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

2. Hiram Genomics Initiative High school Students Hiram Students Recruiting Collaborations TeachingResearch Chromohalobacter salexigens (w/ Purdue Univ. & DOE-JGI) Sphingomonas elodea (w/ Monsanto Co.) 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)

3. Hiram Genomics Initiative Agrobacterium Other Genome Projects Genome Project SphingomonasChromohalobacter XenorhabdusAzotobacter elodeasalexigens bovienii & nematophila vinelandii Functional Genomes of NativeGenomics of K84 (bv. 2) Tumor Strain C58 & S4 (bv. 3)Genetic/Genetic/Genetic/GapGenetic/ Survey (biovar 1) Physical MapPhysical MapPhysical 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) GeneMutant Gap Sequence DisruptionsScreens Closure Annotation (MolCell & (MolCell &(Independent (Genetics & Independent Independent Research) Independent Research) Research) Research)

4. 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

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

6. plant cell bacterium DNA hormones 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

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 ChrI Chr2 Chr1 Chr2 gpm1 12 glk11 1 pyk12 pck12

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 OM PM Sucrose 3-keto-sucrose Sucrose ? Agl transporter 3-keto- glucose fructose glucose  -glc I  -glc II Sucrose hydrolase ? fructose + glucose 3GDH ? ?

12. Growth Assays on Single Gene Knockouts

13. Osmotic Stress Assays

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

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

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

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

18. 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       Actinobacteria Firmicutes           

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  AcnB leads to hypomotility, while  AcnA has normal motility

20. Agrobacterium C58 Aconitase Gene Knockouts AcnA is Major Player!

21. wildtype AcnA -

22. Agrobacterium C58 Aconitase Gene Knockouts AcnA is Major Player!

23. Weird Pigmentation on Certain Media (Contains Selenite) UK1 C58 Schroth Minimal Medium

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

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

26. Mutants Impacted in Response to Selenite MutantSite of Tn Insertion C58sel1Atu0238 thioredoxin reductase family member C58sel6Atu3466 ArsR TF family member C58sel9Atu0284 TspO (regulates response to many stresses)

27. Hiram Genomics Initiative High school Students Hiram Students Recruiting Collaborations TeachingResearch Chromohalobacter salexigens (w/ Purdue Univ. & DOE-JGI) Sphingomonas elodea (w/ Monsanto Co.) 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)

28. Bioinformatic Hunt for 3-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:

29. Diversity of Responses to Selenite