Post on 22-Jan-2016
description
Brad GoodnerDepartment of Biology
Hiram College, Hiram, OH
Genomics as a Means, Not an End, to
Understanding the Biology of a “Simple”
Soil Bacterium
Genomics as a Means, Not an End, to
Understanding the Biology of a “Simple”
Soil Bacterium
HiramGenomicsInitiative
High schoolStudents
HiramStudents
Recruiting
Collaborations
Teaching Research
Chromohalobacter salexigens(w/ Purdue Univ. & DOE-JGI)
Sphingomonas elodea(w/ Monsanto Co.)
Agrobacteriumbv. 2 & 3 strains
(NSF grant w/ 7 partners) 2 Xenorhabdus species
(USDA grant w/ 6 partners)Azotobacter vinelandii
(NSF grant w/ 4 partners)
Hiram Genomics Initiative Agrobacterium Other Genome ProjectsGenome Project
Sphingomonas Chromohalobacter XenorhabdusAzotobacter
elodea salexigens bovienii & nematophila vinelandii
Functional Genomes of
Native Genomics of K84 (bv. 2)
Tumor Strain C58 & S4 (bv. 3) Genetic/ Genetic/ Genetic/Gap Genetic/
Survey (biovar 1) Physical Map Physical Map Physical MapClosure Physical Map (high (Genetics) (Genetics) (Genetics & (Independent(Genetics & schools) high
schools) Research) Independent Gap Research)
Closure (Independent Sequence
Sequence Research) Annotation Annotation
(MolCell, Genetics, (Independent
& Biochem)
Research)
Gene Mutant Gap Sequence
Disruptions Screens Closure Annotation
(MolCell & (MolCell & (Independent (Genetics &
Independent Independent Research) Independent
Research) Research) Research)
Using Research to Bridge Teaching-Learning Gaps
Within Courses
• What prevents us from incorporating original research into the lab component of courses?
• Must excite students – move into independent research projects
• Must excite us• Must teach key skills & concepts• Must be doable within time, space,
& budget constraints• Must be successful as measured
by the norms of science – effective training for the future, presentations at conferences, & publications
Genome
Random Pieces Shotgun
GenomicLibraries
6-8XSequencingCoverage
Overlaps in Small Piecesto Form Contigs
Join LargePieces intoSequenced Genome
Genetic/Physical
Map
Annotation ofContig Ends
Gap Closure
Functional Genomics
Annotation
Basics of a Genome Project
plant cell
bacterium
DNAhormones
Example of Success:Agrobacterium Genome Project
food
• Has involved >300 students within course research projects as well as in independent projects (at Hiram College & University of Richmond) since 1996• 19 student authors on publications in Journal of Bacteriology & Science• >50 student authors on >30 posters presented at research conferences• Successful involvement in collaborations with companies & larger universities
Examples I Will Cover Today
• 1) Sequence annotation by >70 students in MolCell & Genetics courses
• 2) Sucrose metabolism (Jen Hardesty, Mandy Reed, Ginny Mateo)
• 3) Aconitases (DaJuan Whiteside, Terrence Johnson, Razan Yasin, Gina Dottle, John Mark Kuhns, Torrie Ohlin, Telisha Law)
• 4) Selenite tolerance & reduction (Frank Arnold, Dan Arnold, Josh Collins)
Bioinformatic Analysis of Pathways
in C58 (bv1) & S4 (bv3)Part I = Identify players in pathway & note any absences or redundancies
12 proteins for glycolysis & gluconeogenesis
Avi5336 is most likely glucokinaseAvi0235 is likely phosphoglycerate mutase
role of phosphofructoskinase (pfk) is apparently performed by pyrophosphate--fructose-6-phosphate 1-phosphotransferase (fbp)
Agrobacterium has never been shown to have fructose 1,6-bisphosphatase and it was absent in both biovars
C58 S4Redundancies ChrI Chr2 Chr1 Chr2gpm 1 1 2glk 1 1 1pyk 1 2pck 1 2
Bioinformatic Analysis of Pathways
in C58 (bv1) & S4 (bv3)Part II – Identify potential operons
There was one potential operon in both biovars. They both involved a phosphoglycerate kinase gene and a fructose bisphosphate aldolase gene. In this instance, the genes in the operon were on ChrI of S4 but were on ChrII of the C58 genome
Part III – Identify potential lateral gene transfer events
None of our genes appear to have arrived in the genome via lateral gene transfer
Functional Uniqueness Hiding Under Redundancy
of Sucrose Metabolism • Sucrose is major transportable
form of organic carbon in plants
• Role of sucrose metabolism by Agrobacterium in interactions with plants before & after tumorigenesis is unknown
• Failure of standard genetic approach to find sucrose-nonutilizing mutants
• Enzymatic work of 60’s & 70’s suggest at least 2 routes for sucrose degradation
• Genome sequence shows that the situation is much more complicated
Putative Sucrose Metabolic Routes
OM
PM
Sucrose 3-keto-sucrose
3-keto-sucrose Sucrose
?Agl
transporter
3-keto-glucose
fructose
glucose
-glc I-glc II
Sucrose hydrolase
?
fructose + glucose
3GDH
?
?
