N E W S L E T T E R O F F E M S F E D E R AT I O N O F E U RO P E A N M I C RO B I O L O G I C A L S O C I E T I E S

With the launch of the first synthetic genome in May this year, it makes one realize how far and even farther science can reach. Indeed, in this discipline, one’s imagination is the only possible limitation. Faced with all this, the innate sentimentality of man suggests looking back and contemplating on how life on Earth arose and how evolution has developed, also generating the cur-rent plethora of microbial species. With this in mind, FEMS Focus de-cided to take a step back and trace the scientific Tree of Life, also known as the Phylogenetic Tree or Evolutionary Tree. This concept relies on the notion that all life on Earth is connected and could have come from a single origin. More than a century ago, it was Charles Darwin himself who claimed that all modern species evolved from a limited set of ancestors, which existed from forefathers of a lesser number. The consequent implications for microbial evolution, phylogeny and systematics are facts that FEMS Focus would like to elucidate. We hope that you will not only join us in this issue, but enjoy it as well. For what’s more interesting than knowing the frontiers of science, but understanding the origin of it all?

Tone Tønjum & Chared Verschuur

Editors

To add expert views on this subject and the consequences for microbial phylo-geny and systematics, FEMS Focus in-terviewed two authorities in the field, Prof Karl Heinz Schleifer and Prof Mil-ton da Costa. Schleifer has focused on the identification and classification of bacteria since the beginning of his sci-entific career. He received the FEMS-Lwoff Award at the 3rd FEMS Congress in Gothenburg, Sweden 2009. Da

September 2010 No.8

When we consult science in matters of evolution, it points to a common ancestor branching out to different organisms which eventually leads to us, man. Charles Darwin proposed this in his “Origin of the Species”, saying, “There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one”.

Defining the microbial terrain

From the Editorial Team

Costa, on the other hand, is studying biodiversity in specialized niches such as deep sea brine and is the outgoing Presi-dent of FEMS. Because of their work, it is possible today to detect and identify bacteria in waste water, food, clinical specimens and environmental samples without first cultivating them in a labo-ratory – thereby acquiring a whole new insight into the microbial world.

subsequent phylogenetic analysis. There is also good agreement between the topology of phy-logenetic trees derived from conserved, mostly informational genes (involved in translation or transcription). Moreover, trees inferred from whole genome data are largely consistent with rRNA-derived trees.

Milton da Costa (MDC): SSU change slowly over time, with distinct conserved sequences, ena-

Here’s what they have to say about the Phylogenetic Tree of Life:

What is the nature of the molecular clock used as a basis for 16S rRNA gene-based phylogeny of bacteria and shaping the Tree of life?

Karl Heinz Schleifer (KHS): Small subu-nit (SSU; 16S/18S) rRNA genes fulfill

all properties of a useful phylogenetic marker. They are present in all living organisms, functionally constant, suf-ficiently conserved and orthologous markers from acommon ancestor. rRNA genes are rather easy to sequence and align. SSU rRNAs are stable in character, are less subjected to lateral gene transfer than other genes and can be employed for comparative sequence analysis and

Haloplasma contractile from a brine-filled deep of the Red Sea. Credits: André Antunes, Fred Rainey, Robert Huber and Milton da Costa.

TREE OF LIFE POSTER INSIDE

bling their use as a molecular clock and ba-sis for phylogeny. How fast/slowly species evolve, and how useful SSU rRNA genes are as a molecular clock, depend on the phylum of the organism. How has this revolutionalised classification, taxonomy and identification of bacteria?

KHS: Bacterial and archaeal taxonomy at the higher ranks are based on SSU rRNA se-quence data1. The road map of Bergey’s Man-ual of Systematic Bacteriology is also based on SSU rRNA. However, we have to be aware that the conserved status of SSU rRNA is usu-ally not sufficient to distinguish bacteria at the species level. It is now also possible to identify bacteria prior to their cultivation by applying rRNA-targeted oligonucleotide probes2. MDC: The SSU-based phylogeny represents a gold-mine for systematic taxonomy for defin-ing novel species and entities that are discov-ered in various niches.

How stable and variable is this target? Are there alternative or new targets coming up that you would recommend?

