ABSTRACTBacillus subtilisforms biofilms in response to internal and external stimuli. I previously showed that thecysLdeletion mutant was defective in biofilm formation, but the reason for this remains unidentified. CysL is a transcriptional activator of thecysJIoperon, which encodes sulfite reductase, an enzyme involved in cysteine biosynthesis. Decreased production of sulfite reductase led to biofilm formation defects in the ΔcysLmutant. The ΔcysLmutation was suppressed by disruptingcysHoperon 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 ΔcysLmutant. ThecysHgene encodes phosphoadenosine phosphosulfate reductase, which requires a reduced form of thioredoxin (TrxA) as an electron donor. High expression oftrxAinhibited biofilm formation in the ΔcysLmutant but not in the wild-type strain. Northern blot analysis showed thattrxAtranscription was induced in the ΔcysLmutant in a disulfide stress-induced regulator Spx-dependent manner. On the basis of these results, I propose that the ΔcysLmutation causes phosphoadenosine phosphosulfate reductase to consume large amounts of reduced thioredoxin, inducing disulfide stress and activating Spx. Thespxmutation restored biofilm formation to the ΔcysLmutant. The ΔcysLmutation reduced expression of theepsoperon, which is required for exopolysaccharide production. Moreover, overexpression of theepsoperon restored biofilm formation to the ΔcysLmutant. Taken together, these results suggest that the ΔcysLmutation activates Spx, which then inhibits biofilm formation through repression of theepsoperon.IMPORTANCEBacillus subtilishas been studied as a model organism for biofilm formation. In this study, I explored why thecysLdeletion mutant was defective in biofilm formation. I demonstrated that the ΔcysLmutation 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 ofB. 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.