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

Abstract

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.

Real-time PCR detection of Fe-type nitrile hydratase genes from environmental isolates suggests horizontal gene transfer between multiple genera

“…Of the soil isolates, 13 were previously shown to carry a nitrilase gene (Coffey et al 2009). Although the remaining isolates must harbour genes involved in nitrile metabolism, the aim of this study was to identify Fe-type NHases only.…”

“…Mutations may have caused sequence changes, but perhaps only the genes that still encode a functional NHase are laterally transferred due to functional selection (Coffey et al 2009). This would explain the higher predicted protein sequence identity between the NHases, where many single DNA residue changes have not caused a change in amino acid sequence.…”

“…Soil samples and seaweed marine broth cultures were enriched in M9 minimal media broth (Sambrook et al 1989) with modifications (O'Mahony 2004) with different nitriles (10 mM) as the N source (acetonitrile, benzonitrile, adiponitrile, mandelonitrile, acrylonitrile or phenylacetonitrile), and pure isolates were obtained from the enrichment cultures as described previously (Coffey et al 2009), with the following exception; agar-agar ultrapure (Merck, Darmstadt, Germany) was used for all M9 agar.…”

Characterization of α-Nitrile Hydratase and Amidase of Rhodococcus aff. qingshengii from Indonesia

“…Until now, the majority of bacteria producing NHase and amidase enzymes have been reported from shallow marine sediment (Langdahl et al 1996), deepsea sediments (Heald et al 2001), geothermal habitats (Pereira et al 1998), and various soils (Blakey et al 1995;Brandão et al 2003). Polluted environment (Cahill 2004;Kabaivanova et al 2005;Coffey et al 2009) have also been explored in the discovery of novel bacteria producing NHase and amidase enzymes. In the current study, two strains of α-NHase and amidase producing bacteria isolated from polluted river in Cibinong, West Java (Indonesia) were determined using sequence analyses generated from 16S rRNA region.…”

The use of clade‐specific PCR assays to identify novel nitrilase genes from environmental isolates

“…Nitrilase enzymes (EC 3.5.5.1) are involved in the direct conversion of nitriles to their corresponding carboxylic acids and ammonia through a one‐step reaction (Banerjee et al, ). Microbial nitrilases are abundant in nature due to their participation in degradation pathways for both natural and xenobiotic nitriles (Baxter & Cummings, ; Coffey et al., ; Prasad & Bhalla, ).…”