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J Bacteriol. 2019 Mar 26;201(8). pii: e00712-18. doi: 10.1128/JB.00712-18. Print 2019 Apr 15.

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

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Division of Biological Science, Nara Institute of Science & Technology, Ikoma, Nara, Japan


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.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 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.


Bacillus subtilis ; Spx; biofilms; cysteine biosynthesis; disulfide stress

[Available on 2019-09-26]

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