Growth Assays on Single Gene Knockouts
Strain sucrose trehalose raffinose cellobiosepalatinosemaltuloseC58 1.25 1.19 1.13 0.84 1.08 1.25
C58 suc hyd- 0.71 0.77 0.91 0.71 0.53 0.95C58 glcI- 0.85 0.84 0.87 0.69 0.76 0.84C58 glcII- 0.78 0.72 0.86 0.65 0.71 0.71
Growth ratio compared to glucose in M9 minimal:
C58 wildtype growth in M9 minimal
00.10.20.30.40.50.60.7
1 2 3 4 5 6 7 8 9 10 11
Time (hr)
OD
600 GlucoseSucrose
C58 suc hyd- growth on M9 minimal
0
0.1
0.2
0.3
0.4
0.5
1 2 3 4 5 6 7 8 9
Time (hr)
OD
600 Glucose
Sucrose
Osmotic Stress Assays
Strain Water 0.1M NaCl0.5M NaClC58 1 1 0.77
C58 suc hyd- 1 0.02 0C58 glcI- 1 0.37 0.01C58 glcII- 1 0.63 0.01
Growth in M9glucose + 1 mM sucrose:
Strain 0.1M NaCl 0.5M NaCl 0.1M NaCl 0.5M NaCl 0.1M NaCl 0.5M NaCl 0.1M NaCl 0.5M NaCl
C58 1 0.77 0.88 1.24 0.95 0.58 0.91 0.48C58 suc hyd- 0.02 0 1.01 0.04 1 0.07 0.28 0
M9glucose+1 mM
sucrose:
M9glucose+1 mM
trehalose:
M9glucose+1 mM
glutamate:
M9glucose+1 mM
betaine:
Osmotic Adjustmentin A. tumefaciens
Smith et al., 1990, J. Bact. 172:6849-55
One Enzyme – Multiple Roles?Or
A Different Role Is All There Is?
Glucose + Fructose
Sucrose sucrose hydrolase
Mannose Mannosucrose
Glucose
Glc-II
Glc-I Suc hydSucrose
metabolismOsmotic stress
?
?
Other Genes Involved in Osmotic Adjustment
wildtype Atu4610-
Sugar nucleotide epimerase
Atu3741-
MFS-type sugar permeaseAtu1588-
Homoserine dehydrogenase
Aconitases
Many bacteria have 2
Have roles beyond the TCA Cycle
Agrobacterium bv1 is exceptional within -Proteobacteria to have >1 aconitase
AcnA’s
AcnB’s
Pfu HamA RfeA CpeA CbuA IloA PflA AviA SenA EcoA SilA RspA CcrA BquA MesA BsuA SmeA AtuA AvtA BjaA RpsA EruA WolA ParA Mav KraA ArtA DgeA Bcr Sha Sau BceA AdeA SacA FacA XcaA NmeA Dvu Hma AtuB SilB AdeB SynB NmeB MmaB BceB XcaB ParB IloB PflB AviB SenB EcoB
Actinobacteria
Firmicutes
E. coli Model for Aconitase Functions
• AcnB is major TCA isoform• AcnA is induced during stationary phase• Acn’s lose Fe-S center during Fe starvation or oxidative
stress & act as RNA-binding apo-proteins … impact gene expression
AcnB leads to hypomotility, while AcnA has normal motility
Agrobacterium C58Aconitase Gene
KnockoutsAcnA is Major Player!
Agrobacterium C58Aconitase Gene
KnockoutsAcnA is Major Player!
wildtype
AcnA-
Agrobacterium C58Aconitase Gene Knockouts
AcnA is Major Player!
Weird Pigmentation on
Certain Media (Contains Selenite)
UK1 C58
Schroth Minimal Medium
Pigmentation Due to
Strange Globules (Reduced Se?)
Strain FL Strain UK1(Silver Creek, OH) (stream bank in UK)
Pigmentation Due to Reductionof Selenite to Elemental Se
Strain C58
Strain UK1
Strain S4
Mutants Impacted inResponse to Selenite
Mutants Impaired in Response to Selenite
0
0.5
1
1.5
2
2.5
0 10 20 30 40 50 60
Time (Hours)
Absorbance (600nm)
C58 LB
C58Sel1 LB
C58Sel4 LB
C58Sel6 LB
C58Sel9 LB
C58 Se
C58Sel1 Se
C58Sel4 Se
C58Sel6 Se
C58Sel9 Se
Mutant Site of Tn InsertionC58sel1 Atu0238 thioredoxin reductase family memberC58sel6 Atu3466 ArsR TF family memberC58sel9 Atu0284 TspO (regulates response to many stresses)
HiramGenomicsInitiative
High schoolStudents
HiramStudents
Recruiting
Collaborations
Teaching Research
Chromohalobacter salexigens(w/ Purdue Univ. & DOE-JGI)
Sphingomonas elodea(w/ Monsanto Co.)
Agrobacteriumbv. 2 & 3 strains
(NSF grant w/ 7 partners) 2 Xenorhabdus species
(USDA grant w/ 6 partners)Azotobacter vinelandii
(NSF grant w/ 4 partners)
Bioinformatic Hunt for3-Ketosucrose Pathway
• Pathway is not found in Sinorhizobium, Meso-rhizobium, & Brucella
• G3DH is FAD-dependent dehydrogenase
• G3DH is periplasmic• G3DH gene near genes for
associated chemo-taxis, transport, ETC, hydrolysis, & reduction enzymes
Inferences from Literature:
• Used TIGR Comprehensive Microbial Genome Database to identify dehydrogenases unique to Agrobacterium
• 2 appeared to be FAD-dependent
• 1 appeared periplasmic• Nearby genes for MCP,
cytochrome, & reductase• Mutant is still G3DH+!
• Now have G3DH- mutants from large-scale random screen … implicate 2 separate ABC-type sugar transport systems
Bioinformatic Analysis of C58:
Diversity of Responses to Selenite
A. C58
0.010
0.100
1.000
0 10 20 30 40 50 60
Time (hours)
Cell Density (OD600)
B. UK1
0.010
0.100
1.000
0 10 20 30 40 50 60
Time (hours)
Cell Density (OD600)