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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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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.
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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.

Mentions: The tryptic digests were injected to a LC-MS/MS system equipped with a long monolithic silica capillary column; 1,658 proteins were successfully identified by efficient separation (Additional file 1). Specifically, 1,533 proteins were identified under the free-living condition, and 847 proteins were identified by the analytes extracted from nodules without bacteroid isolation and prefractionation (FigureĀ 1). 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 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 conditions, although the 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.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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.
Mentions: The tryptic digests were injected to a LC-MS/MS system equipped with a long monolithic silica capillary column; 1,658 proteins were successfully identified by efficient separation (Additional file 1). Specifically, 1,533 proteins were identified under the free-living condition, and 847 proteins were identified by the analytes extracted from nodules without bacteroid isolation and prefractionation (FigureĀ 1). 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 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 conditions, although the 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.

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