Isolation of identical nitrilase genes from multiple bacterial  strains and real-time PCR detection of the genes from soils  provides evidence of horizontal gene transfer.
Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be wide-spread in the environment.
Coffey
Lee
L
Department of Chemical and Life Sciences, Waterford Institute of Technology, Waterford, Ireland. lcoffey@wit.ie
Clarke
Adrienne
A
Duggan
Patrick
P
Tambling
Karen
K
Horgan
Serenia
S
Dowling
David
D
O'Reilly
Catherine
C
D000097:Acetonitriles / Q000378:metabolism; D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D000097:Acetonitriles; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C006725:benzyl cyanide; D006836:Hydro-Lyases; C048831:nitrile hydratase