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Inactivation of cysL Inhibits Biofilm Formation by Activating the Disulfide Stress Regulator Spx in Bacillus subtilis.

ABSTRACT: 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 ?cysL mutant. The ?cysL 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 ?cysL 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 ?cysL mutant but not in the wild-type strain. Northern blot analysis showed that trxA transcription was induced in the ?cysL mutant in a disulfide stress-induced regulator Spx-dependent manner. On the basis of these results, I propose that the ?cysL 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 ?cysL mutant. The ?cysL mutation reduced expression of the eps operon, which is required for exopolysaccharide production. Moreover, overexpression of the eps operon restored biofilm formation to the ?cysL mutant. Taken together, these results suggest that the ?cysL mutation activates Spx, which then inhibits biofilm formation through repression of the eps operon.IMPORTANCE Bacillus 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 ?cysL 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.

SUBMITTER: Kobayashi K

PROVIDER: S-EPMC6436345 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Inactivation of cysL Inhibits Biofilm Formation by Activating the Disulfide Stress Regulator Spx in Bacillus subtilis.

Kobayashi Kazuo K

Journal of bacteriology 20190326 8

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 ...[more]

PMID: 30718304

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Project description:Bacillus subtilis forms robust biofilms in the presence of large amounts of carbon sources, such as glycerol. However, little is known about the importance of the metabolic systems, or the relationship between metabolic systems and regulatory systems, involved in biofilm formation. Glutamate synthase, encoded by gltAB, is an enzyme that converts 2-ketoglutarate (a tricarboxylic acid [TCA] cycle intermediate) and glutamine into glutamate, which is a general amino group donor in metabolism. Here, we show that a ?gltA mutant exhibited early arrest of biofilm formation in complex medium containing glycerol. This phenotype was not due to glutamate auxotrophy. Consistent with its biofilm formation phenotype, the ?gltA mutant exhibited an early decrease in expression of the epsA and tapA operons, which are responsible for production of biofilm matrix polymers. This resulted from decreased activity of their regulator, Spo0A, as evidenced by reduced expression of other Spo0A-regulated genes in the ?gltA mutant. The ?gltA mutation prevented biofilm formation only in the presence of large amounts of glycerol. Moreover, limited expression of citrate synthase (but not other TCA enzymes) restored biofilm-forming ability to the ?gltA mutant. These results indicate that the ?gltA mutant accumulates an inhibitory intermediate (citrate) in the TCA cycle in the presence of large amounts of glycerol. The ?gltA mutant formed biofilms when excess iron was added to the medium. Taken together, the data suggest that accumulation of citrate ions by the ?gltA mutant causes iron shortage due to chelation, which prevents activation of Spo0A and causes defective biofilm formation.IMPORTANCE Bacillus subtilis, a model organism for bacterial biofilm formation, forms robust biofilms in a medium-dependent manner. Although the regulatory network that controls biofilm formation has been well studied, the importance of the underlying metabolic systems remains to be elucidated. The present study demonstrates that a metabolic disorder in a well-conserved metabolic system causes accumulation of an inhibitory metabolic intermediate that prevents activation of the system that regulates biofilm formation. These findings increase our understanding of the coordination between cellular metabolic status and the regulatory networks governing biofilm formation.

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Inactivation of cysL Inhibits Biofilm Formation by Activating the Disulfide Stress Regulator Spx in Bacillus subtilis.

Publications

Inactivation of <i>cysL</i> Inhibits Biofilm Formation by Activating the Disulfide Stress Regulator Spx in Bacillus subtilis.

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