Abstract
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.
Highlights
Environmental pH stress constitutes a limiting factor for S. meliloti survival and development
Identification of sigma factors involved in the pH stress response of S. meliloti To explore the role of sigma factors in S. meliloti under acidic pH stress conditions, marker-free deletion mutants were successfully produced for the sigma factor genes rpoE1, rpoE2, rpoE5, rpoH1 and fecI, with the utilization of gene Splicing by Overlap Extension or gene SOEing technique [31]
In order to determine the growth properties and to test for a role of those sigma factors in pH stress response, the growth of sigma factor mutant and wild type cells in Vincent minimal medium (VMM) medium was monitored at two distinct pH values: pH 7.0 and pH 5.75
Summary
Environmental pH stress constitutes a limiting factor for S. meliloti survival and development. Stress response in bacteria is essential for effective adaptation to changes in the environment, as well as to changes in the bacterial physiological state This response is mediated by global regulatory mechanisms that operate in an effective method of transcriptional control, with the participation of specialized RNA polymerase subunits, the alternative sigma factors [1]. The heat shock response is a widespread phenomenon found in all living cells In bacteria, it is controlled at the transcriptional level by the alternative sigma factor RpoH (s32) [6,7,8]. In E. coli, RpoH controls the expression of about 91 genes [12], including many coding for heat shock proteins, which are important for survival during stress conditions. Major functions of heat shock proteins are to prevent inactivation of cellular proteins, to reactivate once inactivated proteins, and to help degrade nonreparable denatured proteins that accumulate under stress conditions [8]
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