Dataset Information

Deciphering the essentiality and function of the anti-σM factors in Bacillus subtilis.

ABSTRACT: Bacteria use alternative sigma factors to adapt to different growth and stress conditions. The Bacillus subtilis extracytoplasmic function sigma factor SigM regulates genes for cell wall synthesis and is crucial for maintaining cell wall homeostasis under stress conditions. The activity of SigM is regulated by its anti-sigma factor, YhdL, and the accessory protein YhdK. Here, we show that dysregulation of SigM caused by the absence of either component of the anti-sigma factor complex leads to toxic levels of SigM and severe growth defects. High SigM activity results from a dysregulated positive feedback loop, and can be suppressed by overexpression of the housekeeping sigma, SigA. Using a sigM merodiploid strain, we selected for suppressor mutations that allow survival of yhdL depletion strain. The recovered suppressor mutations map to the beta and beta-prime subunits of RNA polymerase core enzyme and selectively reduce SigM activity, and in some cases increase the activity of other alternative sigma factors. This work highlights the ability of mutations in RNA polymerase that remodel the sigma-core interface to differentially affect sigma factor activity, and thereby alter the transcriptional landscape of the cell.

SUBMITTER: Zhao H

PROVIDER: S-EPMC6679829 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Deciphering the essentiality and function of the anti-σ<sup>M</sup> factors in Bacillus subtilis.

Zhao Heng H Roistacher Daniel M DM Helmann John D JD

Molecular microbiology 20190313 2

Bacteria use alternative sigma factors to adapt to different growth and stress conditions. The Bacillus subtilis extracytoplasmic function sigma factor SigM regulates genes for cell wall synthesis and is crucial for maintaining cell wall homeostasis under stress conditions. The activity of SigM is regulated by its anti-sigma factor, YhdL, and the accessory protein YhdK. Here, we show that dysregulation of SigM caused by the absence of either component of the anti-sigma factor complex leads to to ...[more]

PMID: 30715747

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Project description:In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

Project description:RNA-binding protein Hfq is a key component of the adaptive responses of many proteobacterial species including Escherichia coli, Salmonella enterica and Vibrio cholera. In these organisms, the importance of Hfq largely stems from its participation to regulatory mechanisms involving small non-coding RNAs. In contrast, the function of Hfq in Gram-positive bacteria has remained elusive and somewhat controversial. In the present study, we have further addressed this point by comparing growth phenotypes and transcription profiles between wild-type and an hfq deletion mutant of the model Gram-positive bacterium, Bacillus subtilis. The absence of Hfq had no significant consequences on growth rates under nearly two thousand metabolic conditions and chemical treatments. The only phenotypic difference was a survival defect of B. subtilis hfq mutant in rich medium in stationary phase. Transcriptomic analysis correlated this phenotype with a change in the levels of nearly one hundred transcripts. Albeit a significant fraction of these RNAs (36%) encoded sporulation-related functions, analyses in a strain unable to sporulate ruled out sporulation per se as the basis of the hfq mutant's stationary phase fitness defect. When expressed in Salmonella, B. subtilis hfq complemented the sharp loss of viability of a degP hfq double mutant, attenuating the chronic σE-activated phenotype of this strain. However, B. subtilis hfq did not complement other regulatory deficiencies resulting from loss of Hfq-dependent small RNA activity in Salmonella indicating a limited functional overlap between Salmonella and B. subtilis Hfqs. Overall, this study confirmed that, despite structural similarities with other Hfq proteins, B. subtilis Hfq does not play a central role in post-transcriptional regulation but might have a more specialized function connected with stationary phase physiology. This would account for the high degree of conservation of Hfq proteins in all 17 B. subtilis strains whose genomes have been sequenced.

Dataset Information

Deciphering the essentiality and function of the anti-σM factors in Bacillus subtilis.

Publications

Deciphering the essentiality and function of the anti-σ<sup>M</sup> factors in Bacillus subtilis.

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