No abstract

In Bacillus subtilis, several phenolic acids specifically induce expression of padC, encoding a phenolic acid decarboxylase that converts these antimicrobial compounds into vinyl derivatives. padC forms an operon with a putative coding sequence of unknown function, yveFG, and this coding sequence does not appear to be involved in the phenolic acid stress response (PASR). To identify putative regulators involved in the PASR, random transposon mutagenesis, combined with two different screens, was performed. PadR, a negative transcriptional regulator of padC expression, was identified. padR is not located in the vicinity of padC, and the expression of padR is low and appears constitutive. This is

Bacillus subtilis 168 is resistant to phenolic acids by expression of an inducible enzyme, the phenolic acid decarboxylase (PadC), that decarboxylates these acids into less toxic vinyl derivatives. In the phenolic acid stress response (PASR), the repressor of padC, PadR, is inactivated by these acids. Inactivation of PadR is followed by a strong expression of padC. To elucidate the functional interaction between PadR and the padC promoter, we performed (i) footprinting assays to identify the region protected by PadR, (ii) electrophoretic mobility shift assays (EMSAs) with a modified padC promoter protected region to determine the interacting sequences, and (iii) random mutagenesis of padR to identify amino acid residues essential for the function of PadR. We identified an important consensus dyad sequence called IR1-2 (ATGT-8N-ACAT) overlapping a second dyad element (GTGT-8N-ACAT) that we named dIR1-2bis. The entire dIR1-2bis/IR1-2 sequence permits binding of two PadR dimers in EMSAs, which may be observed for bacteria grown under noninduced conditions where the padC promoter is completely repressed. Three groups of modified PadRs giving a PASR phenotype were characterized in vivo. The DNA sequences of certain mutant padR alleles indicate that important residues are all located in the region containing the coiled-coil leucine zipper domain that is involved in dimerization. These substitutions reduce the affinity of PadR binding to the padC promoter. Of particular interest are residue L128, located at the center of the putative coiled-coil leucine zipper domain, and residue E97, which is conserved among all PadRs.

The phenolic acid decarboxylase gene padA is involved in the phenolic acid stress response (PASR) in gram-positive bacteria. In Lactobacillus plantarum, the padR gene encodes the negative transcriptional regulator of padA and is cotranscribed with a downstream gene, usp1, which encodes a putative universal stress protein (USP), Usp1, of unknown function. The usp1 gene is overexpressed during the PASR. However, the role and the mechanism of action of the USPs are unknown in gram-positive bacteria. Therefore, to gain insights into the role of USPs in the PASR; (i) a usp1 deletion mutant was constructed; (ii) the two genes padR and usp1 were coexpressed with padA under its own promoter as a reporter gene in Escherichia coli; and (iii) molecular in vitro interactions between the PadR, Usp1, and the padA promoter were studied. Although the usp1 mutant strain retained phenolic acid-dependent PAD activity, it displayed a greater sensitivity to strong acidic conditions compared to that of the wild-type strain. PadR cannot be inactivated directly by phenolic acid in E. coli recombinant cultures but is inactivated by Usp1 when the two proteins are coexpressed in E. coli. The PadR inactivation observed in recombinant E. coli cells was supported by electrophoretic mobility shift assays. Although Usp1 seems not to be absolutely required for the PASR, its capacity to inactivate PadR indicates that it could serve as an important mediator in acid stress response mechanisms through its capacity to interact with transcriptional regulators.