Diversity of nitrile hydratase and amidase enzyme genes in Rhodococcus erythropolis recovered from geographically distinct habitats

Abstract

A molecular screening approach was developed in order to amplify the genomic region that codes for the alpha- and beta-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.

FIG. 1.

PCR-SSCP profiles of the NHase α-subunit gene (α-nhase) and β-subunit gene (β-nhase) present in R. erythropolis strains. A complete list of the recently recovered R. erythropolis strains used in the PCR-SSCP assay is available elsewhere (9). An asterisk indicates a representative strain to which a PCR-SSCP pattern code was assigned; PCR-SSCP pattern codes for representative strains are shown in Table 3.

FIG. 2.

PRS profiles of the complete NHase genes present in R. erythropolis strains isolated in our laboratory and obtained from other laboratories and culture collections. PCR products were digested with restriction enzyme AccI. The αβ-nhase PRS pattern codes assigned to the strains are shown in Table 1.

FIG. 3.

Neighbor-joining tree showing the phylogenetic relationships of the complete NHase genes (αβ-nhase) from R. erythropolis strains and other Rhodococcus species. The αβ-nhase PRS pattern code for each strain is indicated in parentheses (Fig. 2 and Table 1). The tree was constructed based on an examination of 1,282 NHase gene nucleotide positions. The values at the nodes indicate the levels of bootstrap support based on a neighbor-joining analysis of 1,000 resampled data sets. Scale bar = 0.01 substitution per nucleotide position.

FIG.4.

Alignment of deduced amino acid sequences of the α-subunit (A) and β-subunit (B) of R. erythropolis NHases. The residue numbers are in accordance with those of the Fe-type NHase from strain R312 (32). Note that because the methionine (M) residue at the N terminus is cotranslationally removed in the α-subunit of the strain N-771 enzyme, the residue numbers for this NHase α-subunit should be one less than the numbers for the other enzymes (77). The NHases of the following strains were used: N-774 (33), N-771 (62, 86), AJ270 (DDBJ/EMBL/GenBank accession number AJ278349), ACV2 (DDBJ/EMBL/GenBank accession number Z48769), R312 (49), 122-AN065 (122), ENG-AN033 (33), DSM 13002 (DSM), DSM 43066^T (N11), ARG-AN024 (24), IND-AN014 (14), 871-AN042 (42), 871-AN053 (53), 870-AN019 (19), ANT-AN007 (7), ARG-AN025 (25), 67-BEN001 (67) (this study), R. rhodochrous IFO 15564 (IFO) (DDBJ/EMBL/GenBank accession number E12518), and JCM 6823 (JCM) (22). Different amino acids in a single sequence or in different sequences are indicated by a black background. The residues conserved in Fe- and Co-type NHases and thought to have direct effects in the active site are indicated by boldface type (32, 59). The gray background indicates the residues that form the iron active center of Fe-type NHase (32, 56). Asterisks indicate the conserved residues in Fe- and Co-type NHases and thiocyanate hydrolase (36, 59).