitana Unidad Xochimilco. 2Universidad Nacional Autónoma de México, México D.F. Laboratorio de Microbiología Molecular. UAM-Xochimilco. Calzada del Hueso #1100. Col. Villa Quietud. Delegación Coyoacán. CP 04960. México, D.F. Tel. 54837000 Ext. 3624 Luis Raúl Gutiérrez Lucas. [email protected] Tel. Particular. 42122957. Introduction. The genus Proteus is a member of the Enterobacteriaceae family. Proteus mirabilis is the causative agent of urinary tract infections, acute pyelonephritis, kidney stones formation and occasionally bacteremia. SPATEs proteins are commonly found in some pathogenic members of this family. Objective. Identify genes related to the production of SPATEs and characterization of proteins encoded by these genes in Proteus mirabilis clinical isolates. Methodology. Proteus mirabilis RTX334 was grown in LB medium. Secreted proteins were precipitated with 65% ammonium sulfate following by a 24 h dialysis. Eight percent (8%) SDS-PAGE was used to determine the presence of partially purified proteins. A protein with an approx molecular weight of 107 kDa was sequenced using mass spectrometry. A western blot assay was performed using anti-Pet polyclonal antibodies. PCR using specific primers was performed and the amplicons obtained were sequenced. BLAST alignment was used in order to find identities with reported SPATEs genes. Results. The alignment of the sequences of the amplified products showed an identity of 99% with the reported pet gene. The amino acid sequence corresponds to the Pet protein reported in E. coli 042. Conclusions. Pet toxin gene was identified in chromosomal DNA from Proteus mirabilis strains RTX334. Pet protein was also secreted by this microorganism. It is the first report of Proteus mirabilis carrying Pet a protein member of the SPATEs.



45

Detection of community acquired methicillin resistant Staphylococcus aureus strains in a Mexican community

Hamdan Partida A1, Sainz-Espuñes T1, Bustos-Martínez J2 1Depto. Sistemas Biológicos, 2Depto. Atención a la Salud. Universidad Autónoma Metropolitana-Xochimilco. Mexico City, Mexico. Calzada del Hueso 1100, Col. Villa Quietud. CP 04960. México D.F. [email protected]

Background Staphylococcus aureus has long been recognized as an important pathogen in human diseases. Staphylococcal infections occur regularly in hospitalized patients and have severe consequences. Some of these infections are caused by methicillin-resistant S. aureus (MRSA) strains. In recent years an increasing number of community infections caused by this microorganism are emerging, causing an important public health problem (1). Objectives The aim of this study was to determine the presence of community acquired MRSA strains (CA-MRSA) in a Mexican community. Methods In a previous work 1039 S. aureus strains isolated from a Mexican community were characterized using standard methods (2). The presence of mecA and lukPV genes, were analyzed using standard PCR. The presence of the SSCmec element was performed by multiplex PCR and sequencing. Conclusions One hundred and thirty one strains were MRSA strains. SSCmec type IV was the predominant type found among the strains (69%) followed by the SSCmec type II (20%), and SSCmec type V (2%). SSCmec types I and III were not found. Twenty nine MRSA strains (0.03% from the total strains) that were previously characterized as SCCmec type IV and SCCmec type V, presented the Panton-Valentine leukocidin toxin gene. This results leads to determine for the first time that CA-MRSA strains are circulating in the tested Mexican community.

References 1. DeLeo FR, Chambers HF. 2009. Reemergence of antibiotic-resistant

Staphylococcus aureus in the genomics era. J Clin Invest. 119: 2464-2474. 2. Hamdan-Partida A, Sainz-Espuñes T, Bustos-Martínez J. 2010. Characterization

and persistence of Staphylococcus aureus isolated from the anterior nares and throat from healthy carriers in a Mexican community. J Clin Micriobol. 48(5):1701-1705.



47

Genomic comparison of Bacillus isolates from dissimilar geographic sites

Hernández-González, Ismael L1. Souza, Valeria2 and Olmedo Álvarez, Gabriela1 1Departamento de Ingenieria Genética de Plantas. CINVESTAV-Irapuato. Km 9.6 Libramiento Norte, carretera Irapuato-León. Irapuato, Gto. México. C. P. 36821. Tel.: +52 (462) 623 96 00 ext. 439, email: [email protected] 2Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, CU, AP 70-275 Coyoacán 04510 México DF.

The Bacillus genus comprises Gram-positive bacteria that are aerobic or facultative anaerobic and that have the ability to develop endospores. They have been isolated from a wide range of environments that include soil, hydrothermal vents, and alkaline environments, shallow marine water, which reflects the wide metabolic capabilities of an extensively distributed genus. Although there are close to ~140 genomes sequenced to date, the group is highly biased toward pathogenic isolates and Bacilli used for industrial applications, while the aquatic isolates are poorly represented. The genome sequences of Bacillus coahuilensis m4-4 and Bacillus sp. m3-13, two bacterial isolates from Cuatro Ciénegas Coahuila, were recently reported. Both Bacilli live in the same oligotrophic environment, with extremely low phosphorous levels, but they appear to have different strategies for dealing with the poor nutrient environment. B. coahuilensis produces sulfolipids and replaces membrane phospholipids, whereas Bacillus sp. m3-13 has genes phn, which code for phosphonate ABC importers, permeases, and a phosphonate-lyase, suggesting that both strategies are used by bacteria from Cuatro Ciénegas. On the other hand, a molecular phylogenetic analysis of 16s rRNA gene sequences indicates that B. coahuilensis m4-4 is closely related to Bacillus aquimaris TF12 while the closest strain to Bacillus sp. m3-13 is B. horikoshii DSM8719. Both of these strains were isolated from a different marine environment and with dissimilar geographic origin. B. horikoshii DSM8719 was isolate from Lonar Lake in India and B. aquimaris TF12 was isolated from the Yellow Sea in Korea. In order to gain insight on the genomic differences that would explain the possible adaptations to the oligotrophic conditions of Cuatro Ciénegas, we sequenced the genomes of B. horikoshii DSM8719 and B. aquimaris TF12, and compared their predicted genomic contents with those of Bacillus sp. m3-13 and B. coahuilensis m4-4, respectively. The results from this comparison will be discussed.



49 The Role of the Histidine Kinase CbrA on the Carbon Catabolic

Repression in Azotobacter vinelandii Hernández-Ortiz A. 1, Pando V. Castañeda M., Espin G., Núñez-López C.E. 1Departamento de Microbiología Molecular, Instituto de Biotecnología, UNAM, Av. Universidad 2001, Col. Chamilpa. Cuernavaca, Morelos 62210, México. Tel. (52) (777) 329 16 29. [email protected] Azotobacter vinelandii is a gamma-proteobacteria of the family Pseudomonadaceae, and capable of producing two polymers of important industrial applications: the exo-polysaccharide alginate and the intracellular polymer PHB. In A. vinelandii organic acids are the preferred carbon source even though it also uses sugars and alcohols [1]. Thus, in a diauxic growth of acetate-glucose, the acetate is consumed first. However, the molecular mechanism of this catabolic repression and the glucose transport and metabolism remains unknown. In Pseudomonas species, the carbon catabolic repression (CCR) is exerted by the RNA-binding protein Crc [4] that inhibits the expression of genes involved in the transport and catabolism of the non-preferred carbon sources, while it indirectly favours the assimilation of the preferred ones. Crc binds to a specific sequence (AAUAAUAA) located close to or overlapping the AUG translation start site. The CrcZ small RNA (sRNA) contains five Crc-binding sites antagonizing the repressing effect of Crc. The expression of crcZ requires the CbrA/B two-component system where CbrA is the histidin kinase while CbrB is the response regulator. This fact places CbrA/CbrB on top of the signal transduction cascade controlling the CCR in P. aeruginosa. In A. vinelandii we have previously identified a Tn5 mutant within the cbrA gene showing increased levels of alginate production. It was also showed that the cbrA::Tn5 mutant was unable to use glucose in the presence of acetate which suggested that, similar to Pseudomonas spp., the histidine kinase CbrA was required to release the catabolic repression of glucose by acetate. The main goal of this work is to elucidate the mechanism of the inhibition of glucose assimilation in the cbrA::Tn5 mutant and whether or not this effect is exerted trough the Crc/CrcZ system. An analysis of the genome sequence of A. vinelandii allowed us to identify elements of the Crc/CrcZ posttranscriptional system. The A. vinelandii Crc protein showed 87% identity with its Pseudomonas counterpart while in the sRNA CrcZ the putative sites for Crc binding are conserved. In an attempt to investigate whether Crc was involved in the repression of glucose catabolism, we looked for putative Crc binding sites in the regulatory region of genes for glucose assimilation. The genes gluP (putative involved in the transport of glucose) and one gene of the glucolitic pathway (edd), were found to have such sites. In order to further investigate the effect of CbrA on the assimilation of glucose during the diauxic growth (acetate-glucose), in a more general way, we are conducting proteomic analysis to identify the proteins differentially expressed during the switch from acetate to glucose catabolism in A. vinelandii. [1] Espín, G. 2003. Biología de Azotobacter vinelandii. Microbios en línea. Instituto de Biotecnología, Universidad Nacional Autónoma de México. www.ibt.unam.mx [2] Hernández-Ortíz, A. 2011. Función de la cinasa histidínica CbrA en el metabolismo de glucosa en Azotobacter vinelandii. Tesis de Licenciatura. Facultad. de Ciencias Biológicas. Universidad Autónoma del Estado de Morelos. México. [3] Quiroz-Rocha, E. Master´s proyect. Unpublished data. [4] Rojo F. 2010. Carbon catabolite repression in Pseudomonas : optimizing metabolic versatility and interactions with the environment. FEMS Microbiol Rev. 34:658-84



51

Proteases sequences analysis of Pasteurellaceae Martínez Genis Milton1, Negrete Abascal Erasmo2, Vaca Pacheco Sergio2, Sánchez Alonso María Patricia Georgina1, Vázquez Cruz Candelario1* 1Centro de Investigaciones en Ciencias Microbiológicas, BUAP, Apdo. Postal 1622, Puebla 72570, México; 2Carrera de Biología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de Mexico; Av. de los Barrios # 1, Los Reyes Iztacala, Tlalnepantla, Estado de México 54090, México. Tel 01-222-229-5500 ext 2536, Email [email protected] Bacterial proteases support the central cell metabolism, functioning to destroy temporally unnecessary proteins, degrading denatured proteins or activating proteins needed in a certain moment. Secreted proteases should be active to obtain nutrients from large proteins. Proteases could also destroy antibacterial substances that interfere with bacterial colonization. We have analyzed the sequence of degS, clpB, sohB, sppA, prc, ecf and pepN protease genes to determine the variability of Pasteurellaceae bacteria and to study bacterial phylogenetics. Genebank sequences were used to carry out the comparisons between them by mean of blastn and clustalW. Results showed variability from 1 to 30% from closely related to distant bacteria, respectively. PCR reactions for target genes were positive in various strains of closely related bacteria, for example degS, clpB, sohB, sppA, and prc, genes for Pasteurella multocida strains X73, 3T and 5T and ecf and pepN genes in Actinobacillus pleuropneumoniae. However PCR did not produce amplicons in distantly related bacteria. Thus PCR amplification of protease genes is a fast test to identify closely related bacteria, but it is not recommended for distant ones. Our results show that protease genes sequence analysis can be used to make phylogenetic analysis only in closely related Pasteurellaceae.



53

L and D-PIN-2 proteases degradation by Pseudomonas aeruginosa

Daniel Juárez López (1),Corzo Gerardo(2) Dra. Elba Villegas(3)

(1) Facultad de CienciaBiológicas, (2) IBT-UNAM, (3) CeIB-UAEM. Av. Universidad 1001 Col. Chamilpa Cuernavaca, Morelos. C.P. 62209 Fax 777297030, [email protected] P.aeruginosa is a Gram (-)bacterium of clinical and nosocomial importance(1), with an increasing antibiotic resistance. It grows in soil, wáter bodies, animal and plant tissues(2). Affects mainly people with immunedeficiencies, surgical treatments, severe burns, and cystic fibrosis (3). Its resistance is due to mechanisms such as drug efflux, mutations, acquisition of plasmids, protease production and biofilm formation.P. aeruginosa reported proteases areelastases A and B, alkaline protease and protease IV(4). L-Pin-2 is an antimicrobial peptide, isolated and purified from Pandinus imperator scorpion venom, it contains 24 amino acids forming anphipathic α-helix(5), with antimicrobial a broad activity against Gram (-) and (+) bacteria, forming pores in the cell wall. Both L-Pin-2 and its analogue D-Pin-2 present a bacteriostatic effect at a concentration of>50 M against P. aeruginosa(6). Degradation and inactivation of L and D Pin-2, may be due to the presence of protease enzymes. The aim of this work was to identify which proteases are responsables of this degradation, in order to propose structural changes in L and D Pin-2 amino acid sequence to provide them protease resistance. In this work we used 5 strains of P. aeruginosa two of them from ATCC 9020 and 27853, the other 3 were from clinical isolates named AC-1, AC-2 and AC-3.The las tthree Straits were isolated by selectiveanddifferential media (McConkeyand King B) withvariations in pyoverdin and pyocyan in production and typical characteristics of the genus, such as, Gramnegative, catalase and oxidase positive.They were also identified by molecular techniques and confirmed asP. aeruginosa. Antibiotic susceptibility differences were observed ATCC 9020 strain was resistantto 5 antibiotics, ATCC 27853 to 9, clinical isolates strains AC-2 and AC-3 to 7, and AC-1 to 12 antibiotics. AllP. Aeruginosa proteolítica ctivity was confirmed in Milk, Gelatine an dBloodagars. The culture super natants with higher proteases activity were filtrated in millipore membrane sof 22 microns, precipitated with acetone, and protein zymograms were performed to observe the bands with proteolyticactivity once they were quantified by Bradford assay. ATCC 27853 and AC-3 precipitates showed different bands patterns in zymograms. Two bands for the first one and one single band for the second. Further assay will be conducted to purify the bands that showed proteolític activity to incúbate them with L-Pin-2 and D-Pin2 and identify there action products by HPLC and Mass spectrometry. References 1.Masaadeh et al., 2009. Incident of Pseudomonas aeruginosa in Post-Operative WoundInfection. American Journal of Infectious Diseases, 5 (1): 1553-6203. 2. Lambert P.A. 2002. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa. J. R. SocMed, 95 (Suppl. 41): 22-26. 3 Poole, K. 2001. Multidrugefflux Pumps and Antimicrobial Resistance in Pseudomonas aeruginosa and Related Organisms. J. Mol. Microbiol. Biotechnol., 3 (2): 255-264. 4. Whooley et al.,1983. Effect of Substrate on the Regulation of exoprotease Production by Pseudomonas aeruginosa ATCC 10145. Journal of General Microbiology, 129, 981-988. 5. Corzo G. et al., 2001. Characterization of unique amphipathic antimicrobial peptides from venom of the scorpion Pandinus imperator. Biochem J. 359, 35-45. 6. Carmona, G. 2010. Characterizationof a D of including the first analogue Pandinin 2. Thesis in Progress. Biotechnology Research Center. UAEM.



55

Essential roles of plasmid p42e in Rhizobium etli CFN42

Cristina Landeta, Araceli Dávalos, Susana Brom and David Romero Centro de Ciencias Genómicas, UNAM. Cuernavaca, Mor., México. [email protected] Av. Universidad s/n Col. Chamilpa 62210, Cuernavaca, Mor., México. Tel. +52(777)3175867

Rhizobium etli CFN42 possesses six large plasmids, ranging in size from 184 to 642kb. Five out of the six plasmids are dispensable for cell viability, but plasmid p42e (505kb) is unusually stable. One possibility to explain this stability would be that genes on p42e carry out essential genes for growth. Also, synteny analysis and incompatibility studies revealed highly stable replicons equivalent to p42e in content and gene order in other Rhizobium species. A functional analysis of p42e demonstrated that nearly 11% of the genes of p42e participate in the physiology and primary metabolism of R. etli, such as aminoacid biosynthesis, vitamin and cofactor biosynthesis, membrane lipid formation, sugar utilization, electron transport and respiratory ability, as well as septum location. Additionally, a systematic deletion analysis of plasmid p42e generated ten deletions affecting predefined sectors of p42e and allowed the identification of two genes (RHE_PE00001 and RHE_PE00024) required for cell viability on rich medium. The former, RHE_PE00001, encodes a hypothetical protein with a probable winged helix-turn-helix motif. The second one, RHE_PE00024, encodes a probable two-component sensor histidine kinase/response regulator hybrid protein. Since we have been unable to inactivate these two genes unless we complemented with an extra copy of the gene, we are studying their essential role through conditional gene silencing by using antisense RNA or upon overproduction of the corresponding protein. Furthermore, a morphology analysis by microscopy and flow-cytometry of two deletions of p42e that exhibit a reduced growth phenotype (that lack RHE_PE00024) revealed smaller cells. Suppressor mutant of deletion 6 (from RHE_PE00002 to RHE_PE00362) were found that abolish the slow-growth phenotype and also recuperates its wild-type morphology. Further attempts to eliminate p42e were done by incompatibility with a plasmid containing repABC of p42e; even in this case, no elimination of p42e was seen, only rearrangements fusing p42e with other replicons. Interestingly, attempts to eliminate p42e providing supernumerary copies of RHE_PE00001 and RHE_PE00024 proved to be unsuccesful, suggesting there are additional factors that may have a role in the high stability of this replicon.



57

Identification of fibrinogen-binding proteins in members of the Pasteurellaceae family

Brenda A. López Ruiz1*, Sergio Vaca P1, Candelario Vázquez2 and Erasmo Negrete Abascal1

1Carrera de Biología, Facultad de Estudios Superiores Iztacala, UNAM. Av. de los barrios Nº 1, Col. Reyes Iztacala, Tlalnepantla, México. 2Centro de Investigaciones en Ciencias Microbiológicas, BUAP, Puebla, México *[email protected]. Tel. 0445532347504, 43365614

Avibacterium, Actinobacillus and Gallibacterium genera belong to the Pasteurellaceae family and are well known for causing different diseases in animals. Since during host colonization microorganisms interact with extra cellular matrix components, fibrinogen-binding proteins (FBP) could be important virulence factors for those bacteria. We have identified FBP associated to cells, and in soluble form, in samples from Actinobacillus pleuropneumoniae serotype 1, Actinobacillus suis ATCC 15555, Avibacterium paragallinarum serovar A, ATCC 0083, Gallibacterium anatis: F-149 and 12656-12. Two proteins of approximately 21 and 60 kDa were identified from those microorganisms using biotinylated sheep fibrinogen. An A. pleuropneumoniae 60 kDa protein from outer membrane proteins was purified by affinity assays. Also, the capacity of A. pleuropneumoniae to degrade fibrinogen was observed when secreted proteins were incubated with sheep fibrinogen during 24 hours at 37ºC. The uptake of fibrinogen and the capacity to degrade it, could contribute to the A. pleuropneumoniae and other Pasteurellaceae pathogenicity. Key words: Fibrinogen, Actinobacillus pleuropneumoniae, Avibacterium paragallinarum, Gallibacterium anatis Proyecto apoyado por PAPIIT IN216010 y PAPCA FESI-UNAM



59

Temporal patterns in oligotrophic soil microcosms in Cuatro Cienegas Basin

Nguyen E. López-Lozano1. Felipe García-Oliva2. Celeste Martinez-Piedragil 2. Luis E. Eguiarte1. Valeria Souza1* 1 Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Apartado Postal 70-275, 04510, México.2 Centro de Investigaciones en Ecosistemas, Universidad Nacional Autónoma de México, A.P. 27-3 (Santa María de Guido) 58090, Morelia, Mich., México. *corresponding author: Tel.: 11 52 5556229030; fax: 11 5255 5622 8995 e-mail: [email protected] Soils are among the most diverse environments analyzed to date. Among the ecosystemic role of the soil microbiota it to perform the biogeochemical cycles. Herein we explore the role of perturbation in the taxonomic and functional diversity of two neighboring sites with contrasting moisture in the oasis of Cuatro Cienegas in Coahuila, Mexico. We pyrosequencing the 16S rRNA V6 region of each sample for general taxonomic and community descriptions and studied 3 important genes of the nitrogen cycle in order to get an idea of the role of perturbation in the nitrogen cycle, we analyzed nifH, nirK and nirS clone libraries for functional group assessment. Complementing the genetic data, biogeochemical analyses were performed for each type of soil at different times during the sampling period. Differences in taxonomical composition were observed among soils. The more humid soil showed greater bacterial diversity and nutrient concentration than the drier soil. The greatest functional differences were observed within the denitrifying bacterial guild, while nitrogen fixers shared more taxonomic groups between sites. Our results suggest that differences in bacterial community composition and diversity are produced by nutrient availability and humidity. Interestingly, despite numerous differences between both the sites the changes in diversity during succession are parallel, both sites suggesting an autotrophic succession. If we compared the perturbed sites after a year of succession, none of them have recovered the original diversity observed in adjacent soil samples. Moreover, this study shows how atypical CCB is in relation with other deserts with much lower diversity.



61

Analysis of the polyhydroxybutyrate (PHB) depolymerase system involved in mobilization of accumulated PHB

inAzotobactervinelandii Daniel Segura, Josefina Guzmán, Déborah Yanajara, Adolfo Cosme, Libertad Adaya, Soledad Moreno, y Guadalupe Espín Departamento de Microbiología Molecular, Instituto de Biotecnología, UNAM. Av. Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos. México. CP [email protected] Polyhydroxybutyrate (PHB) is an aliphatic polyester that is synthesized and intracellularly accumulated as a carbon and energy reserve in several bacteria. Because this polymer has thermoplastic properties, it has received much attention as a biodegradable and biocompatible substitute for non-degradable synthetic plastics. In A. vinelandii the genes coding for the PHB biosynthetic enzymes, as well as several regulatory elements involved in the control of its synthesis, have been characterized. However, little is known about the mobilization process allowing the utilization of accumulated PHB when it is needed. The recently finished genome sequence of A. vinelandii DJ revealed seven genes that are thought to encode intracellular PHB depolymerases (the enzymes responsible for the mobilization of PHB).However, direct evidence for the in vivo roles of their gene products is missing. In this study, we selected three candidate genes (Avin27080, Avin34810, andAvin03910), representing the most probable candidates to be involved in the catabolism of PHB, and investigated the physiological function of their gene products: (i) with recombinant Escherichia coli strains expressing the corresponding proteins; (ii) with A. vinelandiinull mutants. Evidence for significant PHB depolymerase activity wasobtained for the products of Avin34810, and Avin03910 expressed in E. coli, but not for Avin27080. Similarly, a clear phenotype of an altered PHB metabolism was obtained only for the A. vinelandii mutants inactivated in Avin34810 and Avin03910. The presence of a signal peptide in the product ofAvin34810, and a high similarity with extracellular PHB depolymerases of other bacteria, suggested that this gene could be encoding an extracellular enzyme that would enable A. vinelandii to use extracellular PHB as a carbon source. However, evidence for the utilization of exogenous PHB, or for the secretion of this depolymerase was not found. Unexpectedly, mutant Avin34810 showed a strong decrease in PHB accumulation, suggesting its participation in the metabolism of PHB intracellularly by an unknown mechanism. On the other hand, mutant Avin03910 showed an increased PHB accumulation. This phenotype, together with its inability to degrade the intracellular PHB under conditions stimulating mobilization in the wild type strain, suggest that the product of Avin03910 is the main PHB depolymerase responsible for intracellular PHB degradation.