KHS: SSU rRNA is a rather stable and con-served marker. Large sub-unit (LSU) rRNA may even be better because it contains more and longer stretches of informative positions. However, the LSU database is by far not as large as that of SSU rRNA. There are some protein-coding genes (RNA polymerases, initiation and elongation fac-tors in protein synthesis, DNA gyrases, some heat shock proteins) but none of them is as well-studied (rather small data-sets) as rRNAs3,4. MDC: Although the redundancy of SSU sequences in some phyla/genera such as Actinobacteria/Mycobacteria and Thermus are a limiting factor, the opportunities for resolution of other entities are enormous. One way to combat this problem is to use multi-locus sequence analysis (MLSA)

of multiple relevant house-keeping genes (HKG). However, bacterial systematics must be based on a combination of se-quence comparisons with metabolic reac-tions of HKG products, lipids/fatty acids and bacterial morphology and physiology. How did you enter this field of science?

KHS: I started my ca-reer by designing a simple method to determine the primary struc-ture of pep-tidoglycan. Using this method it could be s h o w n that gram-pos i t i v e bac t e r i a c o n t a i n different p e p t i -doglycan types and represent a valuable chemotaxo-n o m i c m a r k e r . Together with my

former PhD ad-visor Otto Kan-dler, I published a review on this topic in 19725, which is one of t h e most cited article in the field of microbiology. After my appointment as head of the depart-ment of microbiology at the TUM in 1974, I set up a research group on the taxonomy of gram-positive bacteria applying chemo-taxonomic, biochemical, immunological and genotypic methods (DNA-DNA hybridiza-tion, rRNA-DNA hybridization). In 1979, Erko Stackebrandt joined our research group and together with Wolfgang Ludwig as PhD-student, 16S rRNA cataloging and sequenc-ing was launched. Later on, our group con-centrated on molecular microbial ecology by applying labeled rRNA-targeted oligonucle-otides to detect and identify in situ bacteria prior to their cultivation1, 6-10.

Selected references

MDC: I was introduced to

science at the young age of 5 by my father during a trip

to the zoo. I loved animals right away and took a bachelor and

master in Zoology including snake phylogeny (1966-68) at the University

of Michigan and the University of Arizona in the US. Then I wanted to develop stud-ies on bacterial taxonomy by employing my experience in multi-locus enzyme electro-phoresis (MLEE) at the University of Indi-ana, where I received my Ph.D. in 1978. At that time, I was recruited as a young profes-sor to the University of Coimbra in Portugal, my native country. Since then I have pursued

my interests on biodiversity by systematics/taxonomy, phylogeny, physiology and bio-chemistry of microorganisms from extreme environments and various niches. This has brought me to many exciting places and dis-

coveries. Among the highlights of my work, the discovery of Haloplasma species and de-lineating of the MDAC genes in Legionella pneumophila are the most recent11. What is the newest cutting edge finding in this field?

KHS: Current bacterial taxonomy is based on type strains only, but these are not always the most representative strains. The All-Species Living tree project deals with reconstruction of a single SSU rRNA tree comprising all type strains of Bacteria and Archaea 12. It is based on the following prerequisites:a) Curated SSU rRNA database of all type strains will be generatedb) ARB and SILVA databases and programmes are appliedc) Maximum likelihood are used for tree re- construction

Another important contribution to the field is bacterial characterization and identifica-tion by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrosco-py (MALDI-TOF), a rapid high-throughput method for measuring peptides and other components. MDC: Current discoveries on the biodiversity of microorganisms from extreme environ-ments and various niches include findings on the anaerobic/aerobic interphase in deep sea brine, bore-holes for fresh water and detect-ing contaminants in stem cells from umbili-cal cords.

Prof. Dr. Karl-Heinz Schleifer was Professor of microbiology and Head of Department of Microbiology at Technische Universität München (TUM), Germany and now TUM Emeritus of Excellence. He has focused on the identification and classifica-tion of bacteria since the beginning of his career and was, after his postdoctoral work at Rockefeller University, New York 1969-70, the first academic in Germany to develop the foundation of microbial hybridisation probe analysis. From the US, he was recruited as Assistant Professor (1971) and in 1974 as professor and Head of Microbiology, TUM. His ideas and visions have heavily influenced not only the results of numerous German Research Foundation Collaborative Research Centres and EU research projects, but also the work of his students. Schleifer has made seminal contributions to the field of biology with his exceptional research in microbiology, molecular biology and microbial ecology. He received the Körber European Science Award in 1995 for his scientific achievements. In 2001, he is a frequently-cited scientist and was designated a “highly-cited researcher” by the renowned Institute for Scientific Informa-tion (ISI). From 1986 to 1994, he was the Secretary General of FEMS. From 2005 to 2008, he was president of the International Union of Microbiological Societies (IUMS).

