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Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti.

ABSTRACT: BACKGROUND: The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell. RESULTS: Comparison of tolC mutant and wild-type strains transcriptomes showed 1177 genes with significantly increased expression while 325 had significantly decreased expression levels. The genes with an increased expression suggest the activation of a cytoplasmic and extracytoplasmic stress responses possibly mediated by the sigma factor RpoH1 and protein homologues of the CpxRA two-component regulatory system of Enterobacteria, respectively. Stress conditions are probably caused by perturbation of the cell envelope. Consistent with gene expression data, biochemical analysis indicates that the tolC mutant suffers from oxidative stress. This is illustrated by the elevated enzyme activity levels detected for catalase, superoxide dismutase and glutathione reductase. The observed increase in the expression of genes encoding products involved in central metabolism and transporters for nutrient uptake suggests a higher metabolic rate of the tolC mutant. We also demonstrated increased swarming motility in the tolC mutant strain. Absence of functional TolC caused decreased expression mainly of genes encoding products involved in nitrogen metabolism and transport. CONCLUSION: This work shows how a mutation in the outer membrane protein TolC, common to many bacterial transport systems, affects expression of a large number of genes that act in concert to restore cell homeostasis. This finding further underlines the fundamental role of this protein in Sinorhizobium meliloti biology.

SUBMITTER: Santos MR

PROVIDER: S-EPMC2912261 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti.

Santos Mário R MR Cosme Ana M AM Becker Jörg D JD Medeiros João M C JM Mata Márcia F MF Moreira Leonilde M LM

BMC microbiology 20100623

<h4>Background</h4>The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell.<h4>Results</h4>Co ...[more]

PMID: 20573193

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Project description:Reactive oxygen species such as peroxides play an important role in plant development, cell wall maturation, and defense responses. During nodulation with the host plant Medicago sativa, Sinorhizobium meliloti cells are exposed to H2O2 in infection threads and developing nodules (R. Santos, D. Hérouart, S. Sigaud, D. Touati, and A. Puppo, Mol Plant Microbe Interact 14:86-89, 2001, https://doi.org/10.1094/MPMI.2001.14.1.86). S. meliloti cells likely also experience oxidative stress, from both internal and external sources, during life in the soil. Here, we present microarray transcription data for S. meliloti wild-type cells compared to a mutant deficient in the key oxidative regulatory protein OxyR, each in response to H2O2 treatment. Several alternative sigma factor genes are upregulated in the response to H2O2; the stress sigma gene rpoE2 shows OxyR-dependent induction by H2O2, while rpoH1 expression is induced by H2O2 irrespective of the oxyR genotype. The activity of the RpoE2 sigma factor in turn causes increased expression of two more sigma factor genes, rpoE5 and rpoH2 Strains with deletions of rpoH1 showed improved survival in H2O2 as well as increased levels of oxyR and total catalase expression. These results imply that ΔrpoH1 strains are primed to deal with oxidative stress. This work presents a global view of S. meliloti gene expression changes, and of regulation of those changes, in response to H2O2IMPORTANCE Like all aerobic organisms, the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti experiences oxidative stress throughout its complex life cycle. This report describes the global transcriptional changes that S. meliloti makes in response to H2O2 and the roles of the OxyR transcriptional regulator and the RpoH1 sigma factor in regulating those changes. By understanding the complex regulatory response of S. meliloti to oxidative stress, we may further understand the role that reactive oxygen species play as both stressors and potential signals during symbiosis.

Project description:BackgroundEnvironmental pH stress constitutes a limiting factor for S. meliloti survival and development. The response to acidic pH stress in S. meliloti is versatile and characterized by the differential expression of genes associated with various cellular functions. The purpose of this study was to gain detailed insight into the participation of sigma factors in the complex stress response system of S. meliloti 1021 using pH stress as an effector.ResultsIn vitro assessment of S meliloti wild type and sigma factor mutants provided first evidence that the sigma factor RpoH1 plays a major role in the pH stress response. Differential expression of genes related to rhizobactin biosynthesis was observed in microarray analyses performed with the rpoH1 mutant at pH 7.0. The involvement of the sigma factor RpoH1 in the regulation of S. meliloti genes upon pH stress was analyzed by comparing time-course experiments of the wild type and the rpoH1 mutant. Three classes of S. meliloti genes could be identified, which were transcriptionally regulated in an RpoH1-independent, an RpoH1-dependent or in a complex manner. The first class of S. meliloti genes, regulated in an RpoH1-independent manner, comprises the group of the exopolysaccharide I biosynthesis genes and also the group of genes involved in motility and flagellar biosynthesis. The second class of S. meliloti genes, regulated in an RpoH1-dependent manner, is composed of genes known from heat shock studies, like ibpA, grpE and groEL5, as well as genes involved in translation like tufA and rplC. Finally, the third class of S. meliloti genes was regulated in a complex manner, which indicates that besides sigma factor RpoH1, further regulation takes place. This was found to be the case for the genes dctA, ndvA and smc01505.ConclusionsClustering of time-course microarray data of S. meliloti wild type and sigma factor rpoH1 mutant allowed for the identification of gene clusters, each with a unique time-dependent expression pattern, as well as for the classification of genes according to their dependence on RpoH1 expression and regulation. This study provided clear evidence that the sigma factor RpoH1 plays a major role in pH stress response.

Project description:The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream of aceA were isolated and functionally characterized in Sinorhizobium meliloti. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed. aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants. aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains. aceA and glcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other alpha-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis.

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Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti.

Publications

Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti.

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