63

Cloning and expression of the 19 kDa glycoprotein (Rv3763) of Mycobacterium tuberculosis in Streptomyces lividans.

Daniela Torres Rodríguez, Laura E. Córdova Dávalos, Gabriela González Cerón y Luis Servín González Departamento de Biología Molecular y Biotecnología. Instituto de Investigaciones Biomédicas, UNAM. Apartado Postal 70228. Ciudad Universitaria, D.F, 04510. TF: (55)5622-8929. Correo electrónico: [email protected]

Glycoproteins present in the cell envelope of Mycobacterium tuberculosis have a fundamental role in pathogenesis by this bacterium, since they elicit multiple responses after infection. The antigenic properties of these glycoproteins make their study relevant, in order to understand their role in the infection process, their interaction with the immune system and their possible use in vaccines. The study of their glycosylation might also reveal possible targets for fighting this disease. Streptomyces lividans is a bacterium related to M. tuberculosis, since both belong to the actinobacteria, and in addition they share homologous protein glycosylation mechanisms. S. lividans is non-pathogenic, exhibits fast growth, and is easily manipulated genetically in the laboratory. The object of this work was to clone and express the 19 kDa antigenic protein of M. tuberculosis in S. lividans as a secreted protein, and to study its glycosylation in this host. When this protein was expressed in S. lividans using its own signal peptide it was possible to detect it in the membrane fraction. The protein appeared to be of a smaller size than predicted, suggesting that it had been subject to proteolytic cleavage; in spite of this it was mannosylated since it reacted with concanavalin A. When the signal peptide was replaced for a secreted signal peptide the protein was found in the culture supernatant, but apparently it was not glycosylated. These results confirm the functional conservation of the glycosylation machinery between both species. However, the native signal peptide of this protein appears to be critical for directing it to the glycosylation machinery.



65

Siderophore biosynthesis regulation mediated by Gac/Rsm pathway in Azotobacter vinelandii.

Morales Ruiz Estefanía. López Pliego Liliana, Miguel Castañeda Lucio CICM-ICUAP BUAP Edificio 103-J. CU C.P. 72570 Puebla, México, [email protected] 2 29 55 00 Ext. 2527 Bacterial metabolism requires an adequate iron supply. Although iron in soils is plentiful, it is not soluble at neutral pH, thus many soil microorganisms synthesize small-molecular-weight soluble iron-chelating molecules known as siderophores in order to sequester and solubilize iron before transporting it into the cell. [1] Azotobacter vinelandii is a soil free-living bacteria which optimal growth depends strongly on iron availability. Iron is essential to support its aerobic life as it is for nitrogen fixation and nitrogenase enzyme protection. This microorganism secretes three types of metallophores: 2, 3- dihydroxybenzoic acid (DHBA), catechols (azotochelin, aminochelin, protochelin) and a yellow-green fluorescent pioverdin-like peptide (azotobactin). Azotobactin production is evidenced by fluorescence under UV light radiation [2]. Recently it has been demonstrated that these siderophores not only has Fe (III) as a chelate ligand but Mo and V as well. These two trace elements are required as cofactors of various redox enzymes. [1, 2] There has been described a sequential pattern of siderophores synthesis according to iron concentration: first 2, 3-DHBA facilitates low-affinity iron uptake and when it is unable to provide sufficient iron for A. vinelandii’s optimal growth, the production of the catechols azotochelin, aminochelin (<7µM Fe) and protochelin (low Fe but high Mo) precedes the production of azotobactin (<3 µM Fe). This unusual sequential control suggests the existence of a complex model of iron-regulation of siderophore synthesis not yet completely elucidated. [3] In our laboratory working on A. vinelandii’s Gac/Rsm regulatory pathway we have found that a gacA mutant shows a deficient azotobactin synthesis since it doesn’t fluoresce. Despite the lack of this siderophore, GacA mutants do not show significant growth deficiencies compared with the wild type strain E growing in an iron-limited medium. When an O-CAS method for siderophore detection was performed, we found that the GacA mutant produces the catechol-type siderophores under unusual iron concentrations (<3µM Fe) while the E strain doesn’t. This evidence suggests that the major response regulator GacA is somehow involved in siderophore biosynthesis regulation. [1] Wichard T. Bellenger J. P., Morel Francois and Kraepiel Anne, “Role of Siderophore Azotobactin in the Bacterial Acquisition of Nitrogenase Metal Cofactors”, Environ. Sci. Technol. 2009, 43, 7218–7224 [2] Pierre Cornelis, “Iron uptake and metabolism in pseudomonads”, Appl Microbiol Biotechnol, 2010, 86:1637–1645 [3] M. S. Sevinc and W. J. Page, “Generation of Azotobacter vinelandii strains defective in siderophore production and characterization of a strain unable to produce known siderophores”, Journal of General Microbiology, 1992, 138, 587-596. Work supported by SEP-CONACYT 129525 project grant.



67

Growth in polyurethane and esterase activities of three Alicycliphilus strains

Luz Elena Moreno González y Herminia Loza Tavera Facultad de Química, Departamento de Bioquímica. UNAM Av. Universidad 3000, Copilco México, D.F. 04510 México. [email protected] Polyurethane (PU) is a polymer synthesized by condensation of diisocyanate and polyol. Depending on the structure of these precursors, PU is classified as polyester -PU and polyether -PU, providing a variety of structures and properties, making PU the most versatile polymer. By the same reason, PU waste has became a serious problem because of inappropriate management. Recently, PU biodegradation has started to be considered as an alternative that can be accomplished by microorganisms or purified enzymes. In our lab, we are studying Alicycliphilus sp. BQ1, a strain able to grow in a minimal medium with a commercial PU (Hydroform) (MM-PUh), as sole carbon source. We have shown that Hydroform can undergo hydrolysis of its ester bonds when exposed to a culture of Alicycliphilus sp. BQ1, so that an esterase activity has been suggested to be involved in PU degradation (App. Environ. Microbiol. 2007. 73:6214). In this work we compared the growth in MM-PUh, of two Alicycliphilus sp. strains, BQ1 and BQ5, with the type strain, Alicycliphilus denitrificans K601, which was originally reported as an alicyclic compound degrader. By using zymograms for esterase activity, we compared the protein pattern for esterases present in membrane and cytosol from the different Alicycliphilus strain grown in LB and MM-PUh and also we measured esterase activity during the growth of BQ1 strain by using p-NPA as substrate. Results: K601 was unable to grow in PU. There were no differences in the esterase patterns presented by the three strains. By zymogram three bands with esterase activity of about 60 kD, 31 kD and 20 kD were observed both, in the cytosol and membrane. Total esterase specific activity measured in BQ1 citosol and membrane extracts, showed similar values; it increased in the log phase and reach its maximum at 48 h, during the stationary phase. These results suggest that the ability to grow in PU resides not in an esterase activity, but in other proteins that provides the capacity to assimilate carbon from this polymer.

This work was supported by CONACYT grant 82881 and DGAPA grant 222811 to HLT.



69

The outer membrane protein I contributes to Poly-β-Hydroxybutyrate synthesis in Azotobacter vinelandii.

Muriel Millán LF*., Castellanos Escamilla M., Moreno León MS., Yanajara Parra D., Segura Gonzalez D. Espin Ocampo EG. Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México. *[email protected], Apdo. Postal 510-3, Cuernavaca, Morelos 62250, Mexico Introduction: Azotobacter vinelandii is a nitrogen-fixing soil bacterium able to produce poly-Beta-hydroxybutyrate (PHB) as a carbon and energy reserve, which is accumulated intracellularly in form of granules. PHB is a polymer of industrial and ecological interest because of its characteristics of biodegradable thermoplastic. The PHB biosynthetic operon phbBAC is transcribed from two overlapping promoters, one of which is under the control of PhbR, a transcriptional activator of the AraC family. In an attempt to identify new regulators of PHB synthesis in A. vinelandii, we carried out a random mini-Tn5 mutagenesis of strain phbR-, which presents 70% reduction in PHB accumulation as compared with the reference strain UW136. From this mutagenesis, we obtained a mutant strain with lower levels of PHB as compared with the mutant strain phbR-. The mini-Tn5 insertion was identified in the Avin23100 gene, which has 85% sequence identity with oprI of Pseudomonas aeruginosa. On the other hand, in our group, OprI (outer membrane protein I) was identified to be associated to membrane of PHB granules. In this study we characterized the effect of the oprI mutation on the PHB synthesis and phbR and phbB expression and confirmed the presence of OprI in PHB granules. Methods and materials: We constructed the oprI mutant derived from UW136 strain. The PHB content was determined in Peptone Yeast rich medium supplemented with 2% sucrose (sucrose PY) and the level of phbR and phbB transcripts was determined by Real-time RT-PCR. The content of PHB granules in the strains was evaluated by electron microscopy. The PHB granules were purified by glycerol discontinuous gradients and analyzed by Coomassie-stained SDS-PAGE. OprI was identified by mass spectrometry. Results: The oprI mutant produced lower levels of PHB as compared with the reference strain UW136 in PY sucrose medium. By electron microscopy we determined that the strain oprI- showed less PHB granules than reference strain. The oprI mutation significantly reduced the level of the phbR transcripts and partially reduced the level of the phbB transcripts. The presence of OprI in the PHB granules was confirmed. These results suggest that OprI is associated to the PHB granules and contributes to phbR and phbBAC expression and polymer accumulation. The mechanism by which OprI is associated to PHB synthesis remains to be investigated.



71

Analysis of the flagellar muramidase (sltf) from the photosynthetic bacterium Rhodobacter sphaeroides.

Manuel Osorio-Valeriano1, Javier de la Mora1, Teresa Ballado1, Laura Camarena2 and Georges Dreyfus1* Instituto de Fisiología Celular1, Instituto de Investigaciones Biomédicas2, Universidad Nacional Autónoma de México, 04510, México City, México.*[email protected] Biogenesis of the flagellum from R. sphaeroides is a tightly regulated process that requires the expression of more than 50 genes in a strict hierarchical order. The flagellar rod is composed by four proteins, FlgB, FlgC, FlgF and FlgG. Besides these structural components several more proteins are required for rod assembly among these FlgJ from Salmonella enterica, has been postulated to be a dual function protein: the N-terminal half could function as a scaffold or cap essential for rod assembly and the C-terminal half may function as a muramidase that degrades the peptidoglycan layer to facilitate rod penetration. FlgJ from R. sphaeroides lacks the C-terminal muramidase domain and mutations in this protein render a Fla- phenotype. In previous work we have described the characterization of an open reading frame (orf) RSP0072, which showed the presence of a soluble lytic transglycosylase domain. The deletion of the N-terminal region (112 aa) of this gene renders a non-motile phenotype, in this preceding study we demostred that RSP0072 interacts with the flagellar rod-scaffolding protein FlgJ. Now in this work we continue charactering of RSP0072 (SltF). The amino acid sequence analysis of SltF suggests that is exported to the periplasm via the Sec pathway (SEC). The deletion of the first 30 residues of SltF renders a protein which is unable to complement a sltF strain. On the other hand the C-terminal moiety of SltF showed to be important for flagellum formation. The C-terminal mutants of SltF were unable to restore motility to the sltF strain, this suggests that region is essential for SltF function. Purified C-terminal mutant proteins of SltF showed lytic activity on extracts of M. lysodeikticus. Pulldown interaction assays between C-terminal mutants of SltF with FlgJ showed that SltF mutants interact better than with SltF wild type. This result was confirmed from yeast two-hybrid experiments. We propose that C-terminal region of SltF is a modulatory domain that regulates the interaction with FlgJ. This protein is located strategically in order to start rod formation and subsequently interacts with SltF to complete the rod structure. This study is supported by CONACyT 106081 and DGAPA IN213408.



73 HSL production and regulation in Sinorhizobium fredii NGR234

Francisco Pedraza López, Miguel Ángel Cevallos Gaos Centro de Ciencias Genómicas, UNAM. [email protected] Programa de Genómica Evolutiva; CCG –UNAM. Avenida Universidad s/n, col. Chamilpa, CP: 62210. Cuernavaca, Morelos, (777)3291690 Bacteria have the capacity to communicate between them, through diffusible chemical signals. Chemical communication inside a bacterial population allows the individuals to estimate the cellular density in the medium and in a coordinated way, under a process known as quorum sensing. Quorum sensing system involves the production, liberation and detection of molecules that have been named auto-inducers. The predominant auto-inducers on Gram-negative bacterias are called acyl-Homoserine Lactones (HSL). The enzymes that synthesize the HSL are HSL synthases of luxI-type. The HSLs can trespass freely through the membranes and its detection depends on the union with the LuxR-type receptor proteins. The TraR-HSL complex activates the transcription of genes that are under the control of the quorum system. We are working at the laboratory with the bacteria Sinorhizobium fredii NGR234 as a study model. Recently the complete sequence of the genome of S. fredii NGR234 was published, which permitted to find some elements that could contribute to the detection of quorum in this organism. It was discovered that the S. fredii NGR234 genome counts with two HSL synthases called TraI and NgrI. The Tral protein is codified in the plasmid pNGR234a. In contrast, the Ngrl protein is codified in the chromosome and now we know that it produces an HSL with a large acyl group. In the vicinity of ngrl and tral we find two regulators of response: traR and ngrR. Through thin layer chromatography tests we corroborated that S. fredii NGR234 produces, in cultivation, two HSLs: one of them is of short acyl chain, while the other, one of long chain. To start analyzing the quorum sensing system network, we built mutants in the ngrI and tral genes through the insertion of an omega cassette (Ωsp). The profile of HSLs these derivatives produced was analyzed through diverse tests of TLC. It was observed that the quantities of both HSL produced decrease enormously in the mutant ones: you only have to interrupt any of the two different THLs observed in the wild stump. This suggests us that somehow the regulation of the expression of any of these two HSL-synthases is interconnected. With the finality of producing the HSL molecules in an independent way, the tral gene was over-expressed in the mutant that hasn’t got synthases. To our surprise, the over-expression of this gene not only increases the levels of both HSLs previously described, it also synthetizes other two different HSLs. This indicates us that Tral is an enzyme more lax regarding the production of HSL. This kind of result suggests us that the regulation and production of HSLs in the S. fredii NGR234 genome is more complex than what was previously suggested. To start elucidating the regulation of the genes expression related to the quorum control, we have built transcriptional fusions of the ngrl, tral, ngrR and traR genes with the fluorescent green protein (EGFP) and we have measured their expression in real time during growth kinetics. In these experiments we have observed that these four genes increase notably their expression (fluorescence unities/DO), inasmuch these cultivations get close to the stationary phase, which suggests us that the expression of these genes depends of the growth phase, behavior observed in the genes that depend of the quorum.



75

The repAC replication system of the Rhizobium leguminosarum pRL7 plasmid is functional: implications regarding the origin and evolution of

repABC plasmids Gabriela Pérez Segura, Ángeles Pérez Oseguera, Miguel Ángel Cevallos Gaos Centro de Ciencias Genómicas-UNAM [email protected] Programa de Genómica Evolutiva, , Centro de Ciencias Genómicas, Av. Universidad s/n Col. Chamilpa 62210, Cuernavaca, Mor. Tel: 017773291690 The repABC replication/ partitioning systems are commonly found in alpha-proteobacteria plasmids and in secondary chromosomes. All of the elements required for their replication and stable maintenance are encoded within a single transcription unit, the repABC operon. The repC gene encodes an initiator protein, while RepA, RepB and a centromere-like sequence (parS) direct plasmid segregation. Strains containing two or more repABC plasmids are a common feature in some alpha proteobacteria groups, indicating that the repABC plasmid family embraces several incompatibility groups. Genes encoded within repABC operons are highly dynamic: each one possess its own distinctive phylogeny and homologous recombination events are common within these operons. Additionally, alpha-proteobacterial genomes contain repAB genes not associated with the ctRNA or with repC as well as plasmids whose replication depends on a ctRNA-repC module without the participation of repAB genes. Some alpha-proteobacteria have repC genes clustered with other genes that are not involved in replication/partitioning functions. These atypical associations of genes could have an important role in the origin and diversification of new plasmids. Here we evaluated the functionality and possible evolutionary consequences of one of these atypical gene associations: the repAC genes present in the R. leguminosarum plasmid pRL7. The repAC genes are organized in an operon and they are capable of sustaining replication but in an unstable manner. RepC was essential for replication, and the origin of replication resides within its coding region. In contrast, RepA did not play any relevant roles.



77

Regulation of alginate by the Histidin Kinase CbrA in Azotobacter vinelandii

Quiroz Rocha Elva, Muriel Millan Felipe, Guzmán Aparicio Josefina, Espín Ocampo Guadalupe, Núñez López Cinthia Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca Morelos, México. Contact: Elva Quiroz. Phone: (777)3292629, E-mail: [email protected] Introduction. Azotobacter vinelandii produces secondary metabolites of industrial use, such as the extracellular polysaccharide alginate and the polyester poly-β-hydroxybutyrate (PHB) (1). Alginate is a polysaccharide used as a gelling and viscosifying agent in the industry. At present the alginates used in the industry are extracted from seaweed, which are subjected to variations due to environmental changes (2). For this reason the production of bacterial alginate, of a defined and uniform composition, is an interesting alternative (1, 2). Background. An alginate-overproducing mutant, called GG15 was chosen for further characterization. GG15 mutant carries the Tn5 insertion within the cbrA gene, encoding a paraloge of the CbrA histidin kinase (composed of 981 aa) of the Pseudomonas aeruginosa CbrA/CbrB two-component system. The genetic arrangement of the A. vinelandii cbrA locus (cbrA-cbrB-crcZ-pcnB) is conserved with respect to P. aeruginosa. In Pseudomonas spp. it was recently shown that that CbrA/CbrB is necessary for the transcription of the small RNA (sRNA) CrcZ, which counteracts the negative effect of the Crc protein. The Crc/CrcZ system is involved in the jerarquical assimilation of carbon sorces trough a process named carbon catabolic repression. Crc inhibits the expression of genes by binding to the target mRNA blocking its translation. The CrcZ small RNA (sRNA) contains five Crc-binding sites antagonizing the repressing effect of Crc. Phenotypic characterization of the GG15 mutant showed an increase of 11-fold in alginate production when compared to the wild type strain. Moreover, the genetic complementation of the mutant with a copy of the cbrA gene reduced alginate production to levels similar to those of the wild type strain, indicating that the histidin kinase CbrA negatively controls the synthesis of alginate. In this study we investigate the molecular mechanism of the negative regulation of alginate by the histidin kinase CbrA and explore the possible involvement of the Crc/CrcZ posttranscriptional system in such control. Since CbrA/CbrB likely activates the transcripcion of the sRNA CrcZ, it is possible that in the GG15 mutant cbrA:.Tn5 the higher activity levels of Crc may account for the increase in alginate production. Results. Several attempts were made to construct mutants in either the crc or the crcZ gene without success, suggesting an essential role of the Crc/CrcZ posttranscriptional system in A. vinelandii. Our results suggest that CbrA controls alginate synthesis in a Crc-independent manner since over-expression of Crc in trans in the wild type background does not result in higher levels of alginate. Current work is focused on exploring the effect of the sRNA CrcZ over-expression in the wild type strain and in the GG15 cbrA::Tn5 mutant. On the other hand, our results suggest that the truncated form of the CbrA Histidin Kinase (aa 1-660) of the GG15, but not its absence, might be exerting the negative effect on alginate synthesis as a cbrA::Sp mutant (truncated at aa 501) failed to overproduce alginate. The reason for such result will be investigated. References: 1. Espin, G. (2003). Biología de Azotobacter vinelandii. IBT. UNAM. 2. Galindo, E., Pena, C., Nunez, C., Segura, D., and Espin, G. (2007). Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Fact 6, 7. 3. Li, W., and Lu, C. D. (2007). Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa. J Bacteriol 189, 5413-20. 4. Nishijyo, T., D. Haas, et al. the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa." Mol Microbiol 40(4): 917-3



79

Functional analysis of the two-component regulatory system ActSRin R. etli CFN42

Alma Reyes-González1,2, David Zamorano-Sánchez2, Patricia Rivera2and Lourdes Girard2 1Facultad de Ciencias, UAEM. 2Centro de Ciencias Genómicas, UNAM. Ap. Postal 565-A, Cuernavaca Mor, 62271, México. Telephone: 777 3291896. E-mail: [email protected]

The two-component regulatory systems RegSR, RegBA, PrrBA and ActSR are well conserved. The corresponding response regulators control the expression of different regulons that are involved in N2 fixation, CO2 fixation, photosynthesis or acid tolerance. In Rhizobiumetli CFN42 the mechanisms that control the expression of the master regulator FixKf are poorly understood. The histidine kinase FixL and the recently identified response regulator FikR, appears to be the main regulatory system in response to oxygen. The role that the ActSR regulatory pair may has on the regulation of the fixKf gene either in microaerobiosis or in the acid response has not been analyzed. The NifA activator in R. etli, regulates its own expression in response to the microaerobic environment of the nodule. We can speculate that the ActSR system of R. elti also regulates the expression of nifA in microaerobiosis like in B. japonicum and S. meliloti. In this work we analyzed the genetic arrangement of the actS and actR genes. These two genes appear to be encoded in a operon with ocdch1(ornithinecyclodeaminase gene) and helO(HrpAhelicase-like protein).In order to identify the promoters in this region, different segments were fused toapromoterlessuidA gene. Two segments promote activation of the uidA gene in aerobic conditions. One is located upstream of the traductional start site of ocdch1, and the other was located within the codifing region of actS. In both, microaerobic conditions provoke an induction of the expression. With these results we concluded that in contrast to what happens in S. meliloti and B. japonicum, actR has its own regulatory region and actS,helO and ocdch1 are probablytranscribed as an operon. In order to determine ifActRcontrols the expression of fixKf in microaerobiosis we compared the expression of fixKf, in a wild type genetic background and in a strain with a null mutation in the ActR regulator. Our results revealed that the expression of fixKfhas a fivefold reduction in the mutant strain. This suggests that in addition to FixL and FikR, ActR is able to modulate fixKf expression in microaerobic conditions. We are currently analyzing if there is coordination between these three regulatory elements, and if the regulatory activity of ActRis controlled by ActS.

We thank M.P. Elizabeth Salas and Rosa Ma. Ocampo for their excellent technical assistance. A. Reyes-González, and D. Zamorano-Sánchez received a doctoral scholarship from CONACYT.This work is partially supported by grant 202109 (DGAPA, UNAM).



