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This study highlights the effect of heavy metal ions on the expression of cobalt-containing nitrile hydratase (NHase) in Rhodococcus strains, which over-produce this enzyme. Both metal-dependent derepression of transcription and maturation of NHase were considered. We demonstrated that nickel ions can derepress the NHase promoter in several Rhodococcus strains. The cblA gene of a cobalt-dependent transcriptional repressor was shown to be indispensable for nickel-mediated derepression. As for maturation, we showed that nickel ions could not replace cobalt ions during the synthesis of active NHase. We also revealed that the amount of β-subunit decreased during NHase expression without added cobalt. We showed this using three variants of NHase in vivo synthesis: by using nickel- or urea-induced synthesis in cblA+ strains, and by using metal-independent constitutive synthesis in cblA- strains. In all cases, we found that the amount of β-subunit was significantly lower than the amount of α-subunit. In contrast, equimolar amounts of both subunits were synthesized after growth in the presence of added cobalt. Nickel did not affect NHase synthesis in mixtures with cobalt. This suggests that the metal selectivity in cblA-dependent regulation of NHase transcription was too low to discriminate between cobalt and nickel, but the selectivity of the NHase maturation mechanism was high enough to do so. Moreover, we can assume that the β-subunit is more subject to proteolytic degradation without the addition of cobalt, than the α-subunit. This indicates that cobalt ions presumably play an unknown role in the stability of the β-subunit in vivo.

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., (Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)