Many proteins encoded in the symbiosis island were also identified. The symbiosis island of M. loti MAFF303099 is one of the notable features, which occurs by integration of a horizontally transferred DNA segment, and is located on a 610,975-bp DNA segment of the chromosome at coordinates 4,644,702 to 5,255,766 [5]. A total of 582 protein-encoding genes were located on the symbiosis island.

Mapping the identified proteins to the symbiosis island showed that 74 proteins (8.7% of 847 proteins) were produced under the symbiotic condition, whereas only 22 proteins (1.4% of 1,533 proteins) were produced under the free-living condition. From the viewpoint of reproducibility, our data show highly-reproducible result Quisinostat with the strict criteria for protein identification (Additional file 2). As shown in this figure, 87% of proteins were identified from 3 data set under the free-living Sotrastaurin supplier conditions, although the Ruxolitinib ic50 previous report indicated that protein profile of free-living M. loti in stationary phase was not reproducible [9]. And identified proteins under the symbiotic condition also show high-reproducibility because 84% of proteins were identified at all measurements. These results indicated that the protein profile successfully obtained with our system reflected the free-living and the

symbiotic conditions. Figure 1 Venn diagram of proteins identified in M. loti. A total of 1,658 proteins were identified. Although 722 proteins were commonly identified under the free-living and symbiotic conditions, 811 and 125 proteins were uniquely identified under the free-living and symbiotic conditions, respectively. KEGG pathway analysis For further investigation about the lifestyle of rhizobia under each condition, the identified proteins were classified according to the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://​www.​genome.​jp/​kegg/​), and metabolic pathways were compared under the free-living and symbiotic conditions. The number of O-methylated flavonoid classified enzymes in each pathway is shown in Table 1,

and the annotated genes in Table 1 are listed in Additional file 3. Table 1 The number of classified enzymes detected by proteome analysis Pathway Symbiotic condition Free-living condition Genesa) Central carbon metabolism 49 56 77 Nitrogen fixation 8 2 8 Ubiquinone biosynthesis 6 5 9 Nucleotide sugar metabolism 1 6 13 Peptidoglycan biosynthesis 2 7 15 a)The number of genes proposed by KEGG pathway analysis. Central carbon metabolism Most enzymes classified in carbon metabolism, such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate (PP), and Entner-Doudoroff (ED) pathways, were commonly identified (Figure 2). It is assumed that the same pathways located in central carbon metabolism remained largely unchanged, irrespective of conditions. Figure 2 The map of central carbon metabolic pathways under the free-living and/or symbiotic conditions.