81

Identificación de los elementos regulatorios en cis y en trans que modulan la expresión de los genes flagelares fla2 de

Rhodobacter sphaeroides

Rivera Osorio Anet1, Poggio Sebastian1, Osorio Aurora1, Dreyfus Georges2 y Camarena Laura1

1Instituto de Investigaciones Biomédicas, 2Instituto de Fisología Celular, Universidad Nacional Autónoma de México. Instituto de Investigaciones Biomédicas, 3er Circuito Exterior S/N Ciudad Universitaria, México, D.F., C.P. 04510. [email protected], Tel. 56229222 Entre las diferentes formas de motilidad bacteriana conocidas hasta el momento, la mejor estudiada es el nado. Para realizar este tipo de desplazamiento es necesario que la bacteria sea capaz de ensamblar uno o varios flagelos. Rhodobacter sphaeroides es una alpha proteobacteria la cual en condiciones de laboratorio ensambla un solo flagelo de localización subpolar. Los genes responsables de la biogénesis de dicho organelo han sido identificados, y la inactivación de estos provoca que las células sean incapaces de formar flagelo (fenotipo Fla-). A partir de una cepa Fla-, en el laboratorio fue aislada una cepa capaz de nadar en medio líquido gracias a la activación de un conjunto de genes crípticos, los cuales una vez expresados, son responsables de la formación de 3 a 5 flagelos polares por célula. Análisis filogenéticos de estos genes y de los genes flagelares previamente caracterizados mostraron que los genes crípticos pertenecen al linaje de las alpha proteobacterias, mientras que los genes expresados en condiciones de laboratorio se agrupan con el grupo de las gamma-proteobacterias. Estamos interesados en elucidar los mecanismos que llevaron a silenciar los genes flagelares endógenos de R. sphaeroides y definir los elementos regulatorios en cis y en trans que modulan su expresión. A la fecha, el análisis funcional de las diferentes regiones flagelares que se ubican entre las regiones intercistronicas de los genes flgB, fliI, fliL, y fliF nos han llevado a identificar cuatro posibles regiones promotoras; las cuales hemos delimitado en fragmentos de 300 pb. Se ha identificado el posible sitio de inicio de la transcripción para cada una de estas regiones promotoras, mediante experimentos de 5´-RACE. Las posibles cajas -10 y -35 han sido mutagenizadas y su efecto sobre la expresión del gen reportero está siendo evaluada actualmente. En este trabajo se propone una secuencia consenso para los genes flagelares fla-2. Los factores que actúan en trans y que potencian o disminuyen la expresión de los genes fla2, están actualmente siendo evaluados mediante el aislamiento de cepas mutantes.



83

The NprR-NprB system of B. cereus group: evolution and role in B. thuringiensis strain 8741

Jorge Gustavo Rocha Estrada1, Victor Flores López2, Rosina Cabrera Ruiz1, Adriana Soto Guzmán1, Gabriel Guarneros2, Mayra de la Torre*1

1- Centro de Investigación en Alimentación y Desarrollo, A. C. Hermosillo, Sonora, México. 2- Centro de Investigación y de Estudios Avanzados IPN. Zacatenco, D. F. *Corresponding author. Email: [email protected] Centro de Investigación en Alimentación y Desarrollo A.C. Carretera a La Victoria km 0.6 C.P. 83304, Hermosillo, Sonora, México. Teléfono: + 52 (662) 289-2 Quorum sensing (QS) is a bacterial mechanism for regulation of gene expression in response to cell density. In gram positive bacteria, oligopeptides are the signaling molecules to elicit QS. The RNPP protein family (Rap, NprR, PlcR and PrgX) are intracellular QS receptors that bind directly to their specific signaling peptide to regulate the transcription of several genes. NprR was originally described as the activator of a neutral protease in Bacillus subtilis and it has been recently related to sporulation, cry genes transcription and extracellular protease activity in strains from the B. cereus group. In the Bacillus thuringiensis genome, downstream nprR a gene encoding a putative QS signaling propeptide (nprB) was found. We aimed to determine if the nprR-nprB gene cassette is widespread in the B. cereus group, and proposed that putative mature propeptide genes (nprB) co-evolved with their respective receptor gene nprR. We also tested the effect of synthetic peptides encoded in nprB on sporulation and cry transcription in Bacillus thuringiensis. The nprR-nprRB sequences from 21 strains of the B. cereus group were analyzed. A phylogenetic tree of amino acid sequences of NprR revealed six groups, each corresponding to one putative mature NprB sequence: SKPDI, SDIYG, WKPDN, WKPD [T/L], SNPDI and SRPDV. Only the signaling pro-peptides containing the first three sequences are predicted to be exported from the cell. The NprR tree does not match the current taxonomic grouping of the B. cereus group or the phylogenetic arrangement obtained when using 16s rRNA sequences from the same strains. SKPDI and other synthetic peptides encoded in nprB from Bt8741 strain had effect on sporulation and expression of a cry1Aa’lacZ transcriptional fusion in the strain Bt8741, suggesting that both are somehow regulated by QS receptor NprR.



85

LadS2 histidine protein kinase regulates alginate production in Azotobacter vinelandii

Rodríguez Antonio Ana Laura, García García Silvia Ma. del Carmen, Castañeda Lucio Miguel Centro de Investigaciones Microbiológicas, Instituto de Ciencias, BUAP. Edif. 103-J C.P.72570. Puebla, México. [email protected] Tel. (222)2295500 Ext. 2527 Azotobacter vinelandii is a soil bacterium belonging to the γ-proteobacteria. Mucoid strains of this bacteria produce the extracellular polysaccharide alginate and the GacS/A two-component system controls alginate production. Two-component signal transduction systems are composed of a trans-membrane histidine phosphokinase, which senses environmental signals, and a cytoplasmic response regulator, which activates transcription upon being phosphorylated by the sensor. In Pseudomonas aeruginosa and Pseudomonas flourescens two additional histidine phosphokinase (RetS and LadS) are involved in the GacA phosphorylation (1). In Silico analysis carried out in A. vinelandii genome sequence revealed three possible LadS paralogous. The putative LadS homologues were called lads1, lads2 and ladS3. In this work we generated a ladS2 mutant by insertion of streptomycin resistance cassette. The ladS2 mutant showed two-fold increase of alginate production compared to the wild type strain. These results suggest that LadS2 is a negative regulator of alginate production; this might be because of its interaction with GacS. 1. Ventre. I, Andrew L. Goodman, Isabelle Vallet-Gely, Perrine Vasseur, Chantal Soscia, Soren Molins, Sophie Bleves, Andre´e Lazdunski, Stephen Lory, and Alain Filloux. (2006) PNAS. vol. 103. Pp.171–176. This research was founded by VIEP-BUAP /CALM-NAT10-II grant



87

Membrane lipid turnover in Sinorhizobium meliloti by intrinsic phospholipases A and a lysophospholipase

Diana X. Sahonero-Canavesi, Christian Sohlenkamp, Isabel M. López-Lara, Otto Geiger Programa de Ecología Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Morelos, México [email protected]

Phospholipids are well-known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Although much is known about phospholipid signaling, turnover, and remodeling in eukaryotes, few of these aspects are understood in bacteria. Distinct conditions of stress cause major changes in the membrane lipid composition of bacteria. For example, in Sinorhizobium meliloti under phosphate-limiting conditions of growth, phospholipids are largely replaced by phosphorus-free membrane lipids. Upon phosphorus limitation, a phospholipase C (PlcP) is induced that degrades phosphatidylcholine (PC) and phosphatidylethanolamine of the bacterium ’s own membrane to diacylglyceride (DAG)(Zavaleta-Pastor et al., 2010 Proc Natl Acad Sci USA 107: 302-307). DAG in turn serves as membrane anchor during the biosynthesis of phosphorus-free membrane lipids. In another example, a FadD-deficient mutant of S. meliloti is unable to convert free fatty acids to their coenzyme A derivatives and therefore cannot degrade fatty acids by β-oxidation. Surprisingly, such a FadD-deficient mutant accumulates free fatty acids (Pech-Canul, personal communication) comprising up to 20% of the total lipid fraction. Presently, it is not known how these free fatty acids are formed in rhizobia. We have identified potential genes for phospholipases A (SMc00930, SMc01003) and a lysophospholipase (SMc04041) in the S. meliloti genome that might be responsible for the fatty acid release. Expression of SMc04041 and SMc01003 in Escherichia coli or of SMc04041, SMc00930 and SMc01003 in S. meliloti causes increased accumulation of free fatty acids. PC exists in S. meliloti but not in E. coli and therefore we hypothesize that SMc00930 liberates fatty acids specifically from PC forming lyso-PC as the second product. Individual mutants deficient in SMc04041, SMc00930, or SMc01003 of S. meliloti accumulate similar low amounts of free fatty acids as the wild type (around 2%). In contrast, a FadD-, SMc01003-deficient double mutant of S. meliloti accumulates free fatty acids up to 4% of the total lipid fraction showing that much of the fatty acids normally accumulated in the FadD-deficient mutant are due to the action of SMc01003. Presently, we characterize substrate specificities of SMc04041, SMc00930, or SMc01003 as well as their physiological roles in the free-living bacterium and in symbiosis with the legume host plant.



89

Cloning, expression and characterization of soluble inorganic pyrophosphatase of Rhodospirillum rubrum and

Rhodobacter sphaeroides

Sánchez Ortiz Verónica, Pelayo González Lili, Celis Sandoval Heliodoro, Suaste Olmos Fernando Instituto de Fisiología Celular, UNAM. A.P 70-243, 04510 México, D.F. [email protected]. Tel. 56225667 Soluble inorganic pyrophosphatase (sPPase, EC 3.6.1.1) is an essential and a ubiquitous enzyme catalyzing the cation-dependent interconversion of inorganic pyrophosphatase and orthophosphate and it’s central in the phosphorus metabolism. In 1971, Klemme and Gest (1) described significant differences between the sPPase present in some photosynthetic bacteria, but until then there was only one kind of sPPase (Family I, FI). Was until 1998 when it was characterized a different type of sPPasa present in Bacillus subtilis (2, 3). Now it is known that sPPase comprise two families that differ markedly in their sequence and structure but retain similarity in their active site and its catalytic mechanism, FI sPPases are present in all cell types, from bacteria to human, while FII sPPases are present exclusively in bacteria and archeae(4). In the present study sPPases from two purple non-sulfur photosynthetic bacteria, Rhodospirillum rubrum (FI) and Rhodobacter sphaeroides (FII), were cloned in the pCR 2.1 TOPO vector, subcloned in pBAD HisB expression vector and transformed in the E. coli Rossetta strain, it will be reported here a study their catalytic mechanisms from pure enzymes. 1. Klemme, J. and Gest, H. (1971). Proc. Nat. Acad, 68: 721-725.

2. Shintani, T., Uchiumi, T., Yonezawa, T., Salminen, A., Baykov, A., Lahti, R. and Hachimori, A. (1998). FEBS Lett. 439: 263-266.

3. Young, T.W., Kuhn, N. J., Wadeson, A., Ward, S. Burges. D. and Cooke, G.D. (1998). Microbiology. 144: 2563- 2571.

4. Fabrichniy, I., Lehtio, L., Tammenkoski, M., Zyryanov, A., Oksanen, E., Baykov, A., Lahti, R. and Goldman, A. (2007). The Journal of Biological Chemistry. 282: 1422–1431.



91 Genomic Analysis of Rhizobium etli Bacteriophages

Rosa Isela Santamaría1, Patricia Bustos1, Luis Lozano1, Omar Sepúlveda2, César Rodríguez1, José Luis Fernández1, Soledad Juárez1, Gabriel Guarneros2, Guillermo Dávila1, and Víctor González1 1Center for Genomic Sciences UNAM, México 2CINVESTAV-IPN, México. Tel. (777)3291690. e-mail [email protected]

Bacteriophages have an enormous impact in the ecology and in the genomic diversity of bacteria. They are very known agents of gene mobilization across bacterial populations, and it is known that bacterial genomes harbor prophages as well as individual genes related to bacteriophages. In contrapart, bacteriophages often carry bacterial genes as a result of the prophage induction and recombination, In the genomes of Rhizobium etli, a soil bacteria that establishes nitrogen fixation symbiosis with the common bean, about 1% of the total gene coding proteins have a match with some of the Phage Orthologous Groups (POGs), a database which cluster the protein products found in every complete bacteriophage genome already sequenced. To understand the genetic interactions between R. etli and their bacteriophages, we isolated and characterized 14 bacteriophages able to infect different strains of R. etli. They were obtained by the enrichment method from rhizosphere soil of beans in México. Host range of the bacteriophages was variable among a collection of R. etli strains, but limited to this species. We obtained the complete genome sequence of 9 bacteriophages. The rest of bacteriophages were resistant to restriction enzymes and in vivo cloning, probably due to DNA modifications. The genome size of the sequenced phages varies from about 43 Kb the smallest to 115 Kb the largest, but most of them have a medium size from 45 to 50 Kb. They were grouped in four classes according to their genomic similarity. A large proportion of the genome of these bacteriophages consists of hypothetical genes (65-70%), and a minor percentage represents bacterial genes like cobT (cobalamin biosynthesis), parAB, and repC (plasmid replication). So far, we have obtained the electronic micrography for one bacteriophage of the collection. It is a short tailed bacteriophage belonging to the order of the Caudovirales and to the family of the Podoviridae. Current experiments are directed to detect the morphology of the rest of bacteriophages by electron microscopy and to correlate host range with genome variation. Acknowledgments. This project is funded by the PAPPIT IN215908. We thank computational aid of José Espiritu and Víctor del Moral. We also thank Dr. Humberto Peralta, Dr. Esperanza Martínez, Dr. Susana Brom, Dr. Valeria Souza and Dr. Ivonne Toledo for providing some of the soil samples or the strains used.



93

Chromium Kills Bacillus subtilis cells by oxidative-induced-DNA damage

Fernando Santos Escobar, Félix Gutiérrez Corona and Mario Pedraza Reyes* Department of Biology, Division of Natural and Exact Sciences, University of Guanajuato, P.O. Box 187, Guanajuato, Gto. 36050 MEXICO. Phone: (473) 73 2 00 06, ext. 8161. Fax: (473) 73 2 00 06, ext. 8153. Email: [email protected]. Hexavalent chromium, Cr(VI), is highly cytotoxic and genotoxic for all forms of life. It has been proposed that, the deleterious effects of this heavy metal are a consequence of its intracellular reduction to Cr(III) generating highly reactive oxygen species (ROS) that promote damage to lipid, proteins and nucleic acids (1, 2, 3). However, to date, the mechanisms involved in preventing and/or repairing the genotoxicity of Cr (VI/III) have not been completely elucidated. In the present work, the Gram-positive bacterium Bacillus subtilis, an excellent paradigm that counts with highly developed genetic and molecular tools for its manipulation was used to investigate this point. Using a recombinant B. subtilis strain containing a chromosomal recA-lacZ fusion and several knock out strains deficient in DNA repair proteins we present experimental evidence suggesting that 1) Cr(VI) and Cr(III), in a direct or indirect manner interact with DNA and induce the SOS response; 2) The superoxide dismutases SodA and components of the oxidized guanine (GO; 4) system counteract the genotoxic effects of hexavalent chromium. Taking together, our current results suggest that hexavalent chromium generates ROS that modify DNA and kills bacterial cells through oxidative-induced DNA damage. 1. Cervantes et al., 2001. FEMS Microbiology Reviews 25. 335-347. 2. Liu and Shi, 2001. Molecular and Cellular Biochemistry. 222; 41–47. 3. Sumner et al., 2005. Microbiology. 151; 1939–1948. 4. Vidales et al., 2009. Journal of Bacteriology. 506–513. Work supported by grant 84482 from CONACYT to MPR. Fernando Santos is supported with a scholarship from CONACYT.



95

Molecular characterization and antimicrobial susceptibility in Escherichia coli strains isolated from pediatric patients with diarrhea

Toledo Rojas Andrea, Sergio Bárcenas León, Héctor Santos Balbuena, Patricia Arzate Barbosa, Albina Martínez Pérez and Castro Ana María Depto. Salud Pública, Facultad de Medicina, Universidad Nacional Autónoma de México. E-mail: [email protected]. Diarrheal disease is a major cause of illness and death among infants and young children worldwide. The World Health Organization has estimated that diarrheal diseases are responsible for the deaths of 2.5 million children worldwide each year, with an annual mortality rate of 4.9 per 1000 children and an incidence of 3.2 episodes diarrhea per year in children under 5 years (Kosek et al., 2003). Among the Escherichia coli causing intestinal diseases, there a six well-described virotypes: enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enterophatogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), diffusely adherent E. coli (DAEC) and enterotoxigenic E. coli (ETEC). Recently, some authors reports the high resistance of diarrheagenic E. coli against different antimicrobial agents, which favors the emergence of multidrug resistance hindering the management of these diseases. Likewise, it is unclear whether there are differences in age-related susceptibility to specific strains, especially among infants. In the present study we implemented a set of microbiological techniques: cell culture, immunological, molecular and antimicrobial drug susceptibility of diarrheagenic E. coli, which may will determine the distribution of different virotypes of E. coli associated with episodes of diarrhea in children less than five years old in México City. For this propose we carry out a study that involved 500 children aged 0-60 months in México City with diarrhea.



97

Distribution of Chromate- and Mercury-Resistance Genes in Clinical Antibiotic-Resistant Gram-negative Bacteria

Gustavo Caballero-Flores1, Milagros Y. Acosta-Navarrete1, Jesús Silva-Sánchez2, Carlos Cervantes1and Martha I. Ramírez-Díaz1 1Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana, Edificio B3, Cd. Universitaria, Morelia, Mich. 58030. 2Centro de Investigación Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Mor. [email protected] Mercury-resistance genes, as well as genes for resistance to other heavy metals, are often found in plasmids or transposons of clinical bacteria, commonly along with antibiotic-resistance genes (1). Genes conferring chromate (CrO4

2-) resistance are frequently found in environmental bacteria, but no studies on the presence of these genes in clinical bacteria have been conducted. The distribution of chromate-resistance genes was evaluated in a collection of clinical Gram-negative antibiotic-resistant strains isolated in México, and compared with that of mercury-resistance genes. Half of the strains (64/130) showed resistance to either chromate (CrR) or mercury (HgR), and 14% were resistant to both agents. CrR were found in Pseudomonas aeruginosa (67%) and Klebsiella pneumoniae (61%) strains, whereas HgR was determined in strains of Enterobacter cloacae (65%) and P.aeruginosa (60%). The presence of CrR or HgR genes was assayed in strains that showed high resistance to each agent by colony hybridization with probes derived from the chrA gene, encoding the ChrA efflux protein, and the merA gene, encoding the MerA mercuric reductase. DNA from the majority of metal-resistant strains hybridized with chrA (88% of CrR strains) and merA (80% of HgR strains). Southern blot hybridization was employed to further identify chrA sequences in plasmid-containing CrR strains. Plasmids of varied size in P. aeruginosa strains (22-38 kb) and in K. pneumonia and E. cloacae strains (40-100 kb) were found to contain chrA-related sequences. Four plasmids of IncN or IncP incompatibility groups from enterobacterial isolates were each transferred by conjugation to an Escherichia coli recipient strain, where they conferred CrR. This is the first report of clinical enterobacteria bearing plasmids with functional CrR genes. These data suggest that, as widely demonstrated for HgR genes, CrR genes are also broadly distributed in Gram-negative clinical bacteria, probably by means of lateral transfer via conjugative plasmids or transposons. 1. Osborn et al. (1997) FEMS Microbiol. Rev. 19:239-262..



99

Novel chromogenic and fluorogenic culture media as tools for the identification of bacteria

Claudio Rodríguez Martínez, Raisa Zhurbenko, Tamara Lobaina Rodríguez, Marilyn Díaz Pérez Centro Nacional de Biopreparados. [email protected]; Carretera a Beltrán km 1 ½, Bejucal, Mayabeque, Cuba; Apartado postal 6048, Habana 6; Tel. 53-47-682441

Chromogenic and fluorogenic substrates used previously in molecular biology experiments as markers for genetic transformations among other applications have being used as valuable ingredients in culture media for the identification of bacteria. Our research group have developed several chromogenic and/or fluorgenic culture media for the faster isolation identification and enumeration of Gram positive and Gram negative bacteria. Glucuronidase, galactosidase, glucosidase and aminopeptidase activities of different species of microorganisms were detected by combinations of different specific chromogenic and/or fluorogenic substrates as methylumberypheryl, X-, Magenta-, Rose-, paranitrophenyl- substrates, among others and also by their combination with “traditional” biochemical substrates and pH indicators included in culture media. This approach allowed the combination, in one single steep, the isolation, differentiation, identification and enumeration of a significant group of bacteria in a single plate in a reduced time. The best example of this research strategy was the development of a chromogenic culture medium for the isolation, differentiation, identification and enumeration of Gram-negative bacteria in clinical, veterinary, food, cosmetic and water samples. The medium allows the identification of Aeromonas spp., Pseudomonas spp, E. coli, and Salmonella. Coliform bacteria, other than E. coli (Klebsiella, Citrobacter, Enterobacter, among others) could be differentiated as a group. Some species of Shigella and Serratia could also be differentiated. The medium bases its functionality on the combination of glucuronidase, galactosidase and a specific carbohydrate splitting properties of some species of Salmonella, together with the proteolytic activity of several bacteria and the induction of the pigment and fluorescence production by Pseudomonas. Other media were developed with the same approach for reducing the number of plates, time and labor in diagnostic microbiology: two liquid media and two solid media for the isolation, identification and enumeration of E. coli and coliform bacteria, chromogenic media for the isolation, differentiation and enumeration of Enterococcus, media for the detection and differentiation of Salmonella and coliform bacteria and two media for the isolation, differentiation and/or identification and enumeration of up to 6 species of Candida. Further developments include other media such as a composition for the isolation and differentiation of Enterococcus, Streptococcus and Staphylococcus and a miniaturized test for identification of bacteria based on nanostructured matrixes embebed with special media.

ABSTRACTS EVEN POSTER

PRESENTATIONS

Even Poster Session II. Wednesday November 9, 2011


2

Differential expression of virulence factors by clinical atypical Pseudomonas spp. isolations

2Cárdenas-Rodríguez, A.; 1Morales-Espinosa, M.R.; 1Delgado-Sapién, G.; 1Sánchez-Méndez, J. L. y 2*Aguirre-Ramírez, M (1) Departamento de Microbiología y Parasitología de la Facultad de Medicina, UNAM. (2*)Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Chi. Tel. (656) 6881800 ext. 1686 y 1786. [email protected] Pseudomonas aeruginosa is an opportunistic human pathogen Gammaproteobacteria. It is the main Gram-negative bacterium responsible of nosocomial deaths. The quorum sensing (QS) response regulates the expression of several cytotoxic compounds and hydrolytic enzymes that determine the bacteria pathogenesis. The phenazyne derivative pyocyanin, inhibits cellular respiration and produces oxidative stress. Elastase, besides its broad tissue proteolitic activity, is an immunomodulator. Rhamnolipids biosurfactans have haemolytic and neutrophil chemotactic activity. Twenty-two strains were isolated from patients of Hospital “Centro Médico Nacional Siglo XXI_IMSS”. These strains share some characteristics of Pseudomonas aeruginosa (growth at 42ºC, toxA gene, oxidase expression) and were phenotipicaly clasified as they would be the same species. However, they produce diferentially their virulence factors and the genotypification by 16S and 23S do not correspond to PAO1 strain. That is its atipical name. The aim of this project is quantify the production of pyocyanin, elastase and rhamnolipids by the atypical pathogen Pseudomonas spp. For this purpose we grew the bacteria in high rhamnolipids production conditions (PPGAS medium) for 24 hrs. We used spetrophotometric methods to quantify the three types of virulence factors. By the results, we classified the strains in four kind groups according to the expression patterns: 1) the one that do not produce any of the three virulence factors; 2) the strain that over expresses the all three 3) the group that over expresses two of the three virulence factor and 4) the group that over expresses just one of the three virulence factors.



