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A molecular screening approach was developed in order to amplify the genomic region that codes for the ␣-and ␤-subunits of the nitrile hydratase (NHase) enzyme in rhodococci. Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as Rhodococcus erythropolis. A hydratase PCR product was also obtained from R. erythropolis DSM 43066 T , which did not grow on nitriles. Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the R. erythropolis strains used as positive controls. PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the R. erythropolis strains revealed unique patterns that mostly correlated with distinct geographical sites of origin. Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases. The phylogenetic analysis and deduced amino acid sequences suggested that the novel R. erythropolis enzymes belonged to the iron-type NHase family. Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes. A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in R. erythropolis. Our findings indicate that the NHase and amidase genes present in geographically distinct R. erythropolis strains are not globally mixed.Nitrile hydratase (NHase) is a soluble bacterial metalloenzyme (41) that catalyzes the hydration of nitrile compounds to the corresponding amides, which may be converted by an amidase to the corresponding acids plus ammonia (40). NHase consists of two subunits (␣ and ␤), each with a molecular mass of approximately 23 kDa; the amino acid sequences of the subunits are not related, and the structural genes are normally adjacent in the same operon, although the ␣-and ␤-coding sequence order is variable (42). NHase is classified into two groups on the basis of the metal ion composing the catalytic center; an Fe-type NHase has a nonheme iron atom (74), and a Co-type NHase has a noncorrinoid cobalt atom (12) at the catalytic center. The Fe-and Co-type NHase enzymes differ in biotransformation activity and substrate specificity, although their amino acid sequences exhibit significant homology.NHase enzymes have considerable practical importance as biocatalysts for the industrial production of acrylamide and nicotinamide (84) and also for environmental bioremediation, where they have been shown to be effective in the removal of nitrile...

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Mycolic acid-containing actinomycetes capable of metabolizing nitriles were recovered from deep-sea sediments and terrestrial soils by enrichment culture on acetonitrile, benzonitrile, succinonitrile or bromoxynil. A total of 43 nitrile-degrading strains were isolated and, together with previously recovered nitrile-degrading rhodococci, were identified by a polyphasic taxonomic approach, which included mycolic acid profiles, pyrolysis mass spectrometry (PyMS), genomic fingerprinting based on sequence variability of the 16S ribosomal RNA gene using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism, and 16S rRNA gene sequence comparison. Isolates phylogenetically related to Rhodococcus erythropolis dominated the culturable microorganisms from most marine and terrestrial samples. These isolates clustered together in a major pyrogroup that showed high congruence with PRS profiles of the 16S rRNA gene. Such high congruence also was obtained for other recovered isolates that were assigned to species of Rhodococcus and Gordonia. Sequencing data validated the results obtained by PRS analysis and enabled phylogenetic relationships to be established. Some of the recovered bacteria probably represent novel microbial species. The fact that nitrile-metabolizing microorganisms were recovered from a wide range of habitat types suggests that nitrile transforming enzymatic activity is geographically widely distributed in nature.