Prof. Milton S. da Costa is Professor of Microbiology and Head of the Dept. of Biochemistry, the Faculty of Sciences and Technology, University of Coimbra, Portugal. He obtained a bachelor and master in Zoology including snake phylogeny (1966-68) at the University of Michigan and the University of Arizona in the USA. From 1971, he studied at the University of Indiana, USA pursuing his graduate studies on bacterial taxonomy em-ploying multi-locus enzyme electrophoretic (MLEE) and was awarded his Ph.D. from University of Indiana in 1978. He was recruited as a young professor at the Uni-versity of Coimbra in Portugal, from where he originated. Professor Milton S. da Costa’s scientific interests are in the areas of the taxonomy, phylogeny, physiology and bio-chemistry of microorganisms from extreme environments. He pursued his discovery and characterization of new bacterial enti-ties, including anarobes, in environmental sources including fresh waters and deep sea brine. Da Costa has a most productive ca-reer with a publication record of more than 150 published works. He is the President of FEMS from 2008 to 2010.

1. Amann, R., W. Ludwig and K.H. Schleifer. Microbiol. Rev. 59:143-169, 1995 2. Woese, C.R. Bacterial evolution. Microbiol Rev. 51: 221–271, 1987 3. Ludwig, W. and K.H. Schleifer. ASM News 65:752-757, 1999 4. Ludwig, W. and K.H. Schleifer. Microbial Phylogeny and Evolution. Concepts and Con- troversies. J. Sapp ed. Pp. 70-98, Oxford University Press 2005 5. Ludwig, W. et al. Electrophoresis 19:554-568, 1998 6. Ludwig, W. Nucl. Acids Res. 32:1363-1371, 2004 7. Richter, M. and R. Rossello-Mora. Proc. Natl. Acad. Sci. US 106:19126-131, 2009 8. Schleifer, K.H. Syst. Appl. Microbiol. 32: 533-542, 2009 9. Schleifer, K.H. & O. Kandler. Bacteriol. Rev. 36, 407-477, 1972 10. Schleifer, K.H. & E. Stackebrandt. Ann. Rev. Microbiol. 143-187, 1983 11. Costa J, Tiago I, da Costa MS, Veríssimo A. Environ Microbiol. PMID: 20482739, May 13, 2010 12. Yarza ,P. et al. The All-Species Living Tree Project, Syst. Appl. Microbiol. 31, 241-250, 2008

• Incomplete or lowqualitysequencesrendering phylogeneticreconstructionsdifficultorimpossible• Sequencesoftendepositedunderincorrect species names , lack of strain informationormisassigned accession numbers.Therearealsomultipleentries (sequencedifferences!)forasingletypestrain• Correct alignmentrequiresnotonlytheprimarygene sequencebutalsosecondary structureinformation.• Tree reconstructionshouldbeperformedbyusing standardized methods.

Problems with 16S rRNA based classification

• rRNAsarestable markers.Theyarelesssubjectedto lateral gene transfer.• Good congruence of branching patternofphyloge netictreesderivedfromconservedmostlyinforma- tionalgenesinvolvedintranslation(e.g.rRNAs,EF-Tu) andtranscription(rpoB,rpoC),respectively• GenomebasedstudiesareconsistentwiththerRNA data• Facilitateidentificationofuncultivatedprokaryotes

Strength of rRNA based classification

Tree of Life image © 2007 Tree of Life Web Project. Image of rose © 1999 Nick Kurzenko. Image of annelid worm © 2001 Greg W. Rouse.