4

The putative ArcB leucine zipper of ArcB is required for proper signaling

Adrián F. Alvarez1, Luis Alberto Núñez Oreza, Dana Días Jiménez, and Dimitris Georgellis Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., México. 1 E-mail: [email protected]. Phone: (52) 55 56225738 The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. In the region connecting the catalytic domains with the transmembrane domain, ArcB contains a putative leucine zipper. Here we report that the ArcB protein segment harboring leucine residues 73, 80, 87, 94, 102, 108 and 115 fulfills the molecular characteristics of a leucine zipper, and that this motif is necessary for proper ArcB signaling.



6

Utilización de la espectroscopia raman y métodos multivariantes para la caracterización y diferenciación de cepas de la bacteria Escherichia Coli.

Avila Rodríguez Raquel1*, Hernández Nava Nereyda1, Martínez Ramírez Alejandro1, Alonso Karla Raquel2, Alvarado Rodríguez Juana2 1Universidad Autónoma de San Luis Potosí Campus Altiplano, Carr. Cedral Km 5+600 Ejido San José de las Trojes Matehuala, S.L.P. C.P. 78700; 2Facultad de Ciencias Químicas de la Universidad Autónoma de San Luis Potosí, 78000 San Luis Potosí, S.L.P., México. [email protected] INTRODUCCIÓN. La gastroenteritis es una enfermedad causada por bacterias, provocando la inflamación de la cubierta del intestino grueso, del intestino delgado, y del estómago, que puede llegar a ser grave en niños, ancianos o personas con un sistema inmune débil, una de las bacterias que puede provocar este problema es la E. coli, la principal dificultad encontrada en el tratamiento de la gastroenteritis es el largo tiempo (4 días) [1] para la identificación del agente etiológico responsable de la infección, actualmente las técnicas para la identificación de agentes patógenos específicos a la gastroenteritis son diversos, como la amplificación de DNA y la inmunoelectroforesis[2]. Actualmente se están desarrollando técnicas de espectroscopias ópticas para el análisis de fluidos biológicos, para la detección de glucosa en sangre, para la caracterización de proteínas y anticuerpos en soluciones acuosas [3] y para la identificación de bacterias causantes de gastroenteritis [4], en el área de odontología se ha utilizado para el análisis de tejido óseo y componentes de calcio incluyendo hidroxiapatita [5]. OBJETIVOS. Determinar la factibilidad de la espectroscopia Raman para la detección de diferentes cepas de la bacteria E. Coli. Identificar grupos funcionales que conforman la estructura química de la bacteria por medio del análisis espectral, y utilizar técnicas especializadas de procesamiento de señal y análisis multivariante para discriminar entre diferentes cepas de la bacteria E.Coli. METODOLOGÍA. El equipo utilizado para las mediciones es un espectrómetro Raman el cual consiste de una fuente de Luz Láser de 300mW, que es la que va a exitar a la muestra, la Luz dispersada por la muestra es colectada por el espectrómetro, donde las frecuencias Raman son colectadas en una Cámara CCD (de 256x1024 pixeles) la cual convierte las señales luminosas a eléctricas, para ser procesadas y observadas finalmente en una computadora. Se tomaron espectros de la bacteria E.Coli de tres cepas diferentes procedentes de pacientes del Hospital Central de SLP, utilizando la escala de MackFarland en 5, se fue cambiando la concentración cada 100uL/mL,hasta 1000uL/mL en una solución salina al 0.85%, tomando tres espectros por concentración, los espectros fueron procesados y se les realizó un análisis de PCA. RESULTADOS Y DISCUSIÓN. Por medio de un análisis espectral las frecuencias Raman encontradas corresponden a asignaciones interesantes como la región (1420-1620cm-1) correspondiente a anillos aromáticos, y en (1150-1900cm-1) la presencia de Carbonos y Oxígenos, así como en 1375cm-1 la presencia de CH2. Después del procesamiento de las señales espectrales, los resultados de análisis de PCA muestran una clara diferencia entre las diferentes cepas de la bacteria E. Coli, los cuales se corroboran con los resultados de las pruebas químicas para las diferentes cepas. CONCLUSIONES. La espectroscopia Raman y el Análisis Multivariante son herramientas útiles para el análisis de muestras biológicas, de tal forma que hemos podido identificar cambios entre las diferentes cepas de la bacteria E. coli, las frecuencias de los espectros Raman encontrados, corresponden a grupos funcionales de anillos aromáticos, C=O, y CH2, dado que las técnicas espectroscópicas son métodos rápidos, fáciles de utilizar, no destructivos, y directos sobre la muestra podemos decir que son técnicas confiables para la identificación de diferentes cepas de E. coli.



8

Implementation of Molecular Methods for Detection and Typing of Salmonella enterica Serotypes

M. en C. Alberto Balderas Martínez1 1Laboratorio Cordobés de Diagnóstico Pecuario, S.C. Av. Las Quintas s/n, Frac. Las Quintas. Córdoba, Veracruz 94543 Tel.: 01 (271) 716 4990. E-mail: [email protected] The genus Salmonella comprises more than 2000 serotypes, many of which can cause severe illness in both humans and animals. The classical diagnostic of Salmonella is based mainly in the evaluation of phenotype properties through many probes (morphology, microscopy, biochemical and serological assays) with results than can be achieved in many days. With the development of molecular biology tools, the identification of bacteria at level of serotype can be performance in less time, from a great variety of samples. Additionally, the selection of virulence factor genes allows differentiates virulent strain from no-virulent. We applied and validated molecular methods such as PCR, Real-Time PCR and RFLP analysis for the detection and typing of Salmonella isolates from many sources and compared efficient advantages and additional results of the implementation of the molecular techniques in the diagnostics of major classes of Salmonella.



10

Analysis of the interaction of GrlA with their target genes in attaching

and effacing Escherichia coli

Bello Díaz Jaime Enrique y Puente García José Luis Instituto de Biotecnología / Universidad Nacional Autónoma de México. Av. Universidad #2001, Col. Chamilpa C.P. 62210 Cuernavaca, Morelos Apdo. Postal 510-3, C.P.62250 045(222)6188422 [email protected] Enteropathogenic E. coli (EPEC) infects mainly children younger than 6 months in developing countries, while enterohemorrhagic E. coli (EHEC) causes hemorrhagic colitis, which can progress to the often deadly hemolytic uremic syndrome, even in industrialized countries. EPEC and EHEC, together with the mouse pathogen Citrobacter rodentium (CR), belong to a group of pathogens that share the ability to form attaching and effacing (A/E) lesions on intestinal epithelia. The A/E lesion is characterized by the localized destruction of apical microvilli of enterocytes and important cytoskeleton rearrangements beneath the adherent bacteria, leading to the formation of actin-rich cup-like structures and intimate bacterium-host cell interactions. The genes required for the formation of the A/E lesion in EPEC, EHEC, and CR are located within a pathogenicity island known as the locus of enterocyte effacement (LEE). Two LEE-encoded regulators, Ler and GrlA, positively regulate the expression of the LEE operons. Ler overcomes H-NS mediated repression, while GrlA plays a dual role as a direct activator of ler expression binding near its promoter sequence and as a GrlR antagonist through protein-protein interactions. GrlR and GrlA have been also involved in regulating the expression of genes outside the LEE. GrlA negatively regulates the expression of flagellar genes in EHEC by reducing transcription of the flhD operon, but it can induce ehx gene expression independently of Ler. Do to its key importance as a virulence regulatory protein of genes within and outside the LEE in A/E pathogens, in this work we are characterizing the GrlA DNA binding sequence in known GrlA targets, seeking to determine whether it recognizes a common sequence motif that may later help us to identify novel genes regulated by GrlA.



12

Molecular characterization of Staphylococcus strains isolated from intranosocomial infected patients and healthy carriers

in a Mexican community Bustos-Martínez J2, Ramírez-Benítez G2, Sainz-Espuñes T1, Hamdan-Partida A1 1Depto. Sistemas Biológicos and 2Depto. Atención a la Salud, Universidad Autónoma Metropolitana-Xochimilco. Mexico City, Mexico. Calzada del Hueso 1100, Col. Villa Quietud. CP 04960. México D.F: [email protected] The infections caused by Staphylococcus spp. are very important to human health, and are one of the main causes of nosocomial infections (NI). An important part of the pathogenicity of these bacteria is its resistence to methicillin, since infections caused by methicillin resistant S. aureus (MRSA) or methicillin resistant S. epidermidis (MRSE) are hard to treat (1). Objectives: In the present work, we characterize Staphylococcus strains isolated from apparently healthy individuals and from patients with NI in a mexican community, also studied the presence of the mecA gene and also compared the genomic profile of the strains. Methods: We determined the oxacillin MIC and the presence of the mecA gene using PCR. The comparison of the genomic profile was carried out with pulse field gel electrophoresis (PFGE). Conclusions: We found a high percentage of Staphylococcus carriers (63%). The most abundant species were S. epidermidis (40%), followed by S. aureus(13%), we also found S. hominis, S. xylosus, S. haemolyticcus, S. saprophyticus and S. capitis strains. Twenty tree percent of the strains were methicillin resistant. Only 4% were MRSA strains and the rest 19% were MRSE. The presence of the mecA gene was found in all MRSA and MRSE strains. Different genomic profiles were found between the healthy volunteer strains and the nosocomial strains. The high percentage of MRSE strains in healthy individuals can be a determinant factor for the horizontal gene transference of the mecA gene towards the sensitive S. aureus strains. References

1. Martins A, Cunha MLRS. 2007. Methicillin resistance in Staphylococcus aures and coagulase-negative Staphylococci: Epidemiologic and molecular aspects. Microbiol Immunol. 51:787-795.



14

Analysis of the use of the Bacillus thuringiensis non-dependent sporulation promoter for for the expression of CryMod toxins

Blanca I. García-Gómez, Helena Porta Ducoing, Alejandra Bravo y Mario Soberón Depto. de Microbiología Molecular, Instituto de Biotecnología de la UNAM. Apdo. Postal 510-3, Cuernavaca 62250, Morelos (México) Fax: 527773291624, e-mail: [email protected] Bacillus thuringiensis (Bt) is a gram-positive bacteria, ubiquitous, is characterized by producing parasporal crystals composed of Cry and Cyt proteins (δ-endotoxins) during sporulation phase. To counter the resistance generated by certain target insects ( Soberón et al., 2011 ), we have designed modified Cry toxins lacking helix α-1 that does not require interaction with the cadherin receptor for oligomer formation. The modified toxins killed cadherin-silenced M.sexta and Bt-resistan Pectinophora gossypiella (Soberón et al., 2007) The first crystal protein gene to be cloned was Cry1Aa. It is expressed during sporulation of B.thuringiensis from overlapping promoters P1and P2 (Wong et al., 1983), this promoter had been used for the expression of several recombinant Cry toxins. Though, because this promoter is recognized by the transcription machinery of E.coli, in some cases turned out to be toxic to E.coli with the outcome of selection of non-toxic mutant proteins. The promoter region of the Cry3A toxin gene of B. thuringiensis is composed of at least three domains and unlike the promoters of other cry genes, this promoter is similar to -dependent promoters rather than sporulation-specific promoters (Baum et al., 1995). In this work we evaluated the use of the three regions of Cry3 promoter for the correct expression of recombinant Cry1AbMod, Cry2AbMod and Cry11AaMod proteins showing that the CryMod toxins retain insecticidal activity. References

1. Baum J.A. and Malvar, T. (1995). Mol Microbiol 18(1), 1-12. 2. Bravo, A., S. Likitvivatanavong, S., Gill, S.S., and Soberón, M. (2011). Insect

Biochem and Molecular Biology (in Press). 3. Soberon, M., Pardo-López, L., López, I., Gómez, I., Tabaschnik, B.E., and Bravo, A.

(2007). Science 318: 1640-1642. 4. Wong, H.C., Schnepf, H.E., and Whiteley, H.R. (1983). J.Biol Chem 258:1960-1967.



16

Adenine-rich codons, irrespective of codon usage, enhance in vitro protein synthesis by promoting mRNA binding to

ribosomal 30S subunit

Castillo Méndez Manuel, Jacinto Loeza Eva, Guarneros Peña Gabriel and Hernández Sánchez Javier Departamento de Genética y Biología Molecular. Centro de Investigación y de Estudios Avanzados del IPN. Av. IPN 2508 Col. San Pedro Zacatenco. C.P. 07360 México, D.F. Tel. 52 55 57 47 38 00. Ext. 5352. [email protected] Adenines downstream the initiation codon promote protein synthesis, however, some adenine-containing codons (AGA and AUA) at early positions inhibit protein synthesis when cognate tRNA is exhausted. It has also been related the presence of adenines with mRNA binding to the ribosome. To understand these apparent inconsistencies we analyzed the effect of these codons on mRNA-ribosome binding strength, mRNA stability, the production of peptidyl-tRNA (pep-tRNA) and protein synthesis. Constructs harboring lacZ derivatives were obtained by site directed mutagenesis where tandems of GGG, AGG, AGA, ATA and AAA codons were inserted at codon positions 2-3 and 3-4. Codons containing more adenines, irrespective of being common or rare, (AAA, ATA and AGA) promoted a higher in vitro synthesis of β-galactosidase (β-gal) in comparison with those rich in guanines (GGG and AGG). β-gal over production diverted cell metabolism and caused a stronger inhibition of in vivo protein synthesis and growth of pep-tRNA hydrolase (pth) defective cells. The higher translation of β-gal correlated with the protection of full length mRNAs from the lacZ variants rich in adenines. With the exception of ATA, only plasmid constructs containing hungry codons generated pep-tRNA (AGA and to a lesser extent AGG) in Pth defective cells and no pep-tRNA was detected in wild type cells. Codons containing more adenines clearly promoted lacZ mRNA binding to 30S subunit. Altogether, these results indicate that mRNA binding to ribosome plays a major role in the enhancement of translation by adenine-rich codons irrespective of codon usage.



18

Contribution of the ribonucleotide reductase (NrdEF) in the generation of stationary-phase-associated mutations in Bacillus subtilis

Karla V. Castro-Cerritos and Mario Pedraza-Reyes Department of Biology, University of Guanajuato, P.O. Box 187, Guanajuato, Gto. 36050, Mexico Adaptive or stationary-phase mutagenesis can be defined as those mutations that permit organisms to grow and divide in response to natural or artificial selection and that occur in non-dividing cells during prolonged nonlethal selective pressure (1). The model system B. subtilis YB955 that measures reversions of the hisC952 metB5 and leuC427 alleles has permitted to demonstrate that this process occurs in this microorganism (2). The oxidized guanine (GO) system has been shown to be implicated in this type of mutagenesis (3, 4). In fact, a report revealed that B. subtilis cells deficient on GO system exhibited a strong propensity to increase the number of adaptive mutants (4). Results of a proteomic analysis performed in cell extracts of these mutant colonies showed the induction of NrdEF, the protein subunits that compose the enzyme ribonucleotide reductase (RNR). RNR catalyzes the reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs). RNR activity is essential during replication and several studies have demonstrated that the expression of its encoding genes is induced after exposure to agents that promote DNA damage (6). In addition, it has been reported that the survival and mutation rates of S. cerevisiae cells exposed to DNA damage is directly related to the dNTPs levels in the cells (7). To investigate the role played by RNR in generating mutations under conditions of limited growth a conditional nrdEF mutant was constructed. Results with this strain revealed that the absence of NrdEF, in the stationary phase reversion assay system, dramatically decreased the production of His+, Met+ and Leu+ revertants in the model system B. subtilis YB955. These results support the idea that RNR is required to promote mutations under conditions that restrict growth. References

1. Bridges. 1998. Mut. Res. 408:1–9. 2. Sung and Yasbin. 2002. J. Bac. 184:5641–5653. 3. Steenken, Jovanovic. 1997. J. Am. Chem. Soc. 119, 617–618. 4. Michaels and Miller. 1992. J. Bac. 174:6321–6325. 5. Vidales, Cárdenas, Robleto, Yasbin, and Pedraza-Reyes. 2009. J. Bac. 191: 506–

513 6. Gon. and Beckwith. 2006. A. Red. Sig. 8: 773-780. 7. Chabes and Thelander. 2003. Cell. 112, 391–401..



20

Characterization of the possible phosphate-specific porin of Caulobacter crescentus

Lilia Colina, Laura Camarena, Sebastián Poggio Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México [email protected] Instituto de Investigaciones Biomédicas 3er Circuito Exterior S/N Ciudad Universitaria México DF 04510 Caulobacter crescentus is a gram-negative oligotrophic bacterium that has a dimorphic cell cycle. Each asymmetric cell division yields a flagellated cell (swarmer cell) and a sessile cell with a polar structure known as stalk or prostheca (stalked cell). The stalk is a thin tubular extension of the cellular envelope with no apparent cytoplasm. The function of the stalk is still unclear, but it has been suggested that it may play a role in nutrient and in particular phosphate uptake. When grown in phosphate-limiting conditions, C. crescentus cells elongate their stalks up to many times the cell length. It has been proposed that this morphologic adaptation allows a more efficient uptake of diluted nutrients. In gram-negative bacteria, the outer membrane is the first barrier for solutes. Porins are outer membrane proteins that assemble into channels that allow the diffusion of small charged molecules into the cell periplasm, and are thus responsible for the permeability properties of the outer membrane. Porins can be nonspecific or highly selective; two phosphate-specific porins have been described: OprP (in Pseudomonas aeruginosa) and PhoE (in Escherichia coli). They are both anion-specific and their expression is induced by the absence of phosphate. An analysis of the C. crescentus genome revealed that this bacterium has a phosphate porin from the OprP superfamily. Given the evidence that indicates a possible role of the stalk in phosphate uptake, the purpose of this work is to characterize the localization and the function of OprP in C. crescentus. Our preliminary results show that this protein does not localize to the stalk and instead is distributed in the outer membrane of the cell body. These results indicate that phosphate uptake through OprP most likely takes place in the cell body and not in the stalk.



22

Implementation of a loss-of-function system to study stationary-phase-associated mutagenesis in Bacillus subtilis

1Norberto Villegas-Negrete, 2Eduardo A. Robleto and 1Mario Pedraza-Reyes 1Department of Biology, Division of Natural and Exact Sciences, Building L, University of Guanajuato, Noria Alta S/N, Guanajuato 36050, México. 2School of life sciences, University of Nevada-Las Vegas, 4505 Maryland Parkway, Box 454004, Las Vegas, Nevada 89154-4004. E-mail: [email protected].

The strain Bacillus subtilis YB955 has been essential in establishing the mechanisms involved in the generation of mutations that occur in non-dividing cells that are subject to nutritional stress, a cellular process that has been termed adaptive mutagenesis [1-3]. Generation of adaptive mutants in this strain occurs through a gain-of-function event that measures reversions of the chromosomal auxotrophies hisC952, metB5 and leuC427. The first two alleles contain non-sense mutations and the third one a missense mutation; therefore, the use of this system may have limitations to analyze the full spectrum of genetic alterations that produce adaptive mutants. However, using this system, it was recently proposed that increases in the transcription rate of the leuC425 allele positively affect the number of stationary-phase–associated Leu+ revertants but not of those associated with growth [4].

To have a wider picture of the mutations involved in this process we are interested in implementing a loss-of-function mutagenesis system in B. subtilis based on the selection of trimethoprim-resistant colonies (TmpR).

To this end, we generated genetic constructs to disrupt the thyA and thyB genes which encode thymidilate synthases A and B in B. subtilis, respectively. After its molecular characterization, the TmpR phenotype and thymine requirement of this strain was corroborated. To make the strain sensitive to Tmp, the thyA gene was cloned downstream of the IPTG-inducible strong Phs promoter in the integrative vector Phyperspank and the Phs-thyA cassette was recombined into the amyE locus of the ThyA, ThyB-deficient strain. This strain will allows us to study the type of mutations that are involved in generating adaptive mutants following the appearance of colonies with a TmpR phenotype under repressed and non-repressed conditions. References 1. Sung, H.-M., and R. E. Yasbin. 2002. J. Bacteriol. 184:5641–5653. 2. Pedraza-Reyes, M and Yasbin, R.E. 2004. J. Bacteriol. 186:6485–6491. 3. Robleto et al. 2007. Crit. Rev. Biochem. Mol. Biol. 42:327–339. 4. Pybus et al. 2010. J. Bacteriol. 192:3321-3328. Work supported by CONACyT (grant 84482). Norberto Villegas is supported by a scholarship from CONACyT



24 Spatial distribution of the bacterial community

in Cotija cheese analyzed by FISH

Q.A. Alejandra Escobar Zepeda, Dra. Maricarmen Quirasco Baruch Depto. de Alimentos y Biotecnología, Facultad de Química, UNAM 04510 D.F. [email protected]

Introduction Cotija cheese is produced in an area between Jalisco and Michoacán. Due to the fact that it is manufactured from raw milk it is ripened by native microorganisms. This work describes the microbial distribution that may result from a differential physicochemical environment inside the entire cheese. The FISH technique was used in order to define the spatial distribution of some bacterial species, since it is a culture-independent method. Methods The three-month ripened cheese analyzed was cylindrical: 26 x 16.5 cm, and it was divided in 5 zones. Physicochemical analysis included: pH, acidity, Cl-, Ca2+, water activity (aW), redox potential (Eh), diacetyl, fatty acid composition and PAGE protein profiles. 16S ARNr was the target for the FISH probes. Sequences were compared with public data bases (NCBI and RDP). The enzymatic permeabilization of bacteria and the specificity of the probes were tested with pure cultures, before experimenting with the cells extracted from the different cheese zones. Results The presence of chemical gradients inside the Cotija cheese, were observed. There was a higher concentration of salt, calcium and higher pH values in the surface than in the central zone. While acidity and aW, had the opposite behavior. The Eh value was more negative in the center than in the surface, in any case it showed an environment with low oxygen content. As for enzymatic activity, proteolysis occurs homogeneously inside the cheese matrix; while there was a higher activity in the rind on caseins of ~24 kDa. Lipolysis was observed in all zones, showing preference for the hydrolysis of C13:0 and C18:2 fatty acids. In accordance to the enzymatic activity results, a uniform distribution of the bacteria responsible for these activities was found: genus Bacillus spp. and Staphylococcus spp. Also, making sense with previous reports of the dominance of Enterococcus spp., such genus was found in a homogeneous way in the matrix. Conclusions The physicochemical gradients observed may be caused more to the gradual moisture loss from the cheese surface, than to bacterial metabolic activities. In spite of the big size of the cheese, gradients did not show enough different values to select a special kind of bacterial population, which is reflected in similar enzymatic activities inside the product. The usefulness of a culture-independent method for the analysis of a bacterial microbiota from a complex matrix was proved.

