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)