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Induction of the Spx regulon by cell wall stress reveals novel regulatory mechanisms in Bacillus subtilis.

ABSTRACT: The transcription factor Spx is the master regulator of the disulfide stress response in Bacillus subtilis. Intriguingly, the activation of Spx by diamide relies entirely on posttranslational regulatory events in spite of the complex transcriptional control of the spx gene. Here, we show that cell wall stress, but not membrane stress, also results in induction of the Spx regulon. Remarkably, two major differences were found regarding the mechanism of induction of Spx under cell wall stress in comparison to disulfide stress. First, transcriptional induction of the spx gene from a ?M -dependent promoter is required for accumulation of Spx in response to cell wall stress. Second, activation of the Spx regulon during cell wall stress is not accompanied by oxidation of the Spx disulfide switch. Finally, we demonstrate that cells lacking Spx have increased sensitivity toward antibiotics inhibiting both early and late steps in peptidoglycan synthesis, suggesting that the Spx regulon plays an important adaptive role in the cell wall stress response. This study expands the functional role of the Spx regulon and reveals novel regulatory mechanisms that result in induction of Spx in B. subtilis.

SUBMITTER: Rojas-Tapias DF

PROVIDER: S-EPMC5820111 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Induction of the Spx regulon by cell wall stress reveals novel regulatory mechanisms in Bacillus subtilis.

Rojas-Tapias Daniel F DF Helmann John D JD

Molecular microbiology 20180118 5

The transcription factor Spx is the master regulator of the disulfide stress response in Bacillus subtilis. Intriguingly, the activation of Spx by diamide relies entirely on posttranslational regulatory events in spite of the complex transcriptional control of the spx gene. Here, we show that cell wall stress, but not membrane stress, also results in induction of the Spx regulon. Remarkably, two major differences were found regarding the mechanism of induction of Spx under cell wall stress in co ...[more]

PMID: 29271514

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Project description:In Bacillus subtilis, the Spx transcription factor controls a large regulon in response to disulfide, heat, and cell wall stresses. The regulatory mechanisms that activate the Spx regulon are remarkably complex and involve changes in transcription, proteolysis, and posttranslational modifications. To identify genes involved in Spx regulation, we performed a transposon screen for mutations affecting expression of trxB, an Spx-dependent gene. Inactivation of ctsR, encoding the regulator of the Clp proteases, reduced trxB expression and lowered Spx levels. This effect required ClpP, but involved ClpC rather than the ClpX unfoldase. Moreover, cells lacking McsB, a dual function arginine kinase and ClpCP adaptor, largely reverted the ctsR phenotype and increased trxB expression. The role of McsB appears to involve its kinase activity, since loss of the YwlE phosphoarginine phosphatase also led to reduced trxB expression. Finally, we show that Spx is itself a regulator of the ctsR operon. Altogether, this work provides evidence for a role of CtsR regulon members ClpC, ClpP, and McsB in Spx regulation and identifies a new feedback pathway associated with Spx activity in B. subtilisIMPORTANCE In Bacillus subtilis, the Spx transcription factor is proteolytically unstable, and protein stabilization figures prominently in the induction of the Spx regulon in response to oxidative and cell envelope stresses. ClpXP is largely, but not entirely, responsible for Spx instability. Here, we identify ClpCP as the protease that degrades Spx under conditions that antagonize the ClpXP pathway. Spx itself contributes to activation of the ctsR operon, which encodes ClpC as well as the McsB arginine kinase and protease adaptor, thereby providing a negative feedback mechanism. Genetic studies reveal that dysregulation of the CtsR regulon or inactivation of the YwlE phosphoarginine phosphatase decreases Spx activity through mechanisms involving both protein degradation and posttranslational modification.

Project description:Bacillus subtilis forms biofilms in response to internal and external stimuli. I previously showed that the cysL deletion mutant was defective in biofilm formation, but the reason for this remains unidentified. CysL is a transcriptional activator of the cysJI operon, which encodes sulfite reductase, an enzyme involved in cysteine biosynthesis. Decreased production of sulfite reductase led to biofilm formation defects in the ΔcysL mutant. The ΔcysL mutation was suppressed by disrupting cysH operon genes, whose products function upstream of sulfite reductase in the cysteine biosynthesis pathway, indicating that defects in cysteine biosynthesis were not a direct cause for the defective biofilm formation observed in the ΔcysL mutant. The cysH gene encodes phosphoadenosine phosphosulfate reductase, which requires a reduced form of thioredoxin (TrxA) as an electron donor. High expression of trxA inhibited biofilm formation in the ΔcysL mutant but not in the wild-type strain. Northern blot analysis showed that trxA transcription was induced in the ΔcysL mutant in a disulfide stress-induced regulator Spx-dependent manner. On the basis of these results, I propose that the ΔcysL mutation causes phosphoadenosine phosphosulfate reductase to consume large amounts of reduced thioredoxin, inducing disulfide stress and activating Spx. The spx mutation restored biofilm formation to the ΔcysL mutant. The ΔcysL mutation reduced expression of the eps operon, which is required for exopolysaccharide production. Moreover, overexpression of the eps operon restored biofilm formation to the ΔcysL mutant. Taken together, these results suggest that the ΔcysL mutation activates Spx, which then inhibits biofilm formation through repression of the eps operon.IMPORTANCEBacillus subtilis has been studied as a model organism for biofilm formation. In this study, I explored why the cysL deletion mutant was defective in biofilm formation. I demonstrated that the ΔcysL mutation activated the disulfide stress response regulator Spx, which inhibits biofilm formation by repressing biofilm matrix genes. Homologs of Spx are highly conserved among Gram-positive bacteria with low G+C contents. In some pathogens, Spx is also reported to inhibit biofilm formation by repressing biofilm matrix genes, even though these genes and their regulation are quite different from those of B. subtilis Thus, the negative regulation of biofilm formation by Spx is likely to be well conserved across species and may be an appropriate target for control of biofilm formation.

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Induction of the Spx regulon by cell wall stress reveals novel regulatory mechanisms in Bacillus subtilis.

Publications

Induction of the Spx regulon by cell wall stress reveals novel regulatory mechanisms in Bacillus subtilis.

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