Acknowledgments PAPIIT IN230511. Dr. Jesús García V. and Dra. Hilda Calderón V., Fac. Química-UNAM



26

Characterization of outer membrane vesicles from different

Pasteurella multocida serogroups

Fernández Rojas Miguel Ángel1*, Vaca Sergio1, Reyes Magda2, Aguilar Francisco3 & Negrete Abascal Erasmo1

Facultad de Estudios Superiores Iztacala, UNAM. Av. De los Barrios, # 1, Colonia Reyes Iztacala, Tlalnepantla, Edo de México1. Departamento de Biología Celular, CINVESTAV-IPN2. INIFAP-Microbiología, Cuajimalpa México, DF3. *[email protected] Tel. (044)5527401555, 57914309.

Pasteurella multocida (Pm), the causal agent of infectious diseases mainly in animals, releases outer membrane vesicles (OMV) containing different virulence factors and immunogenic proteins. Negatively stained OMV in the range of 50 to 250 nm were observed by transmission electron microscopy. Proteins between 25 to 180 kDa sizes were visualized by Coomassie stained 10% polyacrilamide gels. Among those proteins, a 35 kDa immunogenic heat-modified protein was observed in OMV of the 5 different Pm strains evaluated in this work. The 35 kDa protein was identified as OmpA by mass spectrophotometric analysis and was observed enriched in OMV from Pm 43017 (serogroup B) and Pm 12948 (serogroup D). Proteins, in the range of 20 to 80 kDa, were immune recognized by different hyperimmune sera from animals infected with Actinobacillus pleuropneumoniae, Avibacterium paragallinarum or Gallibacterium anatis, suggesting the presence of common proteins among these different microorganisms. The presence of proteolytic activity bands was visualized in zimograms of Pm 43020 (serogroup E) and in a rabbit field isolate of serogroup A, using porcine gelatin or casein as substrates. Bands of approximately 35 kDa and higher molecular masses were identified in all the Pm tested when a rabbit anti-protease from A. pleuropneumoniae was used in a western blot. Key words: Pasteurella multocida, Outer membrana vesicles, proteases

Proyecto apoyado por PAPIIT IN216010 y PAPCA FESI-UNAM



28

Can a Trojan horse be tamed? Resistance to gallium nitrate in Pseudomonas aeruginosa

Rodolfo García-Contreras1, Abril Ruiz1, Elizabeth Lira-Silva1, Ricardo Jasso-Chávez1, Toshinari Maeda2, Fred Boogerd3 and Thomas K. Wood4,5 1Department of Biochemistry, National Institute of Cardiology, México DF 14080, MEXICO 2Department of Biological Functions and Engineering, Kyushu Institute of Technology,

Kitakyushu 808-0196, JAPAN 3Department of Molecular Cell Physiology, VU University, Amsterdam, THE

NETHERLANDS 4Department of Chemical Engineering and 5Department of Biology, Texas A&M University, College Station, Texas 77874-3122, USA

[email protected], phone: 55732911 ext. 1517

Background: Ga exerts bacteriostatical and bactericidal effects against the opportunistic pathogen Pseudomonas aeruginosa. In addition, Ga inhibits biofilm formation and promotes healing of infections. As Ga(NO3)3 is currently used to treat hypercalcemia in humans, it is likely that in the near future Ga compounds could be used to treat P. aeruginosa. Cells cannot distinguish between Fe3+ and Ga3+, and hence Ga3+ is internalized, and then it disrupts the iron metabolism and homeostasis thus acting as a “Trojan horse”. Knowing that P. aeruginosa has a notorious ability to develop resistance against antimicrobials, it is interesting that high resistance against Ga is absent in several clinical P. aeruginosa isolates. Hence, we analyzed if the generation of resistance against Ga in P. aeruginosa was possible. Methods: Ga-resistant mutants generated by random transposition as well as spontaneous resistant mutants were selected in minimal succinate medium, in the presence of toxic Ga(NO3)3 concentrations (≥100 M). Growth rates and yields, and the MIC 50% of the resistant mutants were determined. In addition, functional phenotypic properties inhibited by Ga as well as phenotypes that could lead to resistance are being studied. Results: Both spontaneous and transposon mutants were isolated after a few rounds of selection. The transposon mutants were able to grow 2 to 3-fold faster, and had yields 6-10 fold higher, than the wt strain in 50 M Ga(NO3)3. The MIC 50% against Ga(NO3)3 of the transposon mutants was 5-fold higher than the wt strain, while the MIC 50% of the spontaneous mutants was 13-fold higher. Regarding the resistance mechanisms the basal production of pyoverdine and of the redox active compound pyocyanin are higher in the spontaneous mutant than in the wt strain and pyocyanin significantly increase in the presence of gallium.

Conclusion: High levels of resistance to Ga(NO3)3 can readily be achieved. Therefore, is advisable to elucidate the resistant mechanims before its utilization in the clinic. Our results also suggest a posibble role of pyocyanin in the resistance.



30

Community Analysis of Methanogens from Natural and Enrichment

Samples of Hypersaline Environements Jose Quinatzin Garcia-Maldonado1, Alejandro Lopez-Cortes1, Brad Bebout2, Adrienne Freesbe2

1 Centro de Investigaciones Biológicas del Noroeste, La Paz, BCS, México, 23090 2Exobiology Branch, NASA Ames Research Center, Moffett Field, CA 94035 Contact: [email protected] Mar Bermejo 195, Col. Playa Palo de Santa Rita. (612) 123 8484 Methane production from hypersaline ecosystems has been well documented; nevertheless there are only few reports about the community composition of methanogens from these environments. Only sequences of uncultured Euryarchaoeta and members of the Family Methanosarcinaceae have been previously reported for thalassohaline hypersaline environments. In this study, we analyzed the methanogenic community by denaturing gradient gel electrophoresis, clone libraries and sequencing of methyl-CoM-reductase A gene (mcrA) and 16S rRNA from non-manipulated and enrichment samples of non-previously studied hypersaline environments, to understand the changes occurring in the community composition after stimulation with non-competitive substrates. Incubation experiments showed that the rates of methane production were higher for samples stimulated with TMA than acetate. Enrichment samples presented an increase in the number and intensity of the mcrA/16S rRNA-DGGE bands, in comparison with natural samples from the same site. Distinctive 16S rRNA-DGGE bands were detected in enrichment samples and were closely related with the genera Methanococcoides, which shows the modiffication of the archaeal community that ocurr when TMA or acetate are used as substrates. Different band patterns were observed when TMA and acetate were used as substrate. Sequences retrieved by mcrA-DGGE showed high similarity with Methanohalobium sp., which had not been previously reported for similar hypersaline sites in Mexico. Contributing with the knowledgment of methanogens from hypersaline environments, this is the first study of methanogens comparing different geographical sites with similar natural conditions.



32

Caulobacter crescentus: a sphingolipid synthesis pathway

Daniela A. García-Soriano, Diana X. Sahonero-Canavesi, Isabel M. López-Lara, and Otto Geiger Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico. Av. Universidad #708 Edif. C Depto. 305, Col. Chamilpa, CP 62210; Cuernavaca, Morelos. 3174091 [email protected]

Caulobacter crescentus is a Gram-negative fresh water bacterium that shows an unequal binary fission during its cell cycle and therefore serves as a model organism for the study of cell division and bacterial development. Major membrane lipids of Caulobacter are phosphatidylglycerol, cardiolipin, lysyl-phosphatidylglycerol, and glycolipids. Few bacteria are known to have sphingolipids in their membranes and many of them have “Sphingo” as prefix in their genus name. Surprisingly, the genome of C. crescentus seems to contain a cluster of 8 genes involved in sphingolipid biosynthesis (Geiger et al. 2010. Prog Lipid Res 49:46-60). Specifically, a gene presumably encoding for a serine palmitoyl transferase (SPT, CC_1162) is preceeded by a gene potentially encoding an acyl carrier protein (ACP, CC_1163). SPT catalyzes the first step in sphingolipid biosynthesis, the condensation of L-serine to palmitoyl-CoA forming the sphingolipid precursor 3-oxo-sphinganine. Also, the cluster harbors a gene predicted to encode an acyl-CoA synthetase (ACS, CC_1165). CC_1163 overexpressed in Escherichia coli was labeled in vivo with [3H]β-alanine, a biosynthetic building block of the 4’-phosphopantetheine prosthetic group of ACPs. Overexpression of combinations of SPT, ACP, and ACS in E. coli causes the formation of new lipid products, but only caulobacterial SPT and ACS are strictly required for their formation in the host. Initial analysis of C. crescentus lipid extracts suggests the presence of sphingolipids

.



34

Induction of lateral flagella of Vibrio shilonii in liquid medium*

Jareth Vázquez1, Javier de la Mora1, Laura Camarena2 and Georges Dreyfus1 1Instituto de Fisiología Celular. 2Institúto de investigaciones Biomédicas. Universidad Nacional Autónoma de México 04510 Mexico City, Mexico. [email protected] Tel: 56 22 56 18

The coral pathogen Vibrio shilonii is a marine Gram-negative bacterium. Its motility depends on two flagellar systems: a sodium-driven polar flagellum, which is sheathed and is used in liquid media; and proton-driven lateral flagella, that are expressed in semisolid surfaces. The surface mediated induction of lateral flagella is well known, but is still not clear if the mechanical stress in the polar flagellum is the signal to start expression of lateral flagella. We hypothesized that if we block the sodium channels of polar flagellum with amiloride (a sodium channel inhibitor) in liquid medium, then the polar flagellum will not rotate and bacteria would express lateral flagella. To detect the presence of lateral flagella we used light microscopy (staining flagella with fuchsin), and transmition electron microscopy (TEM).

Some filaments attached to the cell body were observed by optical microscopy, these structures appeared about 4 minutes after treatment, and were seen until the last observation 30 minutes after the amiloride addition. In the TEM the same structures were observed.

In addition we are immunizing rabbits with recombinant lateral flagellin (lafA), the principal component of lateral flagella filament, with the aim to obtain specific antibodies. We expect that using immunoblotting with specific anti-lafA antibodies we will be able to detect the expression of lateral flagella in liquid media in cells treated with amiloride.

*This work is supported by CONACyT: 106081 and DGAPA: IN213408.



36

Microevolutionary analysis of Bacillus coahuilensis through comparative genomics

Gómez Lunar Zulema, Hernández González Ismael, Olmedo Álvarez Gabriela Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Unidad Irapuato, Departamento de Ingeniería Genética, Laboratorio de Biología Molecular de Bacterias II. Km. 9.6 Libramiento Norte Carr. Irapuato-León 36821, Irapuato, Gto, México. Tel. 52 (462) 623 96 00 Ext. 462. [email protected]. Bacterial genomes have a set of essential “core” genes, encoding functions relating to central metabolism and informational processing, but they also have “accessory” genes that commonly encode supplementary metabolic pathways that may have important roles in the adaptation to specific environments, and have had different evolutionary paths. Bacterial genomes suffer numerous events of gene losses and gains that increase genetic variability among strains of the same species (microevolution). Genetic diversity is generated by different mechanisms including the activity of mobile genetic elements (MGE). These MGEs are DNA segments that encode enzymes and other proteins, which mediate the movement of DNA within genomes (intracellular mobility) or between bacterial cells (intercellular mobility). They could increase genetic variability due the genomic rearrangements and adaptive mutations that generate. MGE include transposable elements, plasmids, bacteriophages, integrons and genomic islands. These genetic elements could drive the interspecific genome diversity and eventually the emergency of adaptive traits. The aim of this study is to understand the contribution of MGEs in free-living bacteria to genome dynamics and evolution as well as to the acquisition of potential adaptive traits. To this end we carried out a comparative phenotypic and whole genome analysis of three different Bacillus coahuilensis strains (m4-4, m2-6 and p1.1.43) isolated from a water system in Cuatro Cienegas, Coahuila, Mexico. Our results showed that these strains exhibited differences in auxotrophies and growth rates. The sequences of the three B. coahuilensis strains reveals a genome size between 3.32-3.45 Mpb. Strain m4-4 has a GC content of 37.5%, higher than that of m2-6 and p1.1.43 strains (33 and 32% GC respectively). Genome prediction suggests that the three have similar gene number. Comparison of their metabolic pathways revealed a reduction of the number of genes coding for enzymes in the urea cycle and also in specific amino acid biosynthesis pathways. We are carrying out the comparative analysis, including that of MGEs, to obtain information on their intraespecific genetic diversity. These results will be discussed.



38

Global Identification of small RNAs in Geobacter sulfurreducens

Getzabeth González*, Alfredo Mendoza, Enrique Morett y Katy Juárez Departamento de Ingeniería Celular y Biocatálisis Instituto de Biotecnología. UNAM. Av. Universidad 2001, Cuernavaca, Morelos, 62210, México. Tel. +52 (777) 329-1605 e-mail: *[email protected]

G. sulfurreducens is an anaerobic bacteria capable of producing electricity and remove toxic metals such as uranium from contaminated sites. Although significant progress has been made in the understanding of the electron transfer process in Geobacter sulfurreducens, little is known about the regulatory mechanisms involved in its control. Recently, work with this and other bacteria suggests that the regulation of gene expression by sRNA is widespread. In this sense, we are interested in the identification and analysis of small non-coding RNAs (sRNAs). sRNAs are molecules ranking in size from 50-500 nucleotides that act by pairing to complementary mRNA in an anti-sense orientation (cis-encoded) or by partial complementarity (trans-encoded). Several sRNAs have been predicted to be coded in the G. sulfurreducens genome. In order to obtain a global picture of the sRNAs involved in regulatory processes, we obtained RNA from two different growth conditions. Each RNA sample was size-selected (50-100, 100-300, and 300-500 nucleotides) in a 15% denaturing polyacrylamide gel. Recovered RNA was ligated to a mixture of adapters (multiplexing) to tag the two growth conditions and RNA lengths. The RNA was converted to cDNA for high throughput sequencing using the Illumina GAIIx plataform. Results will be discussed.



40

The catecholamine norepinephrine binds to Streptococcus pneumoniae diminishing adhesion to lung epithelial cells yet promoting systemic

spread Xavier F. Gonzales, Gonzalo Castillo-Rojas, Guillermo Mendoza-Hernández, Elaine Tuomanen and Yolanda López-Vidal Programa de Inmunología Molecular Microbiana, Departamento de Microbiología Y Parasitología, Facultad de Medicina, Universidad Nacional Autonónma de Mexico, Department of Infectious Diseases, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105 Streptococcus pneumoniae (pneumococcus) is one of the primary causes of bacterial pneumonia, partially mediated by the bacteria’s ability to enter and colonize its host. Pneumococcal surface proteins that facilitate colonization have been targets for combating the bacteria. The catecholamine norepinephrine through two component regulation and iron transfer has been described to effect the expression of bacterial virulence genes that aid in colonization. To characterize the role of norepinephrine in S. pneumoniae in vitro and in vivo, we performed binding assays using an antibody for norepinephrine to determine if there was a specific interaction between norepinephrine and S. pneumoniae. Adhesion assays using an epithelial cell line was used to determine if norepinephrine facilitates S. pneumoniae adherence. Further, using transcription analysis, we inquired if norepinephrine regulated pneumococcal virulence genes. Lastly, mice were challenged with norepinephrine treated S. pneumoniae to observe colonization and disease progression. Norepinephrine inhibits pneumococcal adhesion through indirect association to the regulatory system RR/HK06 and the adhesion protein choline binding protein A. Messenger RNA analysis of norepinephrine treated S. pneumoniae revealed the down-regulation of several genes associated to Piu iron uptake ABC transporter. Mice challenged with norepinephrine treated S. pneumoniae displayed a tendency of increased bacterial titers in the lungs and enhanced systemic spread in blood. Our results suggest that inhibitory effects of norepinephrine on pneumococcus and the secondary association of iron transport has the potential to enhance disease under septic conditions.



42

Identification of autotransporter protein genes in Proteus mirabilis strains isolated from clinical cases

Gutiérrez-Lucas Luis Raúl*1; Rosales-Sánchez José Carlos1; Bustos-Martínez Jaime Amadeo1; Hamdan-Partida Aida1; Sainz-Espuñes Teresita del Rosario1. 1Universidad Autónoma Metropolitana Unidad Xochimilco, México D.F. Laboratorio de Microbiología Molecular. UAM-Xochimilco. Calzada del Hueso #1100. Col. Villa Quietud. Delegación Coyoacán. CP 04960. México, D.F. Tel. 54837000 Ext. 3624 Luis Raúl Gutiérrez Lucas. [email protected] Tel. Particular. 42122957. Introduction. Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease- mediated urea hydrolysis. Some SPATEs proteins were recently reported. Objective. Determine the presence of autotransporter protein genes in Proteus mirabilis strains isolated from clinical cases. Methodology. A total of 16 of P. mirabilis strains were isolated from patients from a hospital in Mexico City. Biochemical tests were performed to confirm the species of the strains. DNA extraction was performed following the protocol described in a commercial kit (Qiagen). PCR tests for genes identification was performed using specific primers for SPATEs. Results. Of the 16 strains of P. mirabilis isolated, 10 carried the pic gene, only one the pet gene and 5 carried both genes. Conclusions. Protein analyses are ongoing to determine the expression of these genes.



44

Elucidating Metabolic Pathways and Digging for Genes of Unknown Function in Microbial Communities:

The Riboswitch Approach

Ana Gutiérrez Preciado and Enrique Merino Department of Molecular Microbiology, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, México. Mailing address: Instituto de Biotecnología, UNAM Av. Universidad #2001, Col. Chamilpa, C.P. 62210, Cuernavaca, Morelos, MÉXICO, Telephone number: +52.777.329.1634, Fax number: +52.777.313.8673, Email addresses: [email protected], [email protected]

In the current post-genomic era, only 3% of all available gene sequences have been annotated based on experimental evidence. Even though functions can be predicted for many protein sequences, 25% of these are likely to be wrong. The most widely used methods for gene function prediction rely on sequence similarity, which might not be accurate in many cases. Other methods based on genomic characteristics, such as genomic context or phylogenetic profiles, have been developed to increase gene annotation accuracy; nevertheless these are only efficient when complete genome sequences have been determined. Here we propose a new approach based on locating riboswitches, which are highly conserved regulators of gene expression.



46 Detection of toxin genes in methicillin-resistant Staphylococcus aureus

(MRSA) strains isolated from the anterior nares and throats of healthy carriers in a Mexican community

Bustos-Martínez J2, Espino-García E1, Quiroz-Martínez O1, Sainz-Espuñes T1, Hamdan-Partida A1 1Depto. Sistemas Biológicos and 2Depto. Atención a la Salud, Universidad Autónoma Metropolitana-Xochimilco. Mexico City, Mexico. Calzada del Hueso 1100, Col. Villa Quietud. CP 04960. México D.F. [email protected] Background Staphylococcus aureus is mainly isolated from the anterior nares, nevertheless recent studies highlight the importance of the throat as a common habitat. Human carriers are the major infection source of S. aureus, especially with methicillin-resistant S. aureus strains (MRSA), which can cause nosocomial and community-acquired diseases, wich range from simple abscesses to fatal sepsis. S. aureus can also cause toxinosis such as food poisoning and toxic shock syndrome. For this complex set of diseases, S. aureus produces many pathogenic factors that interfere with the host defenses (1,2). Objectives The aim was to investigate the presence of several toxin genes in community isolated MRSA strains and determine if there were any differences in the toxin gene presence between nose isolated strains compared to those from the throat. Methods One hundred and forty MRSA strains (69 isolated from the nares and 71 from the throat) from a Mexican community were assayed by PCR technique for the presence of staphylococcal enterotoxins genes (SEs): sea, seb, sec, sed, see; exfoliatins (ETs): eta, etb; hemolysins (HLs): hla, hlb, hld, hlg and toxic shock syndrome toxin (TSST-1): tst. Conclusions Hemolysins and tst genes were the most prevalent among the strains. SEs genes were found in a very low rate. Toxin genes were prevalent in the throat isolates except for tst gene, found predominantly among the nares strains. Results showed that MRSA strains circulating in a Mexican community, present one or more toxin genes, the major prevalence of toxin gene presence was found among the throat isolates. References:

1. DeLeo FR, Chambers HF. 2009. Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era. J Clin Invest. 119: 2464-2474.

2. Diep BA, Carleton HA, Chang RF, Sensabaugh GF, Perdreau-Remington F. 2006. Roles of 34 Virulence Genes in the Evolution of Hospital- and Community-Associated Strains of Methicillin-Resistant Staphylococcus aureus. J Infect Dis. 193:1495-1503.



48

Transcription-associated-adaptive mutagenesis in Bacillus subtilis cells deficient in DNA repair

Martha Patricia Hernández-Gómez and Mario Pedraza-Reyes Departament of Biology, Division of Natural and Exact Sciences, University of Guanajuato, P.O.Box 187, Guanajuato, Gto. 36050. [email protected]

Adaptive or stationary phase-associated mutagenesiscan be defined as thosegenetic alterations that allow organisms to grow and divide in response to a natural or artificial mechanism of selection and that occurs in non-dividing cells subject to a non-lethal selective pressure(1). This cellular process has been shown to occur in B. subtilis YB955, a strain that measures reversions to the chromosomal auxotrophies, hisC952, metB5 and leuC427 (2).An important question in the adaptive mutagenesis study focuses in the processes that allow cells which do notdivide and that are under stress, acquire advantageous mutations before being killed by accumulation of deleterious mutations. Recent studies in B. subtilis suggested that, under stressful conditions, stochastic processes may act on highly trascribed genes that could be predisposed to the accumulation of mutations. The selection would favor mutations that increase cell survival, while the transcription associated to the generation of mutations could help cell population to avoid genetic lethal charge(3). On the other, it has been proposed that increased transcriptional rates may promote mutagenesis due to base damage during exposure of DNA strands. In the present work, different B. subtilis strains that overexpress the leuC425 allele in a genetic background deficient on the DNA repair systems that eliminate deaminated bases were constructed. These strains will be used in the adaptive mutagenesis reversion assay under repressed and non-repressed conditions to test the ideas above described.

References 1. Sung H.M. and Yasbin R.E. 2002. J. Bacteriol. 184:5641-5653. 2. Pedraza-Reyes, M and Yasbin, R. E. 2004. J.Bacteriol. 186:6485-6491. 3. Pybus et al. 2010. J. Bacteriol. 192:3321-3328.

Work supported by CONACYT (grant 84482) and University of Guanajuato.



