The complete genome sequence of Mesorhizobium loti strain MAFF303099, a nitrogen-fixing symbiotic bacterium, was determined. The genome consists of a single chromosome (7,036,071 bp) and two plasmids, pMLa (351,911 bp) and pMLb (208,315 bp). The chromosome contains 6,752 potential protein-coding genes, two sets of rRNA genes, and 50 tRNA genes representing 47 tRNA species. Approximately 54% of the protein-coding genes showed sequence similarity to known genes, 21% to hypothetical genes, and 25% had no apparent similarity. A 611-kb DNA segment, likely a symbiotic island, was identified, containing 30 nitrogen fixation genes and 24 nodulation genes. The plasmids contain 320 and 209 potential protein-coding genes, respectively, for various biological functions. The genome sequence was determined using a whole-genome shotgun strategy combined with the bridging shotgun method. The data were assembled using Phrap and validated by walking the entire genome with cosmid clones. The genome was annotated using computer prediction and similarity searches. The symbiotic island, a 500-kb region containing symbiotic genes, was identified in strain ICMP3153 and was found in MAFF303099. The symbiotic island contains 580 protein-coding genes, including genes for nitrogen fixation and nodulation. The plasmids also contain genes for conjugation, biotin synthesis, and thiamine biosynthesis. The genome of M. loti is the first complete genome sequence of a soil bacterium in the alpha-proteobacteria group. The sequence data are available in the RhizoBase database. The study provides insights into the genetic systems involved in symbiotic nitrogen fixation and horizontal gene transfer among natural microsymbionts. The results highlight the importance of the symbiotic island in nitrogen fixation and nodulation, and the role of plasmids in gene transfer and function. The genome sequence also reveals the presence of various genes involved in metabolism, transport, and other biological processes. The study contributes to the understanding of the genetic basis of symbiotic nitrogen fixation in leguminous plants.The complete genome sequence of Mesorhizobium loti strain MAFF303099, a nitrogen-fixing symbiotic bacterium, was determined. The genome consists of a single chromosome (7,036,071 bp) and two plasmids, pMLa (351,911 bp) and pMLb (208,315 bp). The chromosome contains 6,752 potential protein-coding genes, two sets of rRNA genes, and 50 tRNA genes representing 47 tRNA species. Approximately 54% of the protein-coding genes showed sequence similarity to known genes, 21% to hypothetical genes, and 25% had no apparent similarity. A 611-kb DNA segment, likely a symbiotic island, was identified, containing 30 nitrogen fixation genes and 24 nodulation genes. The plasmids contain 320 and 209 potential protein-coding genes, respectively, for various biological functions. The genome sequence was determined using a whole-genome shotgun strategy combined with the bridging shotgun method. The data were assembled using Phrap and validated by walking the entire genome with cosmid clones. The genome was annotated using computer prediction and similarity searches. The symbiotic island, a 500-kb region containing symbiotic genes, was identified in strain ICMP3153 and was found in MAFF303099. The symbiotic island contains 580 protein-coding genes, including genes for nitrogen fixation and nodulation. The plasmids also contain genes for conjugation, biotin synthesis, and thiamine biosynthesis. The genome of M. loti is the first complete genome sequence of a soil bacterium in the alpha-proteobacteria group. The sequence data are available in the RhizoBase database. The study provides insights into the genetic systems involved in symbiotic nitrogen fixation and horizontal gene transfer among natural microsymbionts. The results highlight the importance of the symbiotic island in nitrogen fixation and nodulation, and the role of plasmids in gene transfer and function. The genome sequence also reveals the presence of various genes involved in metabolism, transport, and other biological processes. The study contributes to the understanding of the genetic basis of symbiotic nitrogen fixation in leguminous plants.