Genome Sequence of the Symbiotic Type Strain Rhizobium tibeticum CCBAU85039T

ABSTRACT Rhizobium tibeticum was originally isolated from root nodules of Trigonella archiducis-nicolai grown in Tibet, China. This species is also able to nodulate Medicago sativa and Phaseolus vulgaris. The whole-genome sequence of the type strain, R. tibeticum CCBAU85039T, is reported in this study.

A vailability of nitrogen in soils is one of the main concerns for crop cultivation in agriculture. Nitrogen is essential for the biosynthesis of proteins, amino acids, vitamins, and other compounds. Chemical fertilizers commonly ensure sufficient nitrogen availability for crop production. However, these compounds may also cause environmental problems (1,2). Alternatively, certain bacteria and archaea are able to fix atmospheric dinitrogen via reduction to ammonia. Rhizobia belonging to the classes Alphaproteobacteria or Betaproteobacteria inhabit soils and are able to enter into nitrogen-fixing symbiosis with leguminous plants. They induce the development of root nodules, where, after colonization and differentiation, bacteroids are able to fix dinitrogen (3). Rhizobia are highly diverse regarding their genetic, metabolic, and taxonomic characteristics (4). Mesorhizobium loti MAFF303099 (5) and Ensifer meliloti 1021 (6) were the first rhizobia for which complete genome sequences became available. Recently, the genome sequences of 163 further root-nodule bacteria were published, extending our knowledge regarding this group of bacteria (7). However, particular and important species/groups were not covered by the study cited above. An important clade within the group of rhizobia comprises the strains Rhizobium mesoamericanum CCG502 T (8), Rhizobium grahamii CCG501 T (9), Rhizobium favelukesii LPU83 T (10), and Rhizobium tibeticum CCBAU85039 T (11). While genome sequence information is available for the first three strains, the genome of R. tibeticum CCBAU85039 T had not yet been sequenced. The latter strain is able to nodulate Phaseolus vulgaris, Medicago lupulina, Medicago sativa, Trigonella archiducis-nicolai, and Trigonella foenum-graecum. With the objective to uncover mechanisms of rhizobial diversification and to complement genome sequence information for rhizobial species, the R. tibeticum CCBAU85039 T genome was sequenced.
Genomic DNA of R. tibeticum CCBAU85039 T was isolated using the GENTRA Pure-Gene kit (Qiagen). A sequencing library was constructed and sequenced on the MiSeq platform applying the Illumina paired-end protocol (Illumina, Inc.). In total, 4,090,570 sequence reads were obtained, yielding a total of 1,102,161,679 bp of sequence information. The Illumina reads were assembled by the GS de novo assembler software (gsAssembler, version 2.8; Roche) with a final outcome of 206 large (Ͼ500 bp) contigs. Of these, 167 contigs were arranged in 128 scaffolds. The estimated genome size is around 7 Mb, and, accordingly, the coverage obtained was approximately 159-fold. The genome features an average GϩC content of 59.72%. The N 50 value for scaffolds was 177,123 bp with an average scaffold size of 54,157 bp.
The genome was annotated applying the Prokka pipeline and GenDB (12,13), which predicted 6,977 protein-coding sequences (CDSs) and 45 tRNA genes. The rRNA operon was found on a 4-fold overrepresented contig, suggesting the presence of four rrn copies within the genome. Genome comparisons were done within the EDGAR version 2.0 platform (14). More than 5,200 CDSs of R. tibeticum (75% of all CDSs) represent orthologs to corresponding R. favelukesii LPU83 T genes (15). Moreover, phylogenetic analysis of the concatenated core genomes confirmed a close relationship between both strains, as previously described (16). Further comparative studies will elucidate the similarities and differences among different groups of sequenced rhizobial strains and refine their taxonomic classification.
Accession number(s). This whole-genome shotgun project has been deposited in the EMBL database under the accession numbers FNXB01000001 to FNXB01000167. The version described in this paper is the first version, FNXB01000000.

ACKNOWLEDGMENTS
The bioinformatics support of the BMBF-funded project "Bielefeld-Gießen Center for Microbial Bioinformatics"-BiGi (grant 031A533) within the German Network for Bioinformatics Infrastructure (de.NBI) is gratefully acknowledged. This investigation was supported by grants PICT-2013-0113 and PICT-2012-0518 to G.T.T. and M.P., respectively. G.T.T., A.L., and M.P. are members of the Research Career of CONICET. G.T.T. received a fellowship from the Alexander von Humboldt Foundation.
We acknowledge the financial support of the German Research Foundation (DFG) and the Open Access Publication Fund of Bielefeld University for the article processing charge.