6533b860fe1ef96bd12c3672

RESEARCH PRODUCT

Genetic and biochemical characterization by random and site-directed mutagenesis, repressors involved in stress response phenolic acids in Bacillus subtilis

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The aim of this work was to characterize the genetic and biochemical mechanisms involved in the PASR, the phenolic acid stress response induced by phenolic acids Bacillus subtilis. By genetics and molecular biology strategies, like reverse genetic, PCR or transposon random mutagenesis, gene deletion, complementation of strains, and gene expression, the main components of the PASR were identified and quantified. Site-directed and random mutagenesis of respectively the promoter of padC encoding the phenolic acid decaroxylase, and PadR, the repressor of the padC expression, allowed us to identify the site of binding of PadR with this promoter, as well as the amino-acid residues involved in the functionality of PadR. Results obtained by the use of reporter strains of B. Subtilis construct for this aim, were confirmed by in vitro foot-printing and EMSA, and heterologous co-expression of the genes in E. Coli. This work provides evidence that a supposed non-functional truncated yveFG gene, co-transcript with padC, encoded a YveF peptid which is a moderator of the padC expression, and that the repaired gene was a non-inactivable repressor of padC expression.