Comparative Genome Sequencing of Sinorhizobium medicae

1. Comparative Genome Sequencing of Sinorhizobium medicae Ruihua Shi, Graham Wiley, Fares Z. Najar, Jim White and Bruce A. Roe Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

2. Abstract Nitrogen fixation in legumes is mediated by a symbiotic relationship between Rhizobia, free-living soil bacteria, and the host plant. It is estimated that biological nitrogen fixation produces twice as much nitrogen as all non-biological processes combined. The rhizobium-legume symbiotic system historically is an ideal model system for studying symbiosis that now is benefiting from the genomic analysis of both the host plant and its symbiotic microbes. Sinorhizobium medicae is a newly discovered species of Rhizobium that was first reported in 1995. Although both Sinorhizobium medicae and Sinorhizobium meliloti infect Medicago truncatula, they differ in their host spectrum, antibiotic resistance and infection efficiency. The complete sequence of Sinorhizobium meliloti was reported in 2001, and that of Mesorhizobium loti and Bradyrhizobium japonicum also recently have been determined. We presently are sequencing the genome of Sinorhizobium medicae in an effort to determine the genomic basis for the difference between S. medicae and S. meliloti and for comparison with the other sequenced plant microbes. To date, ~115,000 sequencing reads of S. medicae have been collected, yielding ~10-fold shotgun coverage in ~70 contigs, and an estimated genome size of ~ 6.7 Mb. About 3,000 ORFs were annotated by comparison to S. meliloti. Preliminary analysis reveals that S .medicae has one Nitrogen fixation operon (fix) and a nodulation operon (nod) with conserved ORF organization observed in S. meliloti.

3. Sequencing Strategy Bacterial genome Physical shearing (hydroshear) Subcloning in pUC18 and electro- transformation into E. coli XL1blue-MRF’ Automated DNA isolation using the Hydra96 or Biomek 2000 DNA sequencing using ABI 3700 Computer-generated contig alignment using Phred-Phrap Primer-walking using large-insert clones, and MPCR. Primers generated using MerMade

4. Libraries Construction Small-insert library (initial shotgun) Hydroshear @ setting of 11 Cloning into pUC18 Large-insert library (walking clone) Hydroshear @ setting of 15 Cloning into pCC1FOS Gentle Shearing with 20 ga. syringe Fosmid library

5. Gap closure strategies Fos-End Sequencing Sequences of the Fosmid ends will be overlaid over the WGS assemble to order and orient contigs. The gap-spanning fosmid then will be picked for primer walking, Multiplex-PCR using split genes MPCR- using multiple pairs of primers Selected based on split genes between the ends of contigs to generate PCR products • Uniplex-PCR using paired reads • Pairs of primes are picked using PrimOU program based on the read pairs

6. 100% >100 Kb 90% 80% 50-100 Kb 70% 30-50 Kb 60% 50% 10-30 Kb 40% 4-10 Kb 30% 2-4 Kb 20% 10% 1-2 Kb 0% May'03 Jun'03 Jul'03 Aug'03 Sep'03 Oct'03 Nov'03 Dec'03 Jan'04 S. Medicae Sequencing Progress Size % Date

7. Overview of metabolic profile of S. medicae 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. Comparative Analysis of Nodulation-related genes in S. medicae and S. meliloti (preliminary analysis) An ACT screen shot of the nodJICBA in S. medicae compared to S. meliloti. * NodA: N-acyl-transferase * NodB: Chitooligosaccharide deacetylase * NodC: N-acetylglucosaminyl transferase * NodI: membrane transport protein * NodJ: membrane transport protein

13. Comparative Analysis of Nodulation-related genes in S. medicae and S. meliloti (preliminary analysis) • An ACT screen shot of the Fix operon in S. medicae compared to S. meliloti. Fix operon os involved in Nitrogen fixation in S. meliloti. • FixT2: transcription regulator • FixK2: transcription regulator • FixN2: cytochrome C oxidase polypeptide • FixO1: c-type cytochrome • FixG: iron sulfur membrane protein • FixH: nitrogen fixation protein

14. Conclusion • Approximately 98% of the S. medicae genome has been sequenced with ~115,000 reads • Roughly 6,500 ORFs were discovered, 2,800 of which were annotated through comparison with S. meliloti • Preliminary analysis illustrates that both S. medicae and S. meliloti share similar gene organization for nif and nod gene clusters. • Currently, we are constructing a Fosmid library to aid in the ~6.7 Mbp genome closure.

15. Blastn Result and Annotation of a selected Region in S.medicae A region spans over 75kb does not have significant with nr data base. Blastx reveals that several ORFs are similar to phage ternimase, phage protein (Lactobacillus gasseri), capsid (Shigella flexneri bacteriophage V) and three unknown proteins in Mesorhizobium loti.