50

Characterization of the site-specific recombinase IntA encoded in plasmid p42a of Rhizobium etli CFN42

Rogelio Hernández1, Christian Sohlenkamp2, Susana Brom1 and David Romero1

Programas de Ingeniería Genómica1 y Ecología Genómica2, Centro de Ciencias Genómicas, UNAM, Apdo. Postal 565-A, Cuernavaca, Morelos, México. tel: 777 317-58-67. [email protected]

Site-specific recombination occurs at short specific sequences mediated by the cognate recombinases. Nearly all site-specific recombinases can be classified either as tyrosine ( integrase family), or as serine recombinases (resolvase/invertase family), based on differences in their aminoacid sequence and mechanisms of catalysis. Rhizobium etli CFN42 is a soil bacterium able to induce nitrogen-fixing nodules on the roots of bean plants. This strain contains six plasmids (p42a to p42f), whose sizes range from 185 to 643 kb. Plasmid p42d is the symbiotic plasmid (pSym), because it carries most of the information required for nodulation and nitrogen fixation; p42a is self-transmissible at high frequency and is indispensable for conjugative transfer of the symbiotic plasmid. Sequence analysis revealed a 53-bp region that is 90% identical among the pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats, reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Our previous data showed that cointegration of the pSym and p42a, mediated by site-specific recombination between the attachment-like sites present on the pSym (attD) and p42a (attA), participates in conjugal transfer of p42d. To characterize this system, two plasmids, harboring only the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at a high frequency. Preliminary data indicate that IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a “bidirectional” integrase. Overproduction and purification of IntA was hindered by the insolubility of the native protein. To circumvent this problem, IntA was fused with MBP (maltose binding protein), in the vector pMAL-c2x. The resulting constructs were verified by sequencing, and their functionality ascertained by in vivo assays. The MBP-tagged IntA derivatives were purified by maltose affinity chromatography and used to setup electrophoretic mobility shift assays (EMSAs) with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD; fragments lacking these regions were not retarded by IntA. These results indicate specific binding of IntA to attA and attD.



52 Does immunizing cows with BCG reduce the presence of

Mycobacterium Bovis in colostrums

Herrera-Rodríguez Sara Elisa; Gordiano-Hidalgo María Alejandra; Higareda de Sales Luis Gonzalo; Estrada-Chávez Ciro Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco. Unidad de Biotecnología Médica y Farmacéutica. Tel +52 (33) 33455200, Ext. 1677, e-mail: [email protected] Mycobacterium bovis (M. bovis) is a slow-growing, aerobic bacterium member of the Mycobacterium tuberculosis complex, that is the primarily cause of tuberculosis in cattle known as bovine TB (bTB). M. bovis may also infect and cause illness in other animals, including humans. Bovine tuberculosis is a major economic problem around the world and represents a risk to public health. Bovine colostrum is the early milk produced by cows during the first several days after the calf's birth. This "early" milk has a nutrient profile, cellular and immunological composition substantially different from "mature" milk as it helps the newborn develop in its first week of life. Colostrum intake is critical for a newborn calf, since its immune system is not fully developed at birth. The calf must rely on colostrum from its mother until its own immune system is developed at 1 to 2 months of age. The presence of M. bovis in colostrums can be a risk for infection of newborn calves. There are some reports that show a high frequency of Mycobacterium bovis DNA in colostrums from tuberculous cattle that had been detected by nested PCR (5). However the question of whether newborn calves can be infected with bTB through colostrum has been poorly studied. One possible mechanism for infection could involve Peyer´s patches (PPs) which are also know as gut associated lymphoid tissues (GALT). These are a possible entry portal that could be used by infectious particles. The tuberculin skin test (TST) for bTB is an effective tool for identifying bovine TB in a herd. However this test can miss up to one third of the infected individuals. The purpose of this work is to determine the difference in the presence of M. bovis in the colostrum of cows vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG) and in the colostrums of non-vaccinated cows. While this experiment is in process, the following steps have been completed: All animals from 8 dairy herds were tested for the response against M. bovis using TST. Based on the results from the TST, cows were divided in two groups: 1.-reactors (cells can respond to PPD stimuli) or 2.- non-reactors. Then all the cattle were vaccinated with (BCG). Next colostrums were obtained, DNA was isolated, and the presence of mycobacterium in the colostrums was determined by PCR. We are analyzing these results to determine their concordance with the tuberculin skin test results. In the future, we will analyze samples of colostrums from non-vaccinated herds with 20 % prevalence, as well as herds with low prevalence. The results of this work will show whether there is a difference in M. bovis in the colostrums of vaccinated versus non-vaccinated cows. This will provide important guidance for dairy herd management that could reduce the prevalence of M. bovis.



54

¿Can Ler, the master regulator of Enterohemorrhagic Escherichia coli virulence genes, regulate E. coli core genome genes?

Labastida Martínez A., Puente García J.L. Departamento de Microbiología Molecular, Instituto de Biotecnología - UNAM, Av. Universidad 2001 Col. Chamilpa. Cuernavaca Morelos. CP. 62250 Tel. (777)3291627. Fax (777)3138673 [email protected], [email protected] Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes hemorrhagic colitis. Its virulence depends on the Locus of Enterocyte Effacement (LEE), a pathogenicity island encoding the components of a type three secretion system (TTSS), The global regulator H-NS represses the expression of all promoters in the LEE. These loci are only expressed in the presence of Ler, a de-repressor protein encoded by the LEE1 operon that overcomes H-NS repression. Ler expression is up-regulated at 37ºC in cell culture media such as MEM. All loci currently known to be de-repressed by Ler in EHEC were acquired by horizontal gene transfer events during its evolution. Whether Ler can regulate genes common to all E. coli strains (core genome genes) remains mostly unexplored, however, recent results obtained by Abe et al strongly suggest that several E. coli core genome loci may be regulated by Ler. In the present work we further characterized the Ler-mediated regulation of 3 of these EHEC loci: ompC, encoding an outer membrane porin, the fimbrial gene yehD and the gene of unknown function ypjC. By means of transcriptional fusions we measured the effect of Ler on the expression of the selected genes. Over-expression of Ler from a multicopy plasmid in E. coli K-12 diminished the expression of the ompC while activating the ypjC fusion. We also used DNA binding assays (EMSA) to prove that Ler directly affects the expression of these genes. EPEC and its Δler derivative where used to further analyze Ler mediated regulation of ompC and ypjC. The presence of ler (under Ler expression conditions) corresponded with a 30% decrease in ompC fusion activity, but Ler did not activate ypjC fusion. Ler also binds to the yehD regulatory region, but it did not activate its expression under any of the studied conditions. The fact that the effect of Ler on the selected gene fusions was mainly observed when the protein was over-expressed, raises doubt about its biological relevance. Repression or activation by Ler and its binding to ompC and ypjC may just be a consequence of the unspecific nature of Ler-DNA binding (which is supposed to depend on AT content or DNA curvature). However, we can not discard the possibility that Ler can regulate this or other core genome genes under conditions different from the ones explored in this study and that this regulation could contribute to EHEC's virulence.



56

GacA controls the flagella synthesis and motility in Azotobacter vinelandii

Lara Flores Norarizbeth, Tzontecomani Pérez Tomas, Carreño López Ricardo and Castañeda Lucio Miguel Centro de Investigaciones Microbiológicas, Instituto de Ciencias, BUAP. Edif. 103-J C.P.72570. Puebla, México. [email protected] Tel. (222)2295500 Ext. 2527 Two-component signal transduction (TCS) systems are composed of a trans-membrane histidine phosphokinase, which senses environmental signals, and a cytoplasmic response regulator, which activates transcription upon being phosphorylated by the sensor. The GacS/GacA TCS is conserved in a variety of gram-negative bacteria. In Erwinia carotovora and some Pseudomonas species, it controls motility. Particularly in E. carotovora GacS/A controls the FlhDC master motility regulator (1). In contrast to others Pseudomonadaceas, in A. vinelandii FlhDC regulates the flagellar biogenesis (2). Therefore we hypothesized that GacS/A controls motility regulating flhDC expression. In turn in A. vinelandii GacS/A controls the post-transcriptional regulatory system Rsm (Csr) and the sigma factor S (RpoS). To establish whether GacA, GacS, RsmA, RsmB and RpoS are involved in this phenomena, motility assays were performed in minimal nitrogen free medium (Burk-Sucrose), containing 0.15 or 0.3% agar. The bacterial strains used were ATCC 9046, ATCC 9046rsmA, ATCC 9046rsmB, ATCC 9046gacA, ATCC 9046gacS and ATCC 9046rpoS. Motility assays showed that the movement was only altered in gacS, gacA and rpoS mutants. Transmission electron Microscopy (TEM) experiments allowed us to observe that the gacA mutant lacked flagella. According to the motility assays the rest of the mutants showed peritrichous flagella. Aditionally, we carried out a Western blot experiment to confirm the data obtained previously. The absence of flagellin in the gacA mutant suggest that GacA controls the flagella synthesis in early stages and to prove this, currently we are working on measuring flhDC and fliC expression in the gacA mutant. REFERENCES 1.- Y. Cui, A. Chatterjee, H. Yang and A. Chatterjee. 2008. J. Microbiology 4610-4623. 2.- R. León and G. Espín. 2008. J. Microbiology 154, 1719-1728. This research was founded by CONACyT grant 129525.



58

Fitness contribution of DEAD box proteins in Bacillus subtilis under

different growth conditions López-Ramírez, Varinia, Olmedo-Álvarez Gabriela Centro de Investigación y de Estudios Avanzados del IPN. Unidad Irapuato. Depto. de Ingeniería Genética. Lab. de Biol. Mol. de Bacterias. [email protected] Km. 9.6 Libramiento Norte Carr. Irapuato-León 36821 Irapuato Gto. México. Tel. (52) 462-623-9600 Ext. 452

The structure and maintenance of functional RNA is an important characteristic that allows organisms to respond rapidly to changing environmental conditions. RNA helicases play a main role in this maintenance. The best studied RNA helicases are grouped into the so called DEAD box proteins, which is a conserved family known well for their ability to unwind secondary RNA structure. In Bacillus subtilis there are four members of this family (YfmL, CshA, CshB and DeaD). In a previous work, we established the phylogenetic relationship between these members, finding that their corresponding genes evolved from two different events of duplication. These genes can be therefore grouped as two pairs of paralogs, one of them comprised by YfmL and CshB, and the other one by CshA and DeaD. We are interested in understanding the role that each of these genes have and how they affect fitness in B. subtilis. To this end we designed and generated insertional mutants for each gene using plasmid pMUTIN and characterized the mutant phenotype under different growth conditions (temperature, minimal medium, rich medium and osmotic stress). In these work, we will discuss how the RNA helicase genes contribute to fitness under different growth conditions.



60

Functional frameshifting in pth gene expression

Marco A Magos C. Eva Jacinto L. and Gabriel Guarneros P. Genetics and Molecular Biology Department. CINVESTAV-IPN. Av. Instituto Politécnico Nacional 2508. Col. San Pedro Zacatenco. C.P. 07360. México, D.F. 14-740, 07000. Phone: 5747-3800 Ext. 5352. E-mail: [email protected]

A functional pth gene is essential for the viability of bacteria. The gene encodes peptidyl-tRNA hydrolase (Pth) whose activity cleaves the ester bond between the 3’ –OH of the terminal adenosine in the tRNAs and the carboxyl group of the last amino acid added to the nascent peptide. Presumably, the enzyme acts on peptidyl-tRNAs drop-off the ribosomes during translation. The essentiality of the gene presumably stems from the need to strip the tRNAs from the peptides in order to be recycled for protein synthesis.

We observed that ∆A117, a +1 frameshift mutation, in pth was suppressed at low rate. Informatic analysis of the possible structures adopted by the pth mRNA predicted a complex stem-loop and pseudoknot structure about 48 nucleotides downstream ∆A117. Indeed, a fusion of a 5’ pth segment, including ∆A117, out of frame (+1) with lacZ gene, expressed some β-galactosidase activity indicating that frameshifting in the correct direction took place. That the complex secondary structure helps to suppress ∆A117 mutation was supported by the reduced β-galactosidase activity recorded with variants carrying partial or complete eliminations of the stem-loop and pseudoknot sequences. Those results are in line with the canonical model for frameshifting which proposes that secondary structures in the mRNAs provoke pausing of the translational processivity. Presumably, a paused ribosome has a propensity to slide out of frame suppressing the frameshift mutations.

However, back to the pth gene, we have created frameshift mutations by deletion or insertion of one bp at different positions in the gene. The results showed suppression of these mutations preceding or within the stem-loop and pseudoknot sequences in the mRNA. If a unique mechanism is responsible of these results, we have to think about changes to the canonical model to explain frameshifting.

Supported by CONACyT grant num. 54206



62

MLST and spa-typing of Staphylococcus aureus from backyard dairy

farms reveals zoonotic potential of local strains

Juan José Valdez-Alarcón1, Daniela Angel-Andrés1, Francisco Ramón García-Rodríguez1, Gerardo Uriel Bautista-Trujillo1, José Luis Solorio-Rivera2, Irma Rentería-Solórzano2, Saul Ignacio Carranza-Germán3, Víctor Manuel Baizabal-Aguirre1, Alejandro Bravo-Patiño1, Marcos Cajero-Juárez4 1Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo. Km 9.5 carretera Morelia-Zinapécuaro, Tarímbaro, C.P. 58893, Michoacán, México. Email: [email protected]. Tel/Fax: (443) 295-8029. 2Unidad de Servicios de Apoyo al Diagnóstico, F.M.V.Z.-U.M.S.N.H. 3F.M.V.Z.-U.M.S.N.H. 4Instituto de Investigaciones Agropecuarias y Forestales, U.M.S.N.H.

Keywords Staphylococcus aureus; MLST, spa-typing, bovine mastitis.

Staphylococcus aureus causes diverse pathologies in human and animal hosts. We report Multilocus Sequence Typing (MLST) and spa-typing of S. aureus isolates related to bovine mastitis from backyard dairy farms in the municipality of Tarímbaro, Michoacán, México. Prevalence of bovine mastitis in 13 farms was 43.3% (41.4% subclinical, 1.9% clinical). Risk factors potentially associated (p<0.2) were: herd size, udder cleaning, milking events per day, time between milking, milking method and number of cows under production. Samples were taken from milk and pharyngeal swab from humans working at farms. Identity of isolates was determined by 16S rDNA sequence. Resistance was found to penicillin or ampicillin (50%) or lyncosamide (45%). Resistance to three or more antibiotics occurred in 73% of the isolates. All isolates were sensitive to methicillin and vancomycin. We found 11 new Sequence-types (STs); in 3 of them new alleles were identified, the rest showed new combinations of alleles. Isolates from milk samples were grouped in Clonal Complexes CC97 (ST97, ST126, ST352, ST1476, and new STs), and CC8 (ST8). One isolate from human origin showed ST188 which groups in the CC97. CC97-related isolates presented spa-types t224, t267, t605, t693, t1965 and t4570. spa-types t008, t189 were found in CC8-related isolates. Several isolates were not spa-typable because of the absence of canonical 5’ and 3’ signature sequences or the presence of new sequence repeats. Minimum spanning tree analysis revealed that one of the new STs from CC97 behaved as a founder clone. Isolates with human-related genotypes (CC8) found in bovine milk samples and with bovine genotypes (CC97) found in human samples, suggest a risk of zoonotic behaviour of backyard farm multiresistant S. aureus isolates.



64

Mannheimia haemolytica expresses curli fibers

Montes-García J. Fernando1, Vaca P. Sergio1, Villamar Tomás1, Vázquez Cruz Candelario2 and Negrete-Abascal Erasmo1 1Carrera de Biología, Facultad de Estudios Superiores Iztacala, UNAM; 2 Centro de Investigaciones en Ciencias Microbiológicas, BUAP. Av. De los Barrios #1, Los Reyes Iztacala Tlalnepantla Edo. De México CP 54090 Tel 56231254. [email protected] Mannheimia haemolytica is the primary bacterial pathogen of bovine respiratory disease complex. This bacterium is a normal inhabitant of the nasal cavity of cattle; however, after stress or viral infections, M. haemolytica replicates quickly and colonize the lungs, producing acute pneumonia. It is still unclear what mechanisms allow M. haemolytica become from a commensal microorganism to a pathogen that overcomes the barriers of the respiratory tract and invades the lung. In this work, a 50-kDa Congo red (CR) binding protein from M. haemolytica was identified and characterized. This protein was resistant to chemical and thermal denaturation. Bacteria grown in the presence of CR express long fibers joining both close and distant bacteria. The 50-kDa CR binding protein was immune recognized by an anti-curli polyclonal serum. CR binding proteins resistant to denaturing and protease treatment are known as amyloid proteins and are produced by mammals, fungi and bacteria. Bacteria such as Escherichia coli and Salmonella spp produce extracellular amyloid fibers called curli. It has been described that curli fibers take part in adhesion to surfaces, cell aggregation, invasion and biofilm formation; among others functions. We feel that the 50-kDa CR binding protein we have identified in M. haemolytica may play a role in its pathogenesis.



66

Standardization of a real time PCR assay to quantify the bacterial density of Helicobacter pylori in gastric mucosa

Stephanie Euridice Morales Guerrero, Gonzalo Castillo Rojas, Yolanda López Vidal Programa de Inmunología Molecular Microbiana, Facultad de Medicina, UNAM. ([email protected], Av. Universidad 3000, Facultad de Medicina, Programa de Inmunología Molecular Microbiana. Edif. de investigación. 4to piso, México DF. CP 04510. Teléfono (55) 5616-0844 Helicobacter pylori is a gram-negative, spiral-shape bacterium, wich colonizes the stomach of almost half of the world’s population, usually acquired on childhood, it can persist over the time. Although the chronic infection remains asymptomatic in most individuals, there is a strong association between long-time infection and the development of several gastric diseases. Determine the bacterial density of Helicobacter pylori in gastric mucosa could help to understand the pathogenesis of the infection. The aim of this work was standardize a real-time PCR assay to subsequently determine the bacterial density of Helicobacter pylori in gastric biopsies. A TOPO-TA cloning plasmid carrying an insertion of the ureB gene from Helicobacter pylori 26695 was extracted from a recombinant E. coli strain; serial dilutions of the plasmid from 100 pg to 1 fg were made. The number of plasmids in each dilution was determined and then these dilutions were used as a standard curve in a real-time PCR assay. The reproducibility and the sensitivity, as well as the amplification efficiency of the assay were determined to further evaluate the bacterial density of Helicobacter pylori in ten biopsies from gastric antrum from patients with non-ulcer dyspepsia. The results of these work showed that the standard curve permit to quantify Helicobacter pylori density in a range of 226 to 22,624,434 bacterial genomes, with an average amplification efficiency of 99.6%. Also, the assay permits to determine the bacterial density of Helicobacter pylori in gastric biopsies, ranging from 27,385 to 289,226 bacterial genomes. We concluded that the real-time PCR assay could be used to determine the bacterial density of Helicobacter pylori in gastric biopsies from infected subjects.



68

Proteome Comparison of Two Disease-related Helicobacter pylori Strains

José Eduardo Mucito-Varela*, Ricardo Cerón-Cardelas, Gonzalo Castillo-Rojas, Yolanda López-Vidal Programa de Inmunología Molecular Microbiana, Fac. de Medicina, UNAM. *[email protected]. Av. Universidad 3000, Fac. De Medicina, Programa de Inmunología Molecular Microbiana. Edificio de Investigación. 4º piso, México, D.F. C.P. 04510. Tel. (55)56160844 Helicobacter pylori is Gram negative spiral-shaped bacterium that colonizes the stomach of approximately the half of the world population. The infection produces chronic gastritis that can progress to peptic ulcer disease (10-20%), gastric adenocarcinoma (1-2 %) or gastric mucosal-associated lymphoid tissue (MALT) lymphoma (<1%). Well known virulence factors such as CagA and VacA proteins are associated to disease development. However a disease-specific bacterial factor hasn’t been identified. In order to try to identify new strain specific disease-associated bacterial factors proteome analysis of two H. pylori strains was performed. Strain 34Ca was isolated from a biopsy of gastric cancer patient and strain 62A9 from a hemorrhagic peptic ulcer patient. Both strains were cagA*, vacA s1b/m1 genotypes. Bacteria biomass was obtained after massive culture in Haemophilus Test medium plates supplemented with 8% of horse serum incubated at 37ºC in a 5% CO2 atmosphere for 48-72hrs. Protein extraction was carried out by acetone precipitation. Proteins of each strain were marked with different fluorophores and analyzed by double-dimension gel electrophoresis. 8 Strain-specific (4 each strain) and 4 common proteins were selected and identified by LC/NSI-MS/MS analysis. Strain specific proteins identified in the gastric cancer related strain were: Chaperonin GroEL (Hsp60), an alkyl hydroperoxide reductase (tsaA) and a hypothetical protein. While in the hemorrhagic peptic ulcer strain: urease A subunit and a thioredoxin were present. Research for disease-associated bacterial factors is of outstanding importance to fully understand H. pylori pathogenesis and identify new potential targets for treatment of H. pylori infection.



70

Cloning, expression and glycosylation of the 38-kDa protein ofMycobacterium tuberculosis in Streptomyces coelicolorA3(2)

Manuel Tavares-Cornejo, Laura E. Córdova-Dávalos, Martí Cadena-Sandoval & Luis Servín González Departamento de Biología Molecular y Biotecnología. Instituto de Investigaciones Biomédicas. Apartado Postal 70228, Ciudad Universitaria, D.F., 04510. TF: 5622-8929. Correo electrónico: [email protected] StreptomycescoelicolorA3(2) is an aerobic, saprophytic and gram-positive actinobacterium, which produces a great variety of antibiotics and secondary metabolites, as well as numerous extracellular enzymes. It has been shown that streptomycetes are good hosts for the cloning and expression of both prokaryotic and eukaryotic genes. The 38-kDa protein ofMycobacterium tuberculosis is used as an antigen for diagnosis of tuberculosis. This protein is encoded in thepstS1gene (Rv0934) and isinvolved in the transport of phosphate into the cell.This protein was also among the first glycoproteins to be described in bacteria. Despite this, there are few studies about the glycosylation properties of this protein, due in part to the technical difficulties of working with a slow growing pathogenic bacterium such as M. tuberculosis. Therefore, to study this protein, we have sought to express it inStreptomyces coelicolorA3(2), since we previously constructed a mutant strain of this organism lacking the gene for the 38-kDa homologue. In order to evaluate the expression and glycosylation of this antigenic protein two constructs were built. The first one consists of the complete Rv0934 gene, which encodes the 38-kDa protein, with its own signal peptide, which contains a cysteine that enables this protein to be anchored to the cell membrane. In the second construct the signal peptide and the cysteine have been replaced with the signal peptide of the Streptomyces exfoliatus M11 extracellular lipase, which allows the protein to be secreted. Expression of the protein was evaluated using a specific antibody directed against the protein, and its glycosylation status by binding to the concanavalinAlectin



72

The two-component system CpxR/A negativelly regulates the expression of the SPI-1 and SPI-2 virulence regulons in

Salmonella enterica serotype Typhimurium Irene Jaquelin Palacios Velázquez, Miguel Ángel De la Cruz Villegas, Luary Carolina Martínez Chavarría, Edmundo Calva Mercado, José Luis Puente García y Víctor Humberto Bustamante Santillán Instituto de Biotecnología de la UNAM. Departamento de Microbiología Molecular. Av. Universidad # 2001. Colonia Chamilpa, C. P. 62210, Cuernavaca, Morelos. Teléfono 777 3291627. Correo electrónico: [email protected]

Salmonella enterica serotype Typhimurium (S. Typhimurium) can cause intestinal or systemic infections in human and animals. Most of the genes that S. Typhimurium requires to produce these infections are clustered in two chromosomal regions denominated Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). SPI-1 genes are necessary for the S. Typhimurium invasion of the intestinal epithelial cells; thus for the intestinal infection, whereas the SPI-2 genes are mainly required for its replication and survival inside macrophages, and thus for the systemic disease. SPI-2 genes also have a role in the development of the intestinal inflammatory disease.

