A CLOSTRIDIUM-ACETOBUTYLICUM REGULATOR GENE (REGA) AFFECTING AMYLASE PRODUCTION IN BACILLUS-SUBTILIS

Plasmid pMET7C containing a 6.05 kb DNA insert from Clostridium acetob utylicum P262 made Escherichia coli F19 cells sensitive to metronidazo le. The nucleotide sequence of the C, acetobutylicum DNA controlling m etronidazole sensitivity in E. coil 519 revealed an ORF of 972 bp whic h encoded a protein of 324 amino acids with a calculated M(r) of 35000 . The amino acid sequence encoded by the ORF contained a helix-turn-he lix DNA-binding domain and was homologous to the catabolite control pr otein, CcpA, from Bacillus subtilis and Bacillus megaterium, a tRNA re pressor of E. coil encoded by the shl gene, and the GalR, Lacl and Pur R repressors of E. coil. The C. acetobutylicum ORF, which was termed r egA, complemented a B, subtilis ccpA mutant and an E. coil shl mutant, but was unable to complement E. coil galR, lacl or purR mutants. To d etermine whether the regA gene product was involved in the regulation of amylase gene expression in C. acetobutylicum, a starch-degrading en zyme gene (staA) from C. acetobutylicum NCIMB 8052 was cloned. The Reg A protein inhibited the degradation of starch by the C. acetobutylicum staA gene product in E. coli.