Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1.
The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).
Komeda
H
H
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Kobayashi
M
M
Shimizu
S
S
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D018274:Electroporation; D004790:Enzyme Induction; D005246:Feedback; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D012329:RNA, Bacterial / Q000302:isolation & purification; D015183:Restriction Mapping; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial
D012329:RNA, Bacterial; D006836:Hydro-Lyases; C048831:nitrile hydratase