Genomics as a Means, Not an End, to Understanding the Biology of a “Simple” Soil Bacterium

1. Genomics as a Means, Not an End, to Understanding the Biology of a “Simple” Soil Bacterium Brad Goodner Department of Biology Hiram College, Hiram, OH

2. Agrobacterium bv. 2 & 3 strains (NSF grant w/ 7 partners) 2 Xenorhabdus species (USDA grant w/ 6 partners) Azotobacter vinelandii (NSF grant w/ 4 partners) Teaching Research Collaborations Hiram Students High school Students Recruiting HiramGenomicsInitiative Chromohalobacter salexigens (w/Purdue Univ. & DOE-JGI) Sphingomonas elodea (w/Monsanto Co.)

3. Hiram Genomics Initiative Agrobacterium Other Genome Projects Genome Project Sphingomonas Chromohalobacter Xenorhabdus Azotobacter elodeasalexigensbovienii& 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 Map Closure Physical Map (high(Genetics) (Genetics)(Genetics & (Independent (Genetics & schools)high schools) Research) IndependentGap 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)

4. 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 Using Research to Bridge Teaching-Learning Gaps Within Courses

5. Overlaps in Small Pieces to Form Contigs 6-8X Sequencing Coverage Genome Genetic/ PhysicalMap Gap Closure Join Large Pieces into Sequenced Genome Random Pieces Annotation of Contig Ends Shotgun Genomic Libraries Annotation Functional Genomics Basics of a Genome Project

6. Example of Success: Agrobacterium Genome Project bacterium hormones DNA food plant cell • 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

7. 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)

8. 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 glucokinase Avi0235 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 S4 Redundancies ChrIChr2Chr1Chr2 gpm 1 1 2 glk 1 1 1 pyk 1 2 pck 1 2

9. 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

10. 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

11. Putative Sucrose Metabolic Routes 3GDH Sucrose 3-keto-sucrose OM Agltransporter ? ? PM Sucrose 3-keto-sucrose ? Sucrose hydrolase 3-keto-glucose fructose a-glc II a-glc I ? fructose + glucose glucose

12. Growth Assays on Single Gene Knockouts

13. Osmotic Stress Assays

14. Osmotic Adjustmentin A. tumefaciens Smith et al., 1990, J. Bact. 172:6849-55

15. One Enzyme – Multiple Roles?OrA Different Role Is All There Is? Glucose + Fructose Sucrose sucrose hydrolase Mannose Mannosucrose Glucose

16. Glc-I Suc hyd Glc-II Sucrose metabolism ? Osmotic stress ?

17. Other Genes Involved in Osmotic Adjustment Atu4610- Sugar nucleotide epimerase wildtype Atu3741- MFS-type sugar permease Atu1588- Homoserine dehydrogenase

18. a a g a a Actinobacteria Firmicutes b d g b d a d b a b g g AconitasesMany bacteria have 2Have roles beyond the TCA CycleAgrobacterium bv1 is exceptional withina-Proteobacteria to have >1 aconitase AcnA’s AcnB’s

19. 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 • DAcnB leads to hypomotility, while DAcnA has normal motility

20. Agrobacterium C58Aconitase Gene KnockoutsAcnA is Major Player!

21. Agrobacterium C58Aconitase Gene KnockoutsAcnA is Major Player! wildtype AcnA-

22. Agrobacterium C58Aconitase Gene KnockoutsAcnA is Major Player!

23. Weird Pigmentation on Certain Media (Contains Selenite) UK1 C58Schroth Minimal Medium

24. Pigmentation Due to Strange Globules (Reduced Se?) Strain FL Strain UK1 (Silver Creek, OH) (stream bank in UK)

25. Pigmentation Due to Reductionof Selenite to Elemental Se Strain C58 Strain S4 Strain UK1

26. Mutants Impacted inResponse to Selenite Mutant Site of Tn Insertion C58sel1 Atu0238 thioredoxin reductase family member C58sel6 Atu3466 ArsR TF family member C58sel9 Atu0284 TspO (regulates response to many stresses)

27. Agrobacterium bv. 2 & 3 strains (NSF grant w/ 7 partners) 2 Xenorhabdus species (USDA grant w/ 6 partners) Azotobacter vinelandii (NSF grant w/ 4 partners) Teaching Research Collaborations Hiram Students High school Students Recruiting HiramGenomicsInitiative Chromohalobacter salexigens (w/Purdue Univ. & DOE-JGI) Sphingomonas elodea (w/Monsanto Co.)

28. 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 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 Hunt for3-Ketosucrose Pathway Inferences from Literature: Bioinformatic Analysis of C58:

29. Diversity of Responses to Selenite