Whole-Genome Sequence of the Nitrogen-Fixing Symbiotic Rhizobium Mesorhizobium loti Strain TONO

Mesorhizobium loti is the nitrogen-fixing microsymbiont for legumes of the genus Lotus. Here, we report the whole-genome sequence of a Mesorhizobium loti strain, TONO, which is used as a symbiont for the model legume Lotus japonicus. The whole-genome sequence of the strain TONO will be a solid platform for comparative genomics analyses and for the identification of genes responsible for the symbiotic properties of Mesorhizobium species.

and strain MAFF303099 is the first nitrogen-fixing rhizobial species whose complete genome was sequenced (1). The M. loti strain TONO, together with the strain MAFF303099, has been used widely as a symbiont for a model legume, Lotus japonicus (2)(3)(4)(5)(6), and these two strains exhibit different symbiotic properties on certain symbiotic mutants of L. japonicus (Umehara Y and Shimoda Y, unpublished data). Here, we present the wholegenome sequence of the M. loti strain TONO for future study of the rhizobial genes responsible for the symbiotic properties of Mesorhizobium species.
The genome of M. loti strain TONO was sequenced using Roche 454 GS-FLX Titanium, and a total of 637.1 Mbp (~75-fold coverage) sequence data were obtained from single and pairedend libraries. Obtained reads were assembled by Newbler Assembler (454 Life Sciences), generating eight scaffolds. Sequences of gapped regions inside scaffolds and connections among the eight scaffolds were determined by PCR amplicon sequencing and direct sequencing of TONO genomic DNA by using the same procedures reported in Shimoda et al. (7). The genome of TONO was finally composed into four circular replicons, a chromosome (7,856,088 bp, 62.8% GϩC content) and three plasmids (294,703 bp with 59.5% GϩC content, 220,869 bp with 58.9% GϩC content, and 80,491 bp with 60.4% GϩC content). Genome annotation was performed using the Microbial Genome Annotation Pipeline (MiGAP) provided by DDBJ (8). A total of 8,149 protein-coding genes, 57 tRNAs, and six rRNAs (two copies of 16S-23S-5S operon) were predicted on the genome. Approximately 83% of protein-coding genes (6,774 genes) are assigned to Clusters of Orthologous Groups (COGs) (9).
We compared the genome components of the strain TONO with those of closely related genome of the M. loti strains MAFF303099 (1) and R7A (10). Average nucleotide identity (ANI) (11) values between TONO and MAFF or R7A are 91.7% and 91.5%, respectively. In our assembled TONO genome, a 508-kb symbiotic island integrated into a phenylalanine-tRNA gene with the duplication of 3= terminal portion of the gene was predicted. The symbiotic island of TONO carries genes involved in Nod factor synthesis (nod genes) and nitrogen fixation (nif and fix genes), and these gene clusters are highly conserved among the three strains in their sequence and order of genes. Also, gene clusters encoding the components of a type III secretion system and conjugative transfer proteins are highly conserved between TONO and MAFF303099.
Dot plot similarity analysis of overall TONO chromosome with that of MAFF303099 and R7A revealed that these three strains share collinear similarity throughout the chromosome, except for symbiotic islands, which show fragmented similarity. Furthermore, a comparison of the predicted gene products revealed that approximately 10% of the predicted TONO genes (834 genes) did not show significant similarity (e-value Ͻ10 -4 ) to any genes of MAFF303099 and R7A, and the majority of these genes show similarity to functionally uncharacterized proteins of other bacteria. Although further analyses are needed to identify the genes responsible for the differences in symbiotic properties of Mesorhizobium loti, the whole-genome sequence of strain TONO we reported here provides a solid platform for future studies in Mesorhizobium species.
Accession number(s). The genome sequences have been deposited at DDBJ/EMBL/GenBank under the accession numbers AP017605 to AP017608. JP25712006) and by the NIAS Strategic Fund from National Institute of Agrobiological Sciences to M.H. The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

FUNDING INFORMATION
This work, including the efforts of Yoshikazu Shimoda, was funded by Japan Society for the Promotion of Science (JSPS) (JP25712006).