A myriad of global and Salmonella-specific regulators have been involved in the expression of the SPI-1 and SPI-2 genes. HilD, encoded in SPI-I, consecutively induces the expression of the SPI-1 and SPI-2 genes, by directly controlling the expression of HilA and SsrA/B, the central positive regulators of these genes. The appropriate concentration and activity of HilD is controlled at multiple ways, by its positive autoregulation at a transcriptional level, by CsrA, which represses its translation, through its inactivation by direct interaction with the HilE protein, as well as through its degradation mediated by the Lon protease.

In this work, by using transcriptional fusions to the cat reporter gene and Western blot assays, and analyzing the protein secretion profiles of S. Typhimurium wild-type and different mutant strains, we demonstrate that the two-component system CpxR/A negatively regulates the expression of HilD and thus the SPI-1 and SPI-2 genes. Our results, together with previous reports, suggest that CpxR/A controls the expression of HilD indirectly, mainly by inducing the expression of the alternative sigma factor RpoH, which in turn increases the expression of Lon that degrades HilD.

Our results expand the complex regulatory cascade controlling the expression of the SPI-1 and SPI-2 genes.



74

Annotation and integration of the transcriptional regulation of carbon sources with their metabolic pathways in Escherichia coli K-12

*Peralta-Gil Martin1, Muñiz-Rascado L. 1, Gama-Castro S.1, Guzmán J.2 and Julio Collado-Vides1

1Program on Computational Genomics, Center for Genomic Science UNAM, Cuernavaca, Mor., México, 2Institut of Biotechnology UNAM, Cuernavaca Mor. Méx.*Corresponding author: *[email protected]

RegulonDB is a database that integrates mainly biological knowledge of the mechanisms that regulate the transcription initiation of Escherichia coli and eventually its complete regulatory network. All the information has been obtained from the published literature, with detailed, well-supported, accurate, and up-to-date bibliographic curation concerning operon organization, binding sites for transcription factors, promoters, terminators, and RNA regulatory elements. The structure of this database is accessible on the internet through seven major navigation paths: genes, operons, regulons, sigmulons, sRNAs, growth conditions, and Gensor units (GUs), combining graphics and information from the published literature. As we have developed this database, we have followed different strategies in order to better fulfill the requirements of the database, such as curating by publication date, on the bases of regulon and sigmulon, by functional class, by biological system, by binding sites of a particular transcription factor, and finally by GUs. A GU represents the biological context that naturally provides a better understanding of gene regulation, integrating mechanisms and concatenated reactions into a physiological unit that is defined by the capacity of the genome to regulate gene expression in response to a signal or stimulus. E. coli is a microorganism that can utilize multiple sources of carbon and select the best to survive. In this sense, currently we have curated about 35 regulons related to carbon sources, and we have made diagrams of 15 GUs related to the uptake and degradation of different carbon sources. Additionally, we have further analyzed the mechanisms of regulation of the 35 local transcription factors and the regulator cAMP receptor protein (CRP). The dual transcriptional CRP regulates the expression of over 180 genes in E. coli. Many of these genes are involved in the catabolism of secondary carbon sources, but this regulator is also involved in many other processes. As a result of our analysis, we found that this global regulator has different mechanisms of action to control the transcription of genes or operons involved in the uptake and degradation of different carbon sources. In conclusion we are integrating the metabolic pathways for the different carbon sources with the information on transcriptional regulation. We are also working to understand the mechanisms of action of the transcription factors involved and their interaction with CRP, and also to define its importance in the assimilation of different carbon sources.



76

The Global Regulator Mta of Bacillus subtilis: Regulon and consequences of his absence

Evangelina Esmeralda Quiñones Aguilar1, Marc Chippaux2 and M. Foglino2 1Dirección actual: Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Av. Normalistas No. 800, Colinas de la Normal. CP 44270, Guadalajara, Jalisco MEXICO. [email protected] / [email protected]. 2Laboratoire de Chimie Bactérienne UPR 9043, CNRS, France. In Bacillus subtilis, two possible roles were proposed for the Mta regulator, a member of the MerR family: either protection of the cell from a specific environmental toxic compound or control of a normal physiological process(es). With the aim to answer this question, transcriptoma analyses were performed that uncovered 7 other transcription units demonstrating that Mta is a Global regulator. However, analysis of the gene products of the Mta regulon failed to reveal any Mta function. Nevertheless, that Mta controls a physiological process was demonstrated by the fact that its absence (∆mta) leads to the filamentation of the cells. This phenotype is due to a secondary mutation, tentatively called mcm that appears during sub culturing. During exponential growth, the mta mcm cells are engaged in long sessile filaments that resume into single cells or doublets when entering the stationary phase. Transcriptome analyses reveals that the mcm mutation negatively affects the expression of many genes among which are those belonging to the regulon of the transcription factor for motility σD as well as those of the mtnKS operon. We have demonstrated that, whatever the growth phase, σD is in a permanent σD -OFF state in the mta mcm cells and does not oscillate between σD -ON and σD -OFF states as is the case for the parental strain. We have determined that none of the genes belonging to the Mta regulon is responsible of the mcm mutation. DNA sequencing revealed that the mcm secondary mutation does not affect sigD, the σD structural gene, or any factor known to directly or indirectly control the transcription from the σD - controlled promoters.



78

Vibrio cholerae bacteriophages isolated from

Endhó dam at Hidalgo-México

Solís-Sánchez GA, De la Cruz-Ortiz E., Hernández-Chiñas U, Navarro-Ocaña A, Eslava-Campos CA. Departamento de Salud pública, Facultad de Medicina-UNAM Circuito Interior S/N, Ciudad Universitaria, Coyoacán, DF. México. C.P. 04510 Tel/Fax: (55)5622-0822 e-mail: [email protected]; [email protected]

Some different studies suggest the Vibrio phages participation, in the cyclic appearance of cholera disease outbreaks. Although, the cholera in the Americas was considered as a controlled disease, the Haiti outbreak in October 2010, and the report of two cholera cases in Mexico (November 2010 and April 2011), suggest the reemergence of this disease. The associated factors with the apparent absence,from the environment,of Vibrio cholerae O1/O139 the etiological agent of cholera disease after 8 years are not clear. In a previous study on the dam Endhó was isolated and characterized a new lytic phage named phiVC8, specific to V. cholerae O1 strains. In the present study we are showing results on the isolation and characterization of new specific phages, with lytic activity on Vibrio cholerae O1 and O139 serogroups. Four water samples collected at different places of Endhó damat Hidalgo, Mexico, were utilized for phage isolation. Water samples (200 ml) were centrifuged at 16000 x g and filtered through 0.22µm membrane. For phage detection, eleven strains were used, eight V. cholerae O1, twoV. cholerae O139 and one Escherichia coli, allobtained from the strains collection of the Public Health Department of the Medicine School, National Autonomous University of México. The assay was performed using 1 ml of filtered water and 100µl of each strain culture in mild-log growth phase and mixed with 4 ml of molten soft nutrient agar (0.7%) and poured over nutrient agar plates. The samples were incubated at 37ºC at 18 h and the appearance of clear plaques on the double layer assays was determined as positive result. Ten different bacteriophages were isolated from the water samples, all of them induced lytic plaques on V. cholerae O1/O139 but not on non O1/non O139 strains. On the other hand from the same samples,V. cholerae No-O1/No-O139 strains were isolated. Our results suggest that the environmental conditions of the Endhó dam are suitable to bacteria survival. However, the V. cholerae O1/O139 absence could be connected with the presence in the environment of specific lytic phages. Are being conducted studies, to determine if these new phages are similar or different to ØVC8, previously identified.



80

Role of csd system in the biogenesis of clusters [Fe-S] and virulence of Pseudomonas syringae pv. phaseolicola 1448A

Gabriel Rincón Enríquez, Esther Angélica Cuellar Torres, Evangelina Esmeralda Quiñones Aguilar and Joaquín Alejandro Qui Zapata Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Av. Normalistas No. 800, Colinas de la Normal. CP 44270, Guadalajara, Jalisco MEXICO. [email protected] / [email protected]. Iron-sulfur centers [Fe-S] are metal cofactors present in a large number of proteins. Protein [Fe-S] involved in cellular processes such as respiration, photosynthesis, gene regulation, etc. The biogenesis of [Fe-S] is performed by three major systems in the cell. These systems are ISC (iron sulfur cluster), SUF (sulfur assimilation) and NIF (nitrogen fixation). All three systems have a protein with activity cysteine desulphurase (IscS, SufS and NifS). In some prokaryotic genomes has been shown one fourth protein with cysteine desulphurase activity, CsdA. This protein is part of the CSD operon consisting of genes cdsAEL. The functions proposed for this operon depends on the physiological context of the cell: 1) CsdA can participate with sulfur donor source in the biogenesis of [Fe-S] via SUF and 2) a CsdAEL pathway involved in synthesis of thiolated compound. The analysis in silico of the genome of P. syringae pv. phaseolicola 1448A (Psph) suggests only the presence of the ISC and CSD systems. CsdAEL operon was determined in silico with high degrees of similarity between proteins of Escherichia coli of 57, 59 and 69% from CsdA, CsdE and CsdL, respectively. The insertion of a mutated copy of csdA with a nonpolar cassette (apha-3) was performed. Preliminary results indicate that the phenotypic analysis of the strain with the insertion csdA::apha-3 showed phenotypes related to the biogenesis of [Fe-S] and effect on the virulence of Psph in bean (Phaseolus vulgaris). Therefore the CSD operon plays a key role in the biogenesis of [Fe-S] and virulence on bean of Psph.



82

Analysis in vivo: role antisense RNA in the incompatibility and negative regulation of plasmid symbiotic of R. etli.

Rivera Urbalejo A. P.*, Cevallos M. A. [email protected], Programa de Genómica Evolutiva; CCG –UNAM. Avenida Universidad s/n, col. Chamilpa, CP: 62210. Cuernavaca, Morelos, (777)3291690. In the past decades, the identification of non-coding RNAs and their role as key regulators of gene expression has caught the attention of diverse research groups. Moreover, in bacteria non-coding RNAs have been discovered to have functions of ribozymes, riboswitches and also of strong incompatibility factors. On the other hand, the basic replicon of R. etli belongs to the repABC operon family which is characterized by the presence of an intergenic region that encodes for an antisense RNA (ctRNA) that controls plasmid replication. Based on in vitro experiments we know that the 5’ region of the ctRNA is required in order to interact with the mRNA and when the ctRNA is absent, the repABC operon mRNA forms several loops by the pairing of its bases and therefore, allowing plasmid replication. However the complex ctRNA-mRNA interaction induces a conformational change in the operon mRNA which allows the formation of a stem-loop structure called “element S” which prematurely terminates transcription of the operon by transcriptional attenuation. Surprisingly there is a paradox in respecting the ctRNA considering that this molecule negatively regulates plasmid replication. Based on this fact, it is expected that mutations that bring down the expression of the ctRNA should induce an increase in the number of copies of the plasmid. However, these mutations prevent plasmid replication. In an aim to have a better understanding of this event, this work is focused in the elucidation of the ctRNA 5’ end nucleotides important for its function in two aspects: incompatibility and as a negative regulator. To analyze the feature of incompatibility, different mutations along the ctRNA 5’ end were generated and the importance of the mutated nucleotides was determined in the interaction with the operon mRNA. Results showed that the first ten nucleotides of ctRNA 5´ end are responsible for the incompatibility phenotype. In addition, each mutant ctRNA was used to determine their effect in the plasmid replication capacity. It was observed that any of the mutants were able to self-replicate in contrast to the strains which contain the wt ctRNA in trans. Furthermore, the presence of the in trans wt ctRNA recovered the plasmid replication ability suggesting a functional complementation between the wt ctRNA and each of our constructions.



84

One redox regulation of the Escherichia coli ArcB sensor kinase:the role of ubiquinones and menaquinones

Claudia Rodríguez Rangel, Adrian Álvarez and Dimitris Georgellis Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México City, México; [email protected], 56225738 The Arc two component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes in response to respiratory growth conditions. Under aerobic conditions, the kinase activity of ArcB is silenced by the ubiquinone electrone carriers, wich oxidize two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we show that the redox potential of ArcB is 0 mV, and that its proper activation upon a shift from aerobic to anoxic conditions require the presence of the menaquinone electron carriers. Thus, an essential link in the Arc signal transduction pathway connecting the redox-state of the quinine pool to the transcriptional apparatus is elucidated.



86

Generation and characterization of E. coli strains lacking the PTS system with modifications at the PEP-PYR node in order to increase the

availability of PEP towards aromatic production coutilizing glucose and acetate

Andrea Sabido Ramos y Francisco Bolívar Zapata Instituto de Biotecnología, UNAM. Apdo. Postal 510-3. Phone:+52-777-3291601. E-mail: [email protected] In Escherichia coli, the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) consumes more than 50% of phosphoenolpyruvate (PEP), which is one of the first precursors of the aromatic pathway. In our group, PTS encoded by the ptsHIcrr operon, was deleted from JM101 to generate strain PB11. An evolved strain, PB12, was derived from PB11, which grows faster using glucose as the only carbon source. We have shown that PB12 has the capability to redirect more PEP into the aromatic pathway. In this context, it has been demonstrated that PTS-Glc+

pykA- pykF- mutants, expressing simultaneously aroGfbr and tktA are capable of channeling high carbon fluxes into the aromatic pathway on a rich medium. However, when the PB12 pykA- pykF- mutant was grown in M9 medium supplemented with glucose, no growth was detected. Taken these results together, we propose that simultaneous utilization of glucose and acetate in minimal medium will restore the growth of E. coli pts-, pykA-, pykF-, ppsA- strains (PB11 and PB12). As expected PB11 pykAF- and PB11 pykAF- ppsA- strains were incapable of growing on glucose as the only carbon source. However if the M9 medium contained acetate as the only carbon source, only the PB11 pykAF- mutant was able of growing with a specific growth rate (µ) 22% lower compared to PB11 (0.18 h-1). When both mutants were grown in the glucose-acetate mixture, PB11 pykAF- strain showed a µ of 0.17 h-1 while PB11 pykAF- ppsA- grew with a µ of 0.16 h-1, representing a recovery of approximately 60% for both strains with respect to their parental strain. On the other hand, PB12 pykAF- and PB12 pykAF- ppsA- strains showed µ’s of 0.16 and 0.18 h-1 respectively when growing on glucose as the only carbon source. The response to pyruvate kinase deletion in those strains might be an increase in the metabolic flux via the phosphoenolpyruvate carboxylase (Ppc), a behavior previously seen in a JM101 pykAF- strain when growing on glucose as the only carbon source. Regarding their performance on M9 medium with acetate, only strain PB12 pykAF-

showed an increase of 33% on its µ compared to PB12. The possible elimination of a futile cycle in the metabolic node PEP-Pyr in this strain, could explain its higher µ. Finally, in the glucose-acetate mixture PB12 pykAF- and PB12 pykAF- ppsA- strains reestablished their growth capacity 88% and 80% respectively as compared to the PB12 strain (0.41h-1). The results presented in this work demonstrate that these PTS- strains are capable of redirecting all the glucose towards the production of PEP, which can be available for the synthesis of aromatic compounds, while acetate catabolism may supply biosynthetic precursor and redox power from TCA.



88

Study of RetS, LadS and GacS histidin kinases Interactions in Azotobacter vinelandii.

Sánchez Cuapio Z. and Castañeda Lucio M Centro de Investigaciones en ciencias microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla. Edif. 103-J. CU C.P. 72570 Puebla, México. [email protected]

Alginates are an important family of biopolymers. These linear polysaccharides are structured as chains of (1-4)-β-D-mannuronic acid and α-L-guluronic acid. Azotobacter vinelandii, a gram-negative non-pathogenic soil bacterium produces such polysaccharides. The GacS/A two component system (TCS) is involved in secondary metabolism regulation and in many other aspects of bacterial physiology; in A. vinelandii this system is involved in the control of alginate production (1). The existence of different sensor kinases very similar to GacS (called RetS and LadS) has been reported in Pseudomas aeruginosa y Pseudomonas flourescens. Recently studies show that they interact with GacS to achieve finer control of the entire regulation pathway (2,3 and 4). However, the mechanism by which the sensor kinases RetS and LadS fit into the GacS/GacA system remains unknown. Protein-protein interactions studies seem to be a key step to understand the interconnections controlling this regulatory system.

In this study, using a genomic approach we identified a RetS orthologous and LadS paralogous on A. vinelandii. We subcloned the coding regions corresponding to the citoplasmic domains of such genes. To further characterize and define the molecular interactions between these domains we utilized a bacterial two-hybrid system. Preliminary data from this in vivo interaction study indicates that RetS directly interacts with GacS suggesting that what was once thought as a two-component system might really be a multiple-component system, an idea that would expand our understanding of regulatory Gac networks.

1. M. Castañeda, J. Guzmán, S. Moreno, G. Espín, Journal Of Bacteriology 182, 2624-2628 (2000). 2. M. L. Workentine, L. Chang, H. Ceri, R. J. Turner, FEMS Microbiology Letters 292, 50-56 (2009). 3. A. L. Goodman et al., Genes & Development 23, 249-259 (2009). 4. B. Humair, N. González, D. Mossialos, C. Reimmann, D. Haas, The ISME journal 3, 955-965 (2009).

This work was supported by VIEP,CALM-NAT10-II ,BUAP



90

Synthesis of cardiolipin derivatives in streptomycetes

Mario Sandoval-Calderón1, Ziqiang Guan2 and Christian Sohlenkamp1* 1Programa de Ecología Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Apdo. Postal 565-A, Cuernavaca, Morelos CP62210, Mexico, 2Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, U.S.A. * To whom correspondence may be addressed. Tel: +52-777-3131697; E-mail: [email protected] Cardiolipin (CL) is an anionic phospholipid found almost ubiquitously among bacteria and eukaryotes. In contrast with other phospholipid species, it has four acyl chains. It is preferentially located within energy transducing membranes and it has been found to be essential for the optimal activity of several integral membrane proteins. Additionally, its presence has been related to some cellular processes, such as the germination of Bacillus subtilis spores and the correct development of the soil-dwelling actinomycete Streptomyces coelicolor.

Previously, we discovered that two CL derivatives, monolysocardiolipin (MLCL) and dilysocardiolipin (DLCL), accumulate in S. coelicolor membranes. We assume that these molecules are the product of the hydrolysis of one (MLCL) or two (DLCL) of the esterified fatty acids from CL. However, none of the sequenced Streptomyces genomes bears genes encoding clear phospholipase A or lysophospholipase homologues, the enzymes that could be responsible for this activity. Thus, our aim is to identify the genes responsible for the synthesis of MLCL and DLCL, and the functional characterization of these two molecular species in streptomycetes.

In order to solve this puzzle, we are working on two parallel strategies. The first is a directed approach consisting in mining the available genome sequence information of actinomycetes. Taking advantage of current bioinformatic tools, a set of eleven candidate genes that might encode phospholipase A or lysophospholipase activities was selected. Currently, they are being tested by means of heterologous expression and insertional mutagenesis. The second approach is a random chemical mutagenesis of the S. coelicolor genome, followed by a lipid phenotypic analysis of the resulting mutant clones.



92

Identifying the looseness elements of the primary-sigma factor (SigA) of Rhizobium etli.

Santillán G. Orlando, Ramírez R. Miguel Angel, López L. Gamaliel, Méndez J. Cyntia, Lozano Luis and Dávila M. Yadira Evolutionary Genomics Program. CCG-UNAM. [email protected]. Universidad Avenue, Chamilpa, P.O.B. 565-A, Cuernavaca, Mor. Zip code 62210, Mexico INTRODUCTION. In simple terms, transcription can be viewed as making a complementary copy of RNA from a template DNA. It is done by the RNA polymerase (RNApol) which is made-up of six proteins (, ’, , two monomers of and factor). The Escherichia coli primary- factor (RpoD or 70) and its orthologs are responsible for transcribing housekeeping genes. Primary- factors are divided into four domains (1, 2, 3 and 4). It has been observed that many primary- factors from different -proteobacterial species can recognize a wide variety of E. coli RpoD-dependent promoters, but RpoD is unable to recognize the -proteobacterial counterparts. In this regard, the primary- factor of R. etli (SigA) is one of the tested proteins that shows this behavior (we have called this the looseness phenomenon). It also recognizes a lax consensus promoter (5’-CTTGAC-N16-TATNNT-3’). Here, we try to find out the mechanism underlying the looseness phenomenon in SigA and if it could be explained by the presence of a certain region (or a combination of them) and/or its consensus promoter structure. RESULTS. In order to explore the possibility that a particular region of SigA could explain the looseness phenomenon we have built chimeric genes combining the four domains of SigA and RpoD. Nowadays we have successfully assembled four chimeras and tested them in their ability to sustain the growth of the strain E. coli UQ285. We observed that two chimeras (both have in common the NCR of SigA and the 3-4 of RpoD) were unable to sustain growth of UQ285 at the restrictive temperature. We also made a bioinformatic analysis of the intergenic regions of the R. etli genome (supported on RNA-seq data) in spite of improving the consensus promoter. About 1000 sequences were analyzed and the preliminary results shows the following structure for the consensus promoter: 5’-YTTGMM-N17-CTATWW-3’. DISCUSSION. The results obtained in the in vivo complementation led us propose that the looseness phenomenon could be explained by a combination of domains (or regions). From the initial analysis of the promoter sequences we concluded that the consensus promoter is less conserved than the reported previously. Together these findings suggest that SigA is capable of recognizing a wide range of promoter sequences because of its particular structure. REFERENCES: Mol. Gen. Genet. 1983;191:492-98. Annu. Rev. Microbiol. 2003; 57:441-66. Nucleic Acids Res. 2003;31(13):3593-6. Nucleic Acids Res. 2006;34(5):1470-80. Microbiol. 2006;152:1751-63 Anal. Biochem. 2008;380(2):338-40



94

Genomic analysis of four genes involved in PU degradation by Alicycliphilus sp. BQ1

Julieta Solís-González1, Nancy Barajas2, Jesús Campos2, Luis Lozano3, Miguel A. Cevallos3 y Herminia Loza-Tavera1 1Facultad de Química, Departamento de Bioquímica, UNAM. Av. Universidad 3000. México, D.F. 04510. [email protected]. 2IIQB. Universidad Michoacana, Morelia, Mich, 58030 3Centro de Ciencias Genómicas, UNAM, Cuernavaca Mor, 62210

Polyurethane (PU) is a synthetic polymer which production, consumption and waste increase every year as result of its high resistance to environmental factors and waste inefficient treatments. Some types of PU are susceptible to microbial attack so biodegradation might be an alternative treatment. The few PU catabolic-related bacterial genes reported until now, encode esterases, and a putative transporter system. No further analysis on the metabolic pathways involved on bacterial PU degradation has been performed. However, for degradation of other artificial polymers, monooxygenases, ether hydrolases, and glycolate oxidase have been reported.

