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Comparative genome-wide transcriptional profiling of Azorhizobium caulinodans ORS571 grown under free-living and symbiotic conditions.

ABSTRACT: The whole-genome sequence of the endosymbiotic bacterium Azorhizobium caulinodans ORS571, which forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, was recently determined. The sizes of the genome and symbiosis island are 5.4 Mb and 86.7 kb, respectively, and these sizes are the smallest among the sequenced rhizobia. In the present study, a whole-genome microarray of A. caulinodans was constructed, and transcriptomic analyses were performed on free-living cells grown in rich and minimal media and in bacteroids isolated from stem nodules. Transcriptional profiling showed that the genes involved in sulfur uptake and metabolism, acetone metabolism, and the biosynthesis of exopolysaccharide were highly expressed in bacteroids compared to the expression levels in free-living cells. Some mutants having Tn5 transposons within these genes with increased expression were obtained as nodule-deficient mutants in our previous study. A transcriptomic analysis was also performed on free-living cells grown in minimal medium supplemented with a flavonoid, naringenin, which is one of the most efficient inducers of A. caulinodans nod genes. Only 18 genes exhibited increased expression by the addition of naringenin, suggesting that the regulatory mechanism responding to the flavonoid could be simple in A. caulinodans. The combination of our genome-wide transcriptional profiling and our previous genome-wide mutagenesis study has revealed new aspects of nodule formation and maintenance.

SUBMITTER: Tsukada S

PROVIDER: S-EPMC2725507 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Comparative genome-wide transcriptional profiling of Azorhizobium caulinodans ORS571 grown under free-living and symbiotic conditions.

Tsukada Shuhei S Aono Toshihiro T Akiba Noriko N Lee Kyung-Bum KB Liu Chi-Te CT Toyazaki Hiroki H Oyaizu Hiroshi H

Applied and environmental microbiology 20090619 15

The whole-genome sequence of the endosymbiotic bacterium Azorhizobium caulinodans ORS571, which forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, was recently determined. The sizes of the genome and symbiosis island are 5.4 Mb and 86.7 kb, respectively, and these sizes are the smallest among the sequenced rhizobia. In the present study, a whole-genome microarray of A. caulinodans was constructed, and transcriptomic analyses were performed on free-living cells grown in ri ...[more]

PMID: 19542345

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Project description:The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37?kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ?cheY1 mutant exhibited decreased frequency of direction reversal, whereas the ?cheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ?cheY1 and ?cheY2 mutants was lower than that in the WT, but the ?cheY2 mutant had more obvious EPS defects that were similar to those of the ?cheY1 ?cheY2 and ?eps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ?cheY1 and ?cheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ?cheY2 mutant was severely impaired compared to that of the ?cheY1 mutant. Taken together, the ?cheY2 phenotypes are more severe than the ?cheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ?cheY2 single-mutant phenotype. These defects of ?cheY1 and ?cheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions.IMPORTANCE Azorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.

Project description:BackgroundSwitchgrass (Panicum virgatum L.) is an important biofuel crop that may contribute to replacing petroleum fuels. However, slow seedling growth and soil salinization affect the growth and development of switchgrass. An increasing number of studies have shown that beneficial microorganisms promote plant growth and increase tolerance to salinity stress. However, the feasibility of inoculating switchgrass with Azorhizobium caulinodans ORS571 to enhance the growth and salt tolerance of its seedlings is unclear. Our previous study showed that A. caulinodans ORS571 could colonize wheat (Triticum aestivum L.) and thereby promote its growth and development and regulate the gene expression levels of microRNAs (miRNAs).ResultsIn this study, we systematically studied the impact of A. caulinodans ORS571 on switchgrass growth and development and the response to salinity stress; we also studied the underlying mechanisms during these biological processes. Inoculation with A. caulinodans ORS571 significantly alleviated the effect of salt stress on seedling growth. Under normal conditions, A. caulinodans ORS571 significantly increased fresh plant weight, chlorophyll a content, protein content, and peroxidase (POD) activity in switchgrass seedlings. Under salt stress, the fresh weight, dry weight, shoot and root lengths, and chlorophyll contents were all significantly increased, and some of these parameters even recovered to normal levels after inoculation with A. caulinodans ORS571. Soluble sugar and protein contents and POD and superoxide dismutase (SOD) activities were also significantly increased, contrary to the results for proline. Additionally, A. caulinodans ORS571 may alleviate salt stress by regulating miRNAs. Twelve selected miRNAs were all upregulated to different degrees under salt stress in switchgrass seedlings. However, the levels of miR169, miR171, miR319, miR393, miR535, and miR854 were decreased significantly after inoculation with A. caulinodans ORS571 under salt stress, in contrast to the expression level of miR399.ConclusionThis study revealed that A. caulinodans ORS571 increased the salt tolerance of switchgrass seedlings by increasing their water content, photosynthetic efficiency, osmotic pressure maintenance, and reactive oxygen species (ROS) scavenging abilities and regulating miRNA expression. This work provides a new, creative idea for improving the salt tolerance of switchgrass seedlings.

Dataset Information

Comparative genome-wide transcriptional profiling of Azorhizobium caulinodans ORS571 grown under free-living and symbiotic conditions.

Publications

Comparative genome-wide transcriptional profiling of Azorhizobium caulinodans ORS571 grown under free-living and symbiotic conditions.

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