Sequencing The Sinorhizobium medicae Genome

1. Sequencing The Sinorhizobium medicae Genome Ruihua Shi, Fares Z Najar, Hongshing Lai, Axin Hua, and Bruce A. Roe Department of Chemistry and Biochemistry, Stephenson Research and Technology Center, University of Oklahoma, 101 David L. Boren Blvd, Norman, Oklahoma, 73019

2. Abstract We presently are sequencing the genome of Sinorhizobium medicae, a symbiont of alfalfa, closely related to Sinorhizobium meliloti, with a different host spectrum, antibiotic resistance and infection efficiency, in an effort to determine the genomic basis for both the common features and the differences between the sequenced nitrogen fixing bacteria. To date, ~117,000 end-paired shotgun sequencing reads have been collected from a small insert (2-4 Kbp) plasmid library, yielding ~10-fold shotgun coverage and an estimated genome size of ~6.7 Kb. A large insert (5-8 Kb) pUC18 library and a fosmid library (40 Kb) now have been constructed for primer walking to close the remaining approximately two dozen gaps. 6280 genes have been predicted by FgenesB, 5900 of which have significant homology in the GenBank nr-protein database, and include 5,500 genes with significant homology in S. meliloti, 4,500 genes in Mesorhizobium loti, and 4,200 genes in Bradyrhizobium japanicum. For the ~700 predicted genes that do not have homology with S. meliloti, about 50 genes are transposases similar to those in Mesorhizobium loti, Nostoc punctiforme, Burkholderia fungorum, Bradyrhizobium japanicum, Rhizobium sp. NGR234 and Brucella suis. And 12 predicted conjugal transfer proteins with orthologs in Agrobacterium tumefaciens. Of the approximately 400 genes that are unique to S. medicae, at least 55 are predicted as membrane proteins and at least 50 have a signal peptide sequence. The results of further comparative analysis of these genomes also will be discussed.

3. Sequencing Strategy Bacterial genome Physical shearing (hydroshear) Subcloning in pUC18 and electro- transformation into E. coli XL1blue-MRF’ Automated DNA isolation using the Vprep and Zymark DNA sequencing using ABI 3700 Computer-generated contig aligment using Phred-Phrap Primer-walking using large-insert clones, and MPCR. Primers generated using MerMade

4. Library Construction Hydroshear @ setting of 11 Small-insert library (initial shotgun) Cloning into pUC18 Large-insert library (walking clone) Hydroshear @ setting of 15 Cloning into pCC1FOS Fosmid library Gentle Shearing with syringe

5. Exgap Presentation of Shotgun and Gap Closure Result • ~117,000 paired shotgun reads • Average length for each read: 602 bp • 21 contigs • ~10-fold coverage • Estimated genome size of ~6.7 Mb

6. Preliminary Annotation Results • 2 sets of rRNA genes • 50 tRNA genes corresponding to each of the 20 amino acids • ~6,280 protein coding genes • ~5,900 genes have homology in Genbank • ~380 genes are unique of which 55 genes are predicted as membrane proteins of which 50 have a signal peptide sequence

7. Overview of metabolic profile of S. medicae genes Metabolism 26% Poorly Characterized 35% Cellular Processes 15% Multiple cog hits 10% DNA/RNA Metabolism 14%

8. DNA/RNA metabolism of S. medicae DNA replication, recombination & repair 21% Translation, ribosomal structure & biogenesis 25% Transcription 54%

9. Cellular Processes Cell motility 20% Inorganic ion transport & metabolism 21% Signal transduction mechanisms 16% Cell wall / membrane / envelope biogenesis 21% Cell cycle control, cell division, chromosome partitioning 4% Posttranslational modification, protein turnover, chaperones 18%

10. Metabolism Carbohydrate transport & metabolism 27% Amino acid transport & metabolism 31% Energy production & conversion 19% Nucleotide transport & metabolism 6% Secondary metabolites biosynthesis, transport & catabolism 6% Coenzyme transport & metabolism 11%

11. Poorly Characterized General function prediction only 24% Unknown Function 76%

12. DotPlot Comparison of the S. medicae and S. meliloti Genomes S. medicae S. meliloti

13. DotPlot Comparison of the S. medicae and Bradyrhizobium japonicum Genomes S. medicae B. japonicum

14. DotPlot Comparison of the S. medicae and Mesorhizobium loti Genomes S. medicae Mesorhizobium loti

15. Comparison with Other 3 Sequenced Rhizobia • 6280 S. medicae genes • Homology in other Rhizobia: S. meliloti: 5500 Mesorhizobium loti: 4500 Bradyrhizobium japonicum : 4100

16. Comparison with Other 3 Sequenced Rhizobia

17. Comparison of S. medicae Nitrogen Fixation Region with Other 3 Rhizobia Main window Blastp nr-protein DB Blastp S. meliloti Blastp M. loti Blastp B. japonicum

18. Conclusions • Because the S. medicae and S. meliloti genomes have both highly conserved and unique regions, it is very likely that they have a common ancestor. • Many of the unique characteristics and the specific biological niche for these two Sinorhizobia result from difference in the genes present in their symbiotic islands (Nod, Nif and Fix genes), their different type IV secretion systems, as well as difference in their exoproteins and multidrug-efflux transporters.