Molecular functional analysis of gene cluster involved in nitrile metabolism

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Actinomycete Rhodococcus rhodochrous J1 has two NHases depending on an inducer in the presence of cobalt ions ; when the strain is cultured in the medium containing urea and cyclohexanecarboxamide, high-Mr- (H-) and low-Mr- (L-) NHases are selectively induced, respectively.H-NHase gene was expressed as an active form in R.rhodochrous ATCC12674 using a Rhodococcus-E.coli host-vector (pK4) system, whereas it was expressed as an insoluble (inactive) form in E.coli. SDS/PAGE of cell-extracts of R.rhodochrous transformant carrying plasmid pHJK19, in which a 6.5-kb sequence covering 4.6-kb upstream region, 1.3-kb H-NHase gene and 0.6-kb downstream region was inserted into pK4, showed that the amount of alpha-and beta-subunits of H-NHase was about 50% of the total soluble protein. Analysis using the host-vector system revealed that the 4.6kb region was required for the expression of H-NHase gene. Sequence analysis clarified that one of OFRs in the region had significantly similarity to AmiC which was the negative regulatory protein involved in the expression of an amidase gene from Pseudomonas aeruginosa. Northern blot analysis showed that the genes for the alpha-and beta-subunits of H-NHase were cotranscribed as a 1.8-kb mRNA.Determination of H-NHase activity and H-NHase mRNA levels indicated that the expression of H-NHase gene was regulated by urea at transcriptional level, but not by cobalt ions.