RecA relieves negative autoregulation of rdgA, which specifies a component of the RecA-Rdg regulatory circuit controlling pectin lyase production in Erwinia carotovora ssp. carotovora

The production of pectin lyase (Pnl) in Erwinia carotovora ssp, carotovora strain 71 is induced by DNA-damaging agents such as mitomycin C (MC). This induction requires functions of recA, rdgA and rdgB genes. Based upon sequence homology, rdgA was predicted to encode a repressor and rdgB was presumed to specify a transcriptional activator, To elucidate the function of rdgA, the gene has been over-expressed in Escherichia coli, and the 30 kDa product purified by ammonium-sulphate precipitation, heparin-agarose chromatography and gel filtration. The results of gel mobility-shift and DNase I protection assays revealed that purified RdgA specifically binds the rdgA operator sequence located between the -10 and -35 boxes. The expression of a rdgA-lacZ gene fusion in E. coli MC4100 is suppressed upon overproduction of RdgA from a Ptac-rdgA construct induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). However, the suppression of rdgA-lacZ expression is relieved by MC in the RecA(+) E. coli strain MC4100, but not in its RecA(-) derivative, MC4160, An immunoblot analysis revealed RecA-dependent in vivo cleavage of the 30 kDa RdgA protein upon MC treatment, These results demonstrate that the transcription of rdgA is autoregulated, and strongly support the idea that proteolytic activity of RecA* is responsible for the derepression of rdgA expression.