A Clostridium acetobutylicum regulator gene (regA) affecting amylase production in Bacillus subtilis

Abstract

Plasmid pMET7C containing a 6.05 kb DNA insert from Clostridium acetobutylicum P262 made Escherichia coli F19 cells sensitive to metronidazole. The nucleotide sequence of the C. acetobutylicum DNA controlling metronidazole sensitivity in E. coli F19 revealed an ORF of 972 bp which encoded a protein of 324 amino acids with a calculated Mr of 35,000. The amino acid sequence encoded by the ORF contained a helix-turn-helix DNA-binding domain and was homologous to the catabolite control protein, CcpA, from Bacillus subtilis and Bacillus megaterium, a tRNA repressor of E. coli encoded by the shl gene, and the GalR, Lacl and PurR repressors of E. coli. The C. acetobutylicum ORF, which was termed regA, complemented a B. subtilis ccpA mutant and an E. coli shl mutant, but was unable to complement E. coli galR, lacl or purR mutants. To determine whether the regA gene product was involved in the regulation of amylase gene expression in C. acetobutylicum, a starch-degrading enzyme gene (staA) from C. acetobutylicum NCIMB 8052 was cloned. The RegA protein inhibited the degradation of starch by the C. acetobutylicum staA gene product in E. coli.