The FEMS Focus is published by the FEMS Central Office. Whom to contact? Prof. Dr. Tone Tønjum (tone.tonjum@medisin.uio.no).Design & production: Zak Princic FEMS is a registered charity (no. 1072117) and also a company limited by guarantee (no. 3565643). © 2010 Federation of European Microbiological Societies

FEMS Central OfficeKeverling Buismanweg 4 2628 CL Delft The NetherlandsTel: +31-15-269 3920 Fax: +31-15-269 3921E-mail: fems@fems-microbiology.org

Online subscription to the full set of FEMS Journals for

David P. Fewer M.Sc. Ph. D. was chosen for the next FEMS-ESCMID joint fellowship award on the side of FEMS. The 35-year-old microbiologist hails from Ireland and currently works at the Department of Ap-

plied Chemistry and Microbiology at the University of Helsinki. He has published 21 scientific papers in 16 international journals in 2002. These papers have been cited 190 times with an average citation of

FEMS Grants board selects next FEMS-ESCMID joint fellowship awardee

http://tolweb.org/tree/http://arb-silva.de/living-treehttp://en.wikipedia.org/wiki/Tree_of_life_(science) http://en.wikipedia.org/wiki/Phylogenetic_tree http://www.open2.net/darwin/treeoflife/index.html http://www.sciencemag.org/feature/data/tol/ http://www.evogeneao.com/tree.html http://www.wellcometreeoflife.org/ http://www.bbc.co.uk/darwin/ http://www.open2.net/life/tree_launcher.html http://www.fossilmuseum.net/TreeOfLife.htm http://www.guardian.co.uk/science/2009/jan/21/charles-darwin-evolution-species-tree-life http://rdp.cme.msu.edu

Tree of Life Links and Resources

10.94 per article. Fewer’s research is titled, “Mining the Nodularia spumigena genome for new natural products and genes encod-ing biosynthetic pathways.” The comple-tion of his research will involve a stay in the laboratory of Prof. Enrique Flores at the University of Seville in Spain. Prof. Flores is one of the foremost cyanobacte-rial molecular biologists of our time. #

Thematic issue now available

FEMS

Grants Deadlines

FEMS

Advanced Fellowship

October 1, 2010

FEMS Research Fellowship

FEMS

Visiting Scientist Grants

December 1, 2010

www.fems-microbiology.org

Register as a

Affiliate

Bacteria “stick” (single cell; no nucleus)

Archaea “old” (single cell; no nucleus)

dinoflagellates “terrible whip” [protozoa]

ciliates [protozoa]

brown algae, kelp (Phaeophyceae)

diatoms “cut in two” [algae; plankton]

radiolarians “small sunbeam” [protozoa]

foraminiferans “hole bearers” [plankton]

green algae (Chlorophyta)

mosses (Bryophyta)

liverworts (Marchantiophyta)

ferns (Filicophyta)

cycads (Cycadophyta) [seeds]

conifers (Coniferae) [cones]

rosids

asterids [most flowers]

cacti (Cactaceae)

poppies (Papaveraceae)

laurels (Lauraceae)

magnolias (Magnolia)

lilies (Lilium)

orchids, irises (Asparagales)

palms (Palmae)

grasses (Graminae)

red algae (Rhodophyta) [some seaweeds]

amoeba (Amoebazoa)

slime molds (Mycetozoa)

fungi [yeasts, molds, mushrooms]

comb jellies (Ctenophora “comb bearer”)

sponges (Hexactinellid, Calcarea)

corals, anenomes (Anthozoa)

jellyfish (Scyphozoa)

spiders (Araneae)

mites, ticks (Acarina)

scorpions (Scorpiones)

horseshoe crabs (Xiphosura)

barnacles (Cirripedia “curl footed”)

copepods, krill

crabs, lobster, shrimp (Decapods “ten footed”)

millipedes, centipedes (Myriapoda “many feet”)

dragonflies (Odonata)

cockroaches (Blattodea)

termites (Isoptera “equal wing”)

grasshoppers (Orthoptera “straight wing”)

true bugs, cicada, aphid (Hemiptera “half wing”)

beetles (Coleoptera “sheath wing”)

ants, bees, wasps (Hymenoptera “membrane wing”)

fleas (Siphonaptera)

flies (Diptera “two wing”)

butterflies, moths (Lepidoptera “scale wing”)

roundworms (Nematoda “thread like”)

flatworms (Platyhelminthies ”flat-worm”)

earthworms, leeches (Annelids “little ring”)

clams (Bivalvia “two door”)

squid, octopus (Cephalopods “head foot”)

snails (Gastropods “stomach foot”)

sea cucumbers (Holothurians)

sea urchins (Echinoids “spiny”)

