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Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis.

ABSTRACT: Matrix production during biofilm formation by Bacillus subtilis is governed by a gene control circuit at the heart of which are three dedicated regulatory proteins, the antirepressor SinI, the repressor SinR and the downstream regulator SlrR. Matrix production is triggered by the synthesis of SinI, which binds to and inactivates SinR, thereby derepressing genes for matrix production as well as the gene for SlrR. Recently, two additional regulators of matrix genes were identified: SlrA, which was reported to be an activator of SlrR, and YwcC, a repressor of SlrA synthesis (Kobayashi, 2008). We present evidence indicating that SlrA, which is a paralogue of SinI, is like SinI, an antirepressor that binds to, and inactivates, SinR. We also show that SlrA does not activate SlrR for expression of matrix genes. Instead, SlrR binds to, and inhibits the activity of, SlrA. Thus, the YwcC-SlrA-SinR-SlrR pathway is a negative feedback loop in which SlrA indirectly stimulates the synthesis of SlrR, and SlrR, in turn, inhibits the activity of SlrA. Finally, we report that under standard laboratory conditions SlrA makes only a small contribution to the expression of genes for matrix production. We propose that in response to an unknown signal recognized by the YwcC repressor, SlrA transiently boosts matrix production.

SUBMITTER: Chai Y

PROVIDER: S-EPMC2805041 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis.

Chai Yunrong Y Kolter Roberto R Losick Richard R

Molecular microbiology 20090928 4

Matrix production during biofilm formation by Bacillus subtilis is governed by a gene control circuit at the heart of which are three dedicated regulatory proteins, the antirepressor SinI, the repressor SinR and the downstream regulator SlrR. Matrix production is triggered by the synthesis of SinI, which binds to and inactivates SinR, thereby derepressing genes for matrix production as well as the gene for SlrR. Recently, two additional regulators of matrix genes were identified: SlrA, which was ...[more]

PMID: 19788541

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Project description:Biofilm formation by Bacillus subtilis requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and ymdB mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from ymdB mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing sinR were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the ymdB mutant strains.IMPORTANCE Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.

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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Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis.

Publications

Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis.

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