The malonate decarboxylase operon of Acinetobacter calcoaceticus KCCM 40902 is regulated by malonate and the transcriptional repressor MdcY

A regulatory gene-like open reading frame oriented oppositely to mdcL, coin ed mdcY, was found upstream from the structural genes of the mdcLMACDEGBH o peron in Acinetobacter calcoaceticus KCCM 40902. To elucidate the function of this gene, mdcY was expressed in Escherichia coil, and the MdcY protein was purified to homogeneity. Its DNA binding activity and binding site were examined by gel retardation and footprinting assays in vitro and by site-d irected mutagenesis of the binding sites in vivo. The regulator bound targe t DNA regardless of the presence of malonate, and the binding site was foun d centered at -65 relative to the mdcL transcriptional start site and conta ins a 12-bp palindromic structure (5'-ATTGTA/TACAAT-3'). Using a promoter f usion to the reporter gene luc, we found that the promoter P-mdcY is negati vely regulated by MdcY independent of malonate, However, the promoter P-mdc L recovered its activity in the presence of malonate. When mdcY was introdu ced into A. calcoaceticus KCCM 40902 in which the gene is inactivated by an IS3 family element, malonate decarboxylase was significantly repressed in cultures growing in acetate, succinate, or Luria-Bertani medium. However, i n cells growing in malonate, malonate decarboxylase was induced, indicating that MdcY is a transcriptional repressor and that malonate or a product re sulting from malonate metabolism should be the intracellular inducer of the mdc operon.