starfish (Asteroidea “star-like”)

sea squirts; tunicates (Urochordates)

lancelets (Cephalochordata)

hagfish ( Myxinoids)

lampreys (Petromyzontids)

sharks, rays (Chondrichthyes “cartilage fish”)

perches, silversides (Percomorphs)

salmon, smelts (Protacanthopterygii)

minnows, catfish (Ostariophysans)

eels, morays (Elopomorphs)

coelacanths

lungfish (Dipnoi “two breath”)

frogs (Anura ”no tail”)

salamanders, newts (Urodela ”tail visible”)

turtles, tortises (Testudines)

snakes (Serpentes)

lizards (Lacertilia)

iguanas, chameleons (Iguania)

crocodiles, alligators (Crocodilia)

chickens, ducks (Galloanserae)

birds, penguins

platypus, echidna (Monotremata “single hole”)

kangaroos, opossums (Marsupials “pouch”)

rats, mice, hamsters (Rodentia)

humans, apes, gorillas (Primates “first”)

bats (Chiroptera “hand wing”)

whales, dolphins (Cetacea “whale”)

pigs, cows, goats, sheep (even-toed Ungulates)

horses, rhinoceros, tapirs (odd-toed Ungulates)

dogs, cats, seals (Carnivora “flesh devour”)

Bilateral symetry (not radial)

Animalia(Metazoa ”beyond

animals”)Eat other organisms

Green Plants

Enclosed SeedsAngiosperms

“receptacle-seed”

Plants

Dinosauria

Amphibians

Mammalia

Cell Nucleus and Mitochondria

(Eukarya) “good kernel”

Eutheria (Placenta)

Egg-laying

Reptilia

Lungs

Jaws(Gnathostomes)

Vertebrates

Four-legged(Tetrapods)

Amniotic Egg

Ray-Finned Fish (Actinopterygii)

Monocots

First Life-form

Arthropods(”joint-foot”)

Opisthokonta

Dicots

Eudicots

Gill slits and notochord

(Chordata)

Second openingin cell cluster

becomes mouth (Deuterostomes)

Tree of Life

VascularPlants

(xylem)

Plants

Mam

mals

Arthropods

Arachnids

(8 legs)

Crustaceans

Insects(6 legs)

Reptiles

and Birds

Echinoderms “spiny skin”

Trilobites (extinct) “three lobed”

Had

ean

Arc

hean

Pro

tero

zoic

Cam

bria

n O

rdov

icia

n S

iluria

n D

evon

ian

Car

boni

fero

us P

erm

ian

Tria

ssic

Jura

ssic

Cre

tace

ous

Cen

ozoi

c

BonySkeleton

Bryophytes

Aves

Embryophytes

First opening in cell cluster becomes mouth(Protostomes)

V3.8 copyright

Neal Olander tellapallet.com

Cellular organisms without cell nuclei are Prokaryotes (”before kernel”)

.

This diagram is a cladogram, a tree-like picture showing how organisms are related. Each sub-tree in a cladogram is called a clade, such as mammals, animals, amphibians. Most branches in a cladogram should split into two sub-trees, but for

simplicity this picture has some branches that split into three. Extinct species are represented as dead-end branches. This cladogram is a high-level overview and does not show individual species. Each clade is defined by a distinguishing

characteristic that sets it apart from neighboring clades. For example, tetrapods have 4 legs. Sometimes that characteristic disappears in later organisms, for example: snakes are in the tetrapod clade, but no longer have legs. Some well-known

groups of organisms are not clades - including reptiles, protists, fish, invertebrates, sponges, and prokaryotes - because they do not include all descendents of the most recent common ancestor.

Fish

Seeds (Spermatophytes)

Organs(Eumetazoa “good

animals”)

Molluscs

External Skeleton

Synapsida

Rhizaria

Unikonta

Chromalveolates

Theria

Meta-theria

Cnidaria(radial symmetry)

Sauria

4.6 3.8 2.5 542 488 443 416 359 299 251 199 145 65BYA BYA BYA MYA MYA MYA MYA MYA MYA MYA MYA MYA MYA

Prokaryotes

The Phylogenetic Tree of Life: Small subunit rRNA genes fulfill all properties of a useful phylogenetic marker. They are present in all living organisms, functionally constant, sufficiently conserved orthologous markers from a common ancestor.