Characterization of the gene cluster of high - molecular - mass nitrile hydratase ( H - NHase ) induced by its reaction product in Rhodococcus rhodochrous Jl ( amide / regulation / AmiC / transposase / cobalt )

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 Jl 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-~5) in addition to HNHase aand 8-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 Jl, 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 HNHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates). Nitrile compounds containing a cyano functional group such as cyanoglycosides, cyanolipids, indole-3-acetonitrile and 03-cyano-L-alanine are formed by a wide range of plants (1). We have been studying microbial degradation of highly toxic nitriles (2) and found that their degradation can proceed through two enzymatic pathways; nitrile hydratase (NHase; EC 4.2.1.84) catalyzes the hydration of a nitrile to the corresponding amide which is then converted to the acid plus ammonia by amidase (3), while nitrilase catalyzes the direct hydrolysis of a nitrile to the corresponding acid plus ammonia (4). Interest in both nitrile-converting enzymes has increasingly focused on their versatile functions: biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile (5-9) and enzymatic production of useful compounds from nitriles (2). In microorganisms that catabolyze nitriles by NHase, an interesting phenomenon is found; this enzyme, if inducible, is generally induced by amides (reaction products), not by nitriles (reaction substrates) (2). Rhodococcus rhodochrous JI produces two kinds of NHases: highand low-molecular-mass NHases (H-NHase and L-NHase, respectively), which exhibit different physicochemical properties and substrate specificities. When this strain is cultured in a medium containing urea The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and cyclohexanecarboxamide in the presence of cobalt ions, H-NHase and L-NHase are selectively induced, respectively (2). We have cloned and sequenced both Hand L-NHase genes from R. rhodochrous Jl (10). In each of the Hand L-NHase genes, an open reading frame (ORF) for the /3 subunit is located just upstream of that for the a subunit. This arrangement of the coding sequences is the reverse of the order found in the NHase genes of Rhodococcus sp. N-774 (11) and Pseudomonas chlororaphis B23 (12). The genus Rhodococcus, a member of the class Actinomycetes (13), has recently received much attention in terms of its high ability on biodegradation and biotransformation (14). Rhodococcus has also been studied as a phytopathogen causing leafy galls (fasciation) on plants (15). However, genetic information of Rhodococcus has been extremely limited. We have examined the expression of both Hand L-NHase genes in Escherichia coli cells under the control of lac promoter, but the level of NHase activity in the cell-free extracts is much lower than those of Hand L-NHases in R. rhodochrous Jl (10), suggesting that an uncharacterized regulatory gene would be present in this strain. Research into the regulatory system of Rhodococcus species has so far been hampered by the lack of systems for genetic manipulation of Rhodococcus. In the present study, genes required for the expression of H-NHase have been identified by using a host-vector system in Rhodococcus.* The H-NHase gene cluster was also characterized to clarify its unusual induction mechanism in R. rhodochrous J1. MATERIALS AND METHODS Microbial Strains and Plasmids. R. rhodochrous Jl was previously isolated from soil (16). E. coli JM109 (17) was the host for pUC plasmids. R. rhodochrous ATCC12674 was the host for a Rhodococcus-E. coli shuttle vector plasmid pK4 (18) and its derivatives and was used for the expression of the H-NHase gene. The plasmid pNHJ10OH (10) carrying the H-NHase gene in a 6-kb Sac I fragment on pUC19 was used for subcloning and sequencing of genes. Transformation of R. rhodochrous ATCC12674 by Electroporation.A mid-exponential culture of R. rhodochrous ATCC12674 was centrifuged at 6500 x g for 10 min at 4°C and washed three times with demineralized cold water. Cells were then concentrated 20-fold in cold water and kept on ice. Ice-cold cells (100 ,ul) were mixed with 1 ,g ofDNA in 1 ,/l ofTE buffer (10mM Tris/1 mM EDTA, pH 8.0) in a 1-mm-gapped electrocuvette (Bio-Rad) and subjected to a 2.0-kV electric pulse from a Gene Pulser (Bio-Rad) connected to a pulse controller (25 ,uF capacitor; external resistance, 400 l). Pulsed cells were diluted immediately Abbreviations: NHase, nitrile hydratase; H-NHase, high molecular mass-NHase; L-NHase, low molecular mass-NHase; ORF, open reading frame. §To whom reprint requests should be addressed. *The sequence reported in this paper has been deposited in the GenBank/DDBJ data base (accession no. D67027).