Quantitative time-course proteome analysis of Mesorhizobium loti during nodule maturation.

Rhizobia are nitrogen-fixing bacteria that establish a symbiotic relationship with leguminous plants. To understand the mechanism by which rhizobia alter their metabolism to establish successful nitrogen-fixing symbiotic relationship with hosts, Lotus japonicus were inoculated with Mesorhizobium loti. Bacteroids were isolated from nodules harvested at 2weeks (the early stage of nodule development), and at 3 and 4weeks (the intermediate stage of nodule development) post-inoculation. Using a quantitative time-course proteome analysis, we quantified the variations in the production of 537 proteins in M. loti bacteroids during the course of nodule maturation. The results revealed significant changes in the carbon and amino acid metabolisms by M. loti upon differentiating into bacteroids. Furthermore, our findings suggested that M. loti enters a nitrogen-deficient condition during the early stages of nodule development, and then a nitrogen-rich condition during the intermediate stages of nodule development. In addition, our data indicated that M. loti assimilated ammonia during the intermediate stages of nodule development. Our results provide new insights into the course of physiological transitions undergone by M. loti during nodule maturation.

Disclosure of the differences of Mesorhizobium loti under the free-living and symbiotic conditions by comparative proteome analysis without bacteroid isolation.

BACKGROUND: Rhizobia are symbiotic nitrogen-fixing soil bacteria that show a symbiotic relationship with their host legume. Rhizobia have 2 different physiological conditions: a free-living condition in soil, and a symbiotic nitrogen-fixing condition in the nodule. The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out. To clarify the lifestyle of bacteria, proteome analysis is necessary because the protein profile directly reflects in vivo reactions of the organisms. In proteome analysis, high separation performance is required to analyze complex biological samples. Therefore, we used a liquid chromatography-tandem mass spectrometry system, equipped with a long monolithic silica capillary column, which is superior to conventional columns. In this study, we compared the protein profile of Mesorhizobium loti MAFF303099 under free-living condition to that of symbiotic conditions by using small amounts of crude extracts. RESULT: We identified 1,533 and 847 proteins for M. loti under free-living and symbiotic conditions, respectively. Pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many of the enzymes involved in the central carbon metabolic pathway were commonly detected under both conditions. The proteins encoded in the symbiosis island, the transmissible chromosomal region that includes the genes that are highly upregulated under the symbiotic condition, were uniquely detected under the symbiotic condition. The features of the symbiotic condition that have been reported by transcriptome analysis were confirmed at the protein level by proteome analysis. In addition, the genes of the proteins involved in cell surface structure were repressed under the symbiotic nitrogen-fixing condition. Furthermore, farnesyl pyrophosphate (FPP) was found to be biosynthesized only in rhizobia under the symbiotic condition. CONCLUSION: The obtained protein profile appeared to reflect the difference in phenotypes under the free-living and symbiotic conditions. In addition, KEGG pathway analysis revealed that the cell surface structure of rhizobia was largely different under each condition, and surprisingly, rhizobia might provided FPP to the host as a source of secondary metabolism. M. loti changed its metabolism and cell surface structure in accordance with the surrounding conditions.