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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. Abstract.

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sequencing the sinorhizobium medicae genome

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

abstract
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.

slide3

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

slide4

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

exgap presentation of shotgun and gap closure result
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
preliminary annotation results
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

slide7

Overview of metabolic profile of S. medicae genes

Metabolism

26%

Poorly Characterized

35%

Cellular Processes

15%

Multiple cog hits

10%

DNA/RNA Metabolism 14%

slide8

DNA/RNA metabolism of S. medicae

DNA replication, recombination & repair 21%

Translation, ribosomal structure & biogenesis 25%

Transcription 54%

slide9

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%

slide10

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%

slide11

Poorly Characterized

General function prediction only 24%

Unknown Function

76%

dotplot comparison of the s medicae and bradyrhizobium japonicum genomes
DotPlot Comparison of the S. medicae and Bradyrhizobium japonicum Genomes

S. medicae

B. japonicum

comparison with other 3 sequenced rhizobia
Comparison with Other 3 Sequenced Rhizobia
  • 6280 S. medicae genes
  • Homology in other Rhizobia:

S. meliloti: 5500

Mesorhizobium loti: 4500

Bradyrhizobium japonicum : 4100

slide17

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

conclusions
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.