RESUMO
Rhodococcus strains are important biocatalysts used for biotechnological production of acrylamide catalysed by a nitrile hydratase (NHase) containing cobalt. This metalloenzyme is present at high intracellular concentrations representing up to 50% of the soluble proteins in Rhodococcus rhodochrous M8 strain. Cobalt ions were formerly reported to be essential for the synthesis of the NHase subunits, encoded by nhmBA structural genes in R. rhodochrous M8. To understand the regulatory mechanisms enabling high expression of the NHase structural genes by cobalt, two reporter genes coding for an acylamidase from Rhodococcus erythropolis TA37 and a nitrilase from Alcaligenes denitrificans C-32 were fused to the nhmBA promoter. It was shown that cobalt-dependent regulation of transcription occurs independently of another regulatory genes, nhmCD, involved in substrate-dependent regulation of transcription. Cobalt ions led to an increase (up to five-fold) in transcription of reporter genes correlated with synthesis of corresponding enzymes in R. rhodochrous recombinant strains. This led to identification of a new transcriptional regulator from the ArsR family, named CblA. Using a cblA mutant strain, it was established that CblA acted as a repressor by preventing transcription of the NHase operon promoter in the absence of cobalt ions.
