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

Tatsukami Y, Nambu M, Morisaka H, Kuroda K, Ueda M - BMC Microbiol. (2013)

Bottom Line: The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT

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.

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The map of metabolic pathways under the symbiotic and/or free-living conditions. The map of metabolic pathways is shown: (a) nitrogen fixation, (b) ubiquinone biosynthesis, (c) amino sugar metabolism, (d) peptidoglycan biosynthesis. Box symbols indicate the same things as in Figure 2. Daggers (†) indicate the reactions that have universally existed but have not been proposed in M. loti by KEGG pathway analysis. Asterisks indicate the focused enzymes discussed in the “Results and discussion” section. Abbreviations are as follows: GlcN, Glucosamine; GlcNAc, N-acetyl-D-glucosamine; MurNAc, N-acetylmuramic acid.
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Figure 4: The map of metabolic pathways under the symbiotic and/or free-living conditions. The map of metabolic pathways is shown: (a) nitrogen fixation, (b) ubiquinone biosynthesis, (c) amino sugar metabolism, (d) peptidoglycan biosynthesis. Box symbols indicate the same things as in Figure 2. Daggers (†) indicate the reactions that have universally existed but have not been proposed in M. loti by KEGG pathway analysis. Asterisks indicate the focused enzymes discussed in the “Results and discussion” section. Abbreviations are as follows: GlcN, Glucosamine; GlcNAc, N-acetyl-D-glucosamine; MurNAc, N-acetylmuramic acid.

Mentions: Nitrogenase complex core subunits (NifH, NifD, NifK) and the electron donor proteins (FixA, FixB, FixC), which transfer electrons to the nitrogenase complex, were detected only under the symbiotic condition (Figure 4a). Fixation of atmospheric nitrogen is a characteristic feature of rhizobia only under the symbiotic condition [7]. The proteins related to nitrogen fixation, such as nitrogenase construction (NifN, NifX, NifS, NifW) [28], electron donation (FixX, FixP), and symbiosis-unique ferredoxins (mlr5869, mlr5930, msl8750), were also found to be unique to the symbiotic condition. In addition, NifA and RpoN, which are known to cooperatively regulate nif and fix genes, were detected only under the symbiotic condition [29]. The protein profile strongly reflected the phenotype that was predicted by transcriptome analysis [7].


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

Tatsukami Y, Nambu M, Morisaka H, Kuroda K, Ueda M - BMC Microbiol. (2013)

The map of metabolic pathways under the symbiotic and/or free-living conditions. The map of metabolic pathways is shown: (a) nitrogen fixation, (b) ubiquinone biosynthesis, (c) amino sugar metabolism, (d) peptidoglycan biosynthesis. Box symbols indicate the same things as in Figure 2. Daggers (†) indicate the reactions that have universally existed but have not been proposed in M. loti by KEGG pathway analysis. Asterisks indicate the focused enzymes discussed in the “Results and discussion” section. Abbreviations are as follows: GlcN, Glucosamine; GlcNAc, N-acetyl-D-glucosamine; MurNAc, N-acetylmuramic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750425&req=5

Figure 4: The map of metabolic pathways under the symbiotic and/or free-living conditions. The map of metabolic pathways is shown: (a) nitrogen fixation, (b) ubiquinone biosynthesis, (c) amino sugar metabolism, (d) peptidoglycan biosynthesis. Box symbols indicate the same things as in Figure 2. Daggers (†) indicate the reactions that have universally existed but have not been proposed in M. loti by KEGG pathway analysis. Asterisks indicate the focused enzymes discussed in the “Results and discussion” section. Abbreviations are as follows: GlcN, Glucosamine; GlcNAc, N-acetyl-D-glucosamine; MurNAc, N-acetylmuramic acid.
Mentions: Nitrogenase complex core subunits (NifH, NifD, NifK) and the electron donor proteins (FixA, FixB, FixC), which transfer electrons to the nitrogenase complex, were detected only under the symbiotic condition (Figure 4a). Fixation of atmospheric nitrogen is a characteristic feature of rhizobia only under the symbiotic condition [7]. The proteins related to nitrogen fixation, such as nitrogenase construction (NifN, NifX, NifS, NifW) [28], electron donation (FixX, FixP), and symbiosis-unique ferredoxins (mlr5869, mlr5930, msl8750), were also found to be unique to the symbiotic condition. In addition, NifA and RpoN, which are known to cooperatively regulate nif and fix genes, were detected only under the symbiotic condition [29]. The protein profile strongly reflected the phenotype that was predicted by transcriptome analysis [7].

Bottom Line: The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus