ABSTRACT
pgsB encodes gamma-poly glutamic acid (gamma-PGA) synthetase and constitutes an operon with pgsC, pgsAA, and pgsE. Genetic analysis revealed that degQ and swrA, the known regulators of pgsB, are not required for pgsB expression when high cellular concentrations of phosphorylated form of the response regulator DegU (DegU-P) are present. However, swrA appeared still to be required for gamma-PGA synthesis under the conditions we tested. Since genetic analysis suggested that DegU-P activates pgsB directly, we performed gel retardation and footprint analyses using purified His-tagged DegU and the pgsB promoter. The in vitro experiments revealed that His-tagged DegU bound to the immediate upstream region of the -35 region of the pgsB promoter. A six-base deletion within the sequence (the -44 to -39 region) abolished DegU-binding to the pgsB promoter and pgsB transcription, confirming the importance of the sequence for DegU-dependent regulation of pgsB. Hence we conclude that DegU is a direct activator of the pgsB operon.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Polyglutamic Acid/analogs & derivatives , Transcription, Genetic/physiology , Bacterial Proteins/physiology , Base Sequence , DNA Primers , Polyglutamic Acid/biosynthesis , Promoter Regions, GeneticABSTRACT
The Bacillus subtilis yrkP gene encodes a response regulator of a two-component regulatory system of unknown function. A previous DNA microarray experiment suggested that multicopy yrkP greatly enhanced the expression of yrkN, the ykcBC operon, and yrkO, which encodes a putative transporter. Here, lacZ fusion analysis confirmed these results and also revealed that YrkP autoregulates the putative yrkPQR operon, indicating that yrkPQR and yrkO form a divergon structure. In addition, real-time PCR analysis revealed that transcription of yrkO, yrkN, and ykcBC was significantly reduced in the yrkP strain. Hence, YrkP positively regulates the expression of these genes. Gel retardation analyses showed that YrkP bound to the promoter regions of yrkO, yrkN, and ykcB, albeit with lower binding affinities to the latter two promoters. The in vitro binding of YrkP to the promoter region of the yrkPQR and yrkO divergon was then analyzed by DNase I footprinting analysis. This revealed that YrkP recognizes three regions containing single-motifs or a direct repeat of the ten-base sequence [T/G]TCA[T/C]AAATT. lacZ fusion analysis of deleted and mutagenized promoter regions of yrkO and yrkPQR divergon confirmed that the three YrkP-binding regions are needed for the YrkP-mediated activation of yrkO and/or yrkPQR.
Subject(s)
Bacillus subtilis/genetics , Gene Expression Regulation, Bacterial , Transcription Factors/genetics , Bacterial Proteins/genetics , DNA Primers , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genotype , Oligonucleotide Array Sequence Analysis , Recombinant Fusion Proteins/metabolism , Regulon/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
During the course of screening for competence-deficient mutants in the mutant collection constructed by the Japan Consortium of Bacillus Functional Genomics, a disruption mutant of sodA encoding superoxide dismutase was identified as a mutant with decreased transformation efficiency. In fact, in the sodA mutant we observed a severe decrease in the expression of srfA required for the development of genetic competence. Northern and primer extension analyses revealed inhibition of the transcription of the comQXP quorum-sensing locus in the sodA mutant, thereby preventing srfA expression. Furthermore, an excess amount of superoxide anion induced by the addition of paraquat also resulted in a decrease in comQXP transcription. Thus, it was concluded that high levels of superoxide are able to inhibit specifically the transcription of the comQXP operon. In support of this conclusion, the effect of added paraquat was significantly alleviated in a comX-independent srfA expression system.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Membrane Proteins/antagonists & inhibitors , Peptide Synthases/genetics , Superoxides/metabolism , Transferases/antagonists & inhibitors , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/genetics , Membrane Proteins/genetics , Oxidative Stress , Paraquat/pharmacology , Peptide Synthases/metabolism , Quorum Sensing/genetics , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Transcription, Genetic/drug effects , Transferases/geneticsABSTRACT
srfA is an operon required for the synthesis of surfactin and the development of genetic competence in Bacillus subtilis. We observed that the expression of srfA is downregulated upon treatment with H2O2. Thus, we examined the involvement of several oxidative stress-responsive transcription factors in srfA expression. Our DNA microarray analysis revealed that the H2O2 stress-responsive regulator PerR is required for srfA expression. This was confirmed by lacZ fusion analysis. A ComX feeding assay and epistatic analyses revealed that the role of PerR in srfA expression is independent of other known regulators of srfA expression, namely, comQXP, rapC, and spx. Gel mobility shift and footprint assays revealed that PerR binds directly to two tandemly arranged noncanonical PerR boxes located in the upstream promoter region of srfA. A transcriptional srfA-lacZ fusion lacking both PerR boxes showed diminished and PerR-independent expression, indicating that the PerR boxes we identified function as positive cis elements for srfA transcription.