We have isolated and identified a bacterial strain, Alicycliphilus sp. BQ1, able to grow on minimal medium with commercial PU (Hydroform) as sole carbon source (MM-PUh). The aim of this work is to identify the genes involved in PU degradation in Alicycliphilus. Four mutant strains unable to grow in MM-PUh were generated by Mariner Himar1: GmR ApR transposon random insertion. The transposon inserted chromosomal regions were cloned and sequenced. In addition, a preliminary version of the Alicycliphilus BQ1 genome has been generated by using Illumina-Solexa technology which produced 21’870,000 36 bases sequences, that were assembled in 486 nodes.

BLAST analysis identified that the disrupted genes encoded a periplasmic substrate binding protein, which is part of an ABC type sulfonate transporter (clone 5), a lytic transglycosilase (clone 8), an hypothetical protein (clone 9), and a glyoxalase (clone 38). Genomic context analysis of BQ1 and genomic comparisons with Alicycliphilus denitrificans BC and K601 showed that the gene mutated in the clone 9, is not present in BC and K601 strains, it is part of a 3 kb insertion which also include three other genes, encoding an hydrolase (possible esterase) and hypothetical proteins. Moreover, the gene mutated in clone 9 is less than 1 kb apart from the gene mutated in clone 38. This arrangement, not detected in other phylogenetically related organisms, might be one of the differences responsible for the capacity of BQ1 to grow in MM-PUh.

Work supported by CONACYT grant 82881 and DGAPA grant IN222811. JSG acknowledges CONACYT for her scholarship.



96

Design, construction and characterization of thermoinducible recombinant Escherichia coli strains

Expressing the immunogenic proteins ESAT-6 and CFP10 from Mycobacterium tuberculosis

1Pedro E. Navarro García, 1Norma A. Valdez-Cruz, 2Clara Espitia, 1Mauricio A. Trujillo-Roldán Instituto de Investigaciones Biomédicas UNAM. 1Unidad de Bioprocesos2, Departamento de Inmunología, Tel: 56229192 [email protected] Human and bovine tuberculosis caused by M. tuberculosis and M. bovis respectively is a global public health problem [1,2]. Specific antigens from pathogenic mycobacteria called ESAT-6 (Early Secretory Antigenic Target-6kDa) and CFP10 (Culture Filtrate Protein-10 kDa), are able to improve the PPD test for rapid screening and diagnosis, turning it more specific and sensitive [3,4,5]. The recombinant production of these antigens in E. coli is a safer alternative that can provide recombinant proteins for being used in biological assays. Thermo-inducible systems in E. coli can generate yields of up to 30% from the total cellular protein [6]. No chemical inducers are added, the depletion of substrates is avoided, the cell culture is easy handled, scale-up processes are simplified and a wide variety of strong and tightly regulated promoters adds some versatility [7]. The thermal induction of recombinant proteins in E. coli activates the heat shock response (HSR), triggered by the sigma factor 32 (σ32) and involves the over-expression of proteins such as GroEL, DnaK and DnaJ. These refold proteins affected by temperature and/or the over-expression of recombinant proteins, allowing the cell to become thermo-tolerant. Additionally, DnaK and DnaJ proteins are able to regulate the HSR itself by promoting the degradation of σ32 [7]. In this study, recombinant E. coli strains expressing the thermo-sensitive cI857 repressor and carrying a thermo-inducible plasmid with ESAT-6 and CFP10 genes under the strong pL promoter were used. As a possible molecular mechanism of regulation, we employed a constitutive plasmid carrying the DnaK and DnaJ genes. Moreover, two culture strategies are planned to involve a phase of culture growth for 3 hours at 29°C and a warm-up to 39°C, in a 1.0 L bioreactor. The first strategy consists of an induction by 1-minute warm-up by 40 pulses, while the other involves periods of 40 minutes at 39°C allowing the HSR to develop and eventually achieve a new steady state. The production of recombinant proteins and GroEL, DnaK, DnaJ expression were followed by electrophoresis and immunodetection. 1.- WHO, 2009 2.- Prabhudesai y Singh, 2009 3.- van Pinxteren et al., 2000 4.- Brock et al, 2001 5.- Aagaard et al, 2010 6.- Vallejo et al., 2002 7.- Valdez-Cruz, et al, 2010 8.- Valdez-Cruz et al., 2011 9.- Caspeta et al., 2009.



98

The Rhizobium etli sigma factors profile

Méndez Jiménez Cyntia Citlali1,2, Alarcón González Ana Yatzin1, Dantán González Edgar2, López Leal Gamaliel1, Santillan Godínez Orlando1 y Ramírez Romero Miguel Angel1. 1Centro de Ciencias Genómicas (UNAM) y 2Centro de Investigaciones en Biotecnología (CEIB) [email protected]. Telephone number 017773291690 Rhizobium etli is an α-proteobacteria that encodes a large number of sigma factors, which represent an important "arsenal" that allow you to respond to stressors and lead to cellular adaptation. This work aims to quantify the intracellular concentrations of sigma factors and their expression profiles under different conditions of stress, to have a clearer picture of how bacteria respond to changes in their environment. In order to meet this objective will be made absolute quantifications of the 23 sigma factors of R. etli in 10 different growth conditions by RT-PCR in real time. The results of the expression levels for 19 sigma factors from RNA samples extracted at six growth conditions, indicating that all sigmas are expressed, although in varying amounts in different conditions. It was also found that the sigma factors RpoE2 and RpoE4 presented higher expression level in all growth conditions, while the RHE_PF00422 sigma factor in most of the conditions presented the lowest levels. In order to identify whether the sigma with the highest expression levels play a role as general regulators of the response to different stress conditions, is intended to generate mutants of the genes encoding these factors and obtain a phenotypic characterization.



100 Isolated Lactic Acid Bacteria from whey cheese production

Mario Domínguez Magaña, Francisco Pulido Barajas, Sandra del Moral Ventura, María García Gómez * Universidad del Papaloapan, Campus Tuxtepec. Circuito Central No. 200. Col. Parque Industrial C.P. 68301. Tel/Fax: 012878759240 * Email: [email protected] Keywords: whey, LAB,CFU/mL Introduction.The whey cheese production is the resulting by-product of the separation and precipitation of caseins during the cheese manufacturing; it has been considered a waste product, although it contain proteins with elevated biological value to possess in more proportion essencial amino acids, sulfur-containing amino acids and branched chain amino acids.(1).Because of nutrients content, it is posible to isolate endogenic microorganisms from whey cheese production like lactic acid bacteria (LAB) for comercial interest on food technology and for other areas. The aim of this work was evaluated the effect of endogenic LAB fermentation time of whey from cheese production on viable cellscount (CFU), on pH and % of titratableacidity to obtain the optimal time for isolation LAB for further characterization and identification. Materials and methods.Two, a 250 mL Erlenmeyer flask containing 150 mL of fresh serum from Loma Bonita, Oaxaca cheese manufacturer,was pushed into the fermentation equip at 37+2°C, 150 rpm stirred. Fermentation samples were obtained at 0, 24, 48 and 72 h for determination CFU, pH and % of titritableacidity.Viable cells coin was obtained with spread plate method (2)employing specific agar: APT, MRS and Elliker. We employed too agar for pathogenic bacteria as control measure: McKonkey and EMB. The sample was centrifugatedat 10,000 rpm, 15 min and at supernatant was evaluated pH (3) and % of titratable acidity expressed like % lactic acid (4). Results.At 48 h of fermentationthe LAB began to lose startind and the coliforms bacteria began to grown in spite of the low acidity of the medium (5.40 to 3.38).The % of lactic acid was increased at 48 h of fermentation (07 to 2.2%), but it was lossing at 72 h. Conclusions.The optimal time for LAB isolation from whey cheese production is 24 h after of the whey recolection References. (1) Alvarado C., Guerra M. (2010). Anales Venezolanos de Nutrición. Vol 23(1):42-49. (2) Vander D., Splittstoesse F. (1992). Compendium of methods for the microbiological examination of foods: 94-135. (3)Hart L., Fisher H. (1991). Determinación de pH. En Análisis de los Alimentos. Ed. Acribia. Zaragoza, España: 15-17. (4) Pereira F. (1988). Acidez como % de ácido láctico. En: Alimentos. Manual de Análisis Fisicoquímicos. Ed. Universidad Autónoma de Yucatán, México: 26-28

AUTHOR INDEX

Author Index


A Acosta Navarrete MY, 125 Adaya L, 107 Aguilar C, 27 Aguilar F, 141 Aguilar JL, 77 Aguilar Martínez CA, 4 Aguilar Piedras JJ, 91 Aguilera S, 36 Aguirre Ramírez M, 73, 129 Alarcón González AY, 177 Alejandrez Martínez N, 78 Alonso Karla R, 131 Alvarado Rodríguez J, 131 Álvarez A, 170 Álvarez AF, 33, 130 Álvarez L, 49 Álvarez Morales A, 36 Andrade A, 40, 46 Ángel Andrés D, 159 Arambula D, 19 Arista Carrera I, 79 Arruebarrena Di Palma A, 80 Arzate Barbosa P, 124 Ávila Rodríguez R, 131 Avitia Cao Romero M, 4, 28 B Baca BE, 80, 91 Baizabal Aguirre VM, 81, 159 Balderas Martínez A, 132 Ballado T, 55, 112 Barajas N, 175 Bárcenas León S, 124 Bautista Trujillo GU, 81, 159 Bebout B, 143 Becerra Rivera VA, 4, 29 Beckmann R, 77 Bello Díaz JE, 133 Bermúdez Barrientos JR, 82 Bermúdez Cruz RM, 56 Bolívar F, 27 Boogerd F, 142

Bravo A, 135 Bravo Patiño A, 81, 159 Brom Klanner S, 4, 30, 86, 104, 153 Bustamante VH, 87, 164 Bustos Martínez J, 99, 134, 149, 151 Bustos P, 30, 122 C Caballero Flores G, 125 Cabellos Avelar T, 29, 83, 92, 93 Cabrera Ruiz R, 4, 31, 118 Cadena Sandoval M, 163 Cajero Juárez M, 81, 159 Calva Mercado E, 164 Camacho Hernández MI, 5 Camacho MI, 33 Camarena L, 14, 47, 55, 72, 89, 97, 112, 117, 138, 145 Campos García J, 37, 175 Cárdenas Rodríguez A, 129 Carranza Germán SI, 81, 159 Carreño López R, 156 Carreón R, 85 Castañeda Lucio M, 78, 101, 109, 119, 156, 172 Castañeda Montes MA, 84 Castellanos Escamilla M, 9, 111 Castellanos M, 50, 74 Castillo Méndez M, 84, 136 Castillo Rojas G, 148, 161, 162 Castro AM, 85, 124 Castro Cerritos KV, 137 Celis Sandoval H, 65, 121 Cerón Cardelas R, 162 Cervantes C, 51, 125 Cervantes L, 30, 86 Cervantes Rivera R, 34 Cevallos Gaos MA, 5, 113, 114 Cevallos MA, 34, 169, 175 Chagoya López A, 92 Chippaux M, 166 Cleary TG, 87 Cobaxin Cárdenas ME, 5, 35

Author Index


Colina L, 138 Collado Vides J, 57, 165 Contreras CA, 87 Córdova Dávalos LE, 79, 108, 163 Corzo G, 103 Cosme A, 107 Cosme Herrera YI, 91 Creus CM, 80 Croda García G, 73 Cruz C, 85 Cuellar A, 61 Cuellar Torres EA, 168 Cuevas Córdoba B, 61 Czornyj E, 19 D Dantán González E, 177 Dávalos A, 104 Dávila G, 30, 122 Dávila M. Y, 174 Dávila S, 40 De Anda R, 27 De la Cruz Ortiz E., 167 De la Cruz Villegas MA, 164 De la Mora J, 55, 112, 145 De la Torre M, 31, 118 De la Torre Zavala S, 5, 36, 88 Del Moral Ventura S, 178 Delgado G, 45 Delgado Sapién G, 129 Días Jiménez D, 130 Díaz Guerrero M, 94 Díaz Pérez AL, 5, 37 Díaz Pérez C, Díaz Pérez M, 126 Domenzain Reyna C, 47, 89 Domínguez Magaña M, 178 Domínguez Malfavón L, 44 Dreyfus G, 47, 55, 89, 97, 112, 117, 145 E Eguiarte LE, 17, 28, 45, 63, 106 Enríquez Rincón F, 41 Escalante A, 27 Escobar Zepeda A, 140

Eslava Campos CA, 98, 167 Espin Ocampo EG, 50, 59, 101, 107, 111, 115 Espino García E, 151 Espinosa N, 46, 94 Espitia C, 176 Estrada Chávez C, 154 F Fabela S, 89 Fernández JL, 122 Fernández Ramírez F, 56 Fernández Rojas MA, 141 Figueroa Arredondo P, 6, 41 Flores López V, 118 Flores N, 27 Foglino M, 166 Bolívar Zapata F, 171 Freesbe A, 143 Funes S, 77 G Galán Vásquez E, 9, 53 Gama Castro S, 165 García Contreras R, 8, 43, 142 García García Silvia MC, 119 García Gómez BI, 135 García Gómez E, 46 García Gómez M, 178 García Hernández DE, 93 García López D, 90 García Maldonado JQ, 143 García Mena J, 8, 44 García Montes de Oca LY, 92 García Oliva F, 106 García RL, 43 García Rodríguez FR, 159 García Soriano DA, 144 Garza Ramos U, 90 Gaytán Enríquez M, 94 Geiger O, 49, 68, 120, 144 Georgellis D, 33, 95, 130, 170 Gingery M, 19 Giono S, 85 Girard L, 30, 60, 116

Author Index


Gómez Barreto R, 90 Gómez D, 85 Gómez Hernández N, 60 Gómez Lojero C, 83 Gómez Lunar Z, 146 Gonzales XF, 148 González O, 61 González A, 45 González Cerón G, 79, 108 González Chávez R, 95 González de la Vara LE, 92 González G, 147 González González A, 8 González Pedrajo B, 8, 46, 94, 98 González Tinoco Y, 97 González V, 122 González Valdez A, 73, 96 González Vera MA, 8, 47 Gordiano Hidalgo MA, 154 Gosset G, 27 Gottesman S, 14, 22 Greenberg P, 11, 21 Grosso Becerra V, 73 Guan Z, 68, 173 Guarneros Peña G, 31,56, 66, 84, 118, 122, 136, 158 Guo H, 19 Gutiérrez Cirlos EB, 29, 83, 92, 93 Gutiérrez Corona F, 123 Gutiérrez Lucas LR, 98, 149 Gutiérrez Preciado A, 150 Guzmán Aparicio J, 115 Guzmán J, 57, 107, 165 Guzmán Soto A, 31 H Hamdan Partida A, 99, 134, 149, 151 Hernández Chiñas U, 167 Hernández Eligio JA, 50 Hernández Flores JL, 36 Hernández Gómez MP, 152 Hernández González IL, 100, 146 Hernández Morales A, 36 Hernández Nava N, 131 Hernández Ortiz A, 101

Hernández R, 153 Hernández Sánchez J, 84, 136 Herrera Rodríguez SE, 154 Higareda de Sales LG, 154 I Ishida C, 56 Islas A, 39, 88 J Jacinto L. E, 158 Jacinto Loaeza E, 84, 136 Jaimes Miranda F, 77 Jasso Chávez R, 142 Jiménez Mejía R, 9 Jiménez R, 51 Juárez K, 40, 147 Juárez López D, 103 Juárez López K, 6 Juárez S, 122 K Kameyama Kawabe L, 66 Kameyama L, 56 Kolter R, 6, 18 Krell T, 67 L Labastida Martínez A., 155 Landeta C, 104 Lara Flores N, 156 Leyva Castillo LE, 83 Leyva Hernández E, 96 Lira Silva E, 142 Lobaina Rodríguez T, 126 Lopez Cortes A, 143 López L. G, 174 López Lara IM, 9, 49, 120, 144 López Leal G, 9, 52, 177 López Lozano NE, 106 López Pliego L, 109 López Ramírez V, 157 López Ruiz BA, 105 López Vidal Y, 148, 161, 162 Loza Tavera H, 110, 175 Lozano L, 122, 174, 175 Luna Arias JP, 41 Luna BC, 53

Author Index


M Maeda T, 43, 142 Magos C. MA, 158 Martínez Antonio A, 53, 62 Martínez Barnetche J, 90 Martínez Chavarría LC, 164 Martínez del Campo AE, 10, 55 Martínez Genis M, 102 Martínez Peñafiel E, 10, 56 Martínez Pérez A, 124 Martinez Piedragil C, 106 Martínez Ramírez A, 131 Martínez Romero E, 90 Mejía M, 59 Méndez J. C, 174 Méndez Jiménez C, 52, 177 Mendoza A, 40, 147 Mendoza Hernández G, 97, 98, 148 Merino Jiménez C, 44 Merino Pérez E, 73, 150 Miller JF, 7, 19 Miranda Molina A, 49 Monjarás J, 46 Montes García JF, 160 Mora L, 63 Morales Espinosa MR, 129 Morales Guerrero SE, 161 Morales Ruiz E, 109 Moreno González LE, 110 Moreno León S, 50, 107, 111 Moreno Ramírez L, 80 Morett E, 40, 147 Mucito Varela JE, 162 Muñiz Rascado L, 57, 165 Muriel Millán LF, 111, 115 N Navarro García PE, 176 Navarro Ocaña A, 167 Negrete Abascal E, 102, 105, 141, 160 Nogales J, 49 Núñez López CE, 78, 101, 115 Núñez Oreza LA, 130

O Ochoa TJ, 87 Olmedo Álvarez G, 39, 88, 100, 146, 157 Osorio A, 47, 89, 117 Osorio Valeriano M, 112 P Palacios Velázquez IJ, 164 Pando V, 101 Pech Canul A, 49 Pedraza Alva G, 41 Pedraza López F, 34, 113 Pedraza Reyes M, 123, 137, 139, 152 Pelayo González L, 121 Peña C, 59 Peralta Gil M, 10, 57, 165 Pereyra C, 80 Pérez Franco V, 41 Pérez Oseguera A, 114 Pérez Segura G, 34, 114 Poggio S, 47, 89, 117, 138 Ponce Y, 86 Porta Ducoing H, 135 Pu M, 43 Puente García JL, 87, 133, 155, 164 Pulido Barajas F, 178 Q Qui Zapata JA, 168 Quiñones Aguilar EE, 166, 168 Quiñones Valles C, 12, 62 Quirasco Baruch M, 140 Quiroz Martínez O, 151 Quiroz Rocha E, 115 R Ramírez Benítez G, 134 Ramírez Díaz MI, 125 Ramírez MI, 51 Ramírez Romero MA, 52, 174, 177 Ramos JL, 67 Rangel W, 43 Rebollar Caudillo EA, 12 Rebollar Eria A, 63 Rentería Solórzano I, 81, 159 Reyes Cortés R, 56

Author Index


Reyes González A, 60, 116 Reyes M, 141 Reyes Prieto M, 90 Rincón Enríquez G, 168 Rios A, 61 River P, 60 Rivera Osorio A, 117 Rivera P, 116 Rivera Urbalejo AP, 169 Robles Burgueño R, 31 Robleto EA, 139 Rocha Estrada JG, 31, 118 Rodríguez Antonio AL, 119 Rodríguez C, 122 Rodríguez Martínez C, 126 Rodríguez Rangel C, 170 Rodríguez Torres MD, 6, 39, 88 Romero D, 14, 30, 74, 104, 153 Romo Castillo M, 46 Rosales Sánchez JC, 149 Ruiz A, 142 S Sabido Ramos A, 171 Sahonero Canavesi DX, 120, 144 Sainz Espuñes TR, 98, 99, 134, 149, 151 Salinas K, 51 Sánchez Alonso MPG, 102 Sánchez Castillo J, 35 Sánchez Cuapio Z, 172 Sánchez L, 45 Sánchez Méndez JL, 129 Sánchez Ortiz V, 121 Sandoval Calderón M, 173 Santamaría RI, 30, 122 Santillán Godínez O, 52, 174, 177 Santos Balbuena H, 124 Santos Escobar F, 123 Sarmina Leonel GA, 13, 65 Segura D, 59, 107 Segura González D, 12, 111 Sepúlveda Robles OA, 13, 56, 66, 122 Servín González L, 14, 71, 73, 79, 108, 163 Sheng L, 43

Silva Jiménez H, 13, 67 Silva Sánchez J, 90, 125 Soberón Chávez G, 14, 73, 96 Soberón M, 135 Sohlenkamp C, 68, 82, 120, 153, 173 Solís González J, 175 Solís Sánchez GA, 167 Solorio Rivera JL, 81, 159 Sosa Aguirre CR, 37 Soto Guzmán A, 118 Soto MJ, 49 Souza Saldívar V, 3, 17, 28, 39, 45, 63, 88, 100, 106 Suaste Olmos F, 65, 121 T Tavares Cornejo M, 163 Tejeda V, 73 Téllez J, 90 Tinoco P, 90 Tirado AL, 40 Toledo Rojas A, 85, 124 Tomás M, 43 Torres Rodríguez D, 108 Trent S, 10, 20 Trujillo Roldán MA, 176 Tuomanen, E 148 Tzontecomani Pérez T, 156 V Vaca Pacheco S, 102, 105, 160 Vaca S, 141 Valdez Alarcón JJ, 81, 159 Valdez Cruz NA, 176 Van der Sluis EO, 77 Vázquez C, 105 Vázquez Cruz C, 102, 160 Vázquez J, 145 Vences Guzmán MA, 13, 68, 82 Venegas D, 97 Villamar T, 160 Villarreal Astorga C, 91 Villegas E, 103 Villegas Negrete N, 139 Vinuesa P, 30

Author Index


W Wood TK, 43, 142 X Xiqui ML 80 Y Yanajara Parra D, 107, 111 Yáñez FO, 74 Z Zamorano Sánchez D, 12, 60, 116 Zenteno Cuevas R, 12, 61 Zhurbenko R, 126

Program & Abstracts Second Meeting of Biochemistry and ... · Guadalupe Espín Dimitris Georgellis...

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