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

Abstract

Nitriles are widespread in the environment as a result of biological and industrial activity. Nitrile hydratases catalyse the hydration of nitriles to the corresponding amide and are often associated with amidases, which catalyze the conversion of amides to the corresponding acids. Nitrile hydratases have potential as biocatalysts in bioremediation and biotransformation applications, and several successful examples demonstrate the advantages. In this work a real-time PCR assay was designed for the detection of Fe-type nitrile hydratase genes from environmental isolates purified from nitrile-enriched soils and seaweeds. Specific PCR primers were also designed for amplification and sequencing of the genes. Identical or highly homologous nitrile hydratase genes were detected from isolates of numerous genera from geographically diverse sites, as were numerous novel genes. The genes were also detected from isolates of genera not previously reported to harbour nitrile hydratases. The results provide further evidence that many bacteria have acquired the genes via horizontal gene transfer. The real-time PCR assay should prove useful in searching for nitrile hydratases that could have novel substrate specificities and therefore potential in industrial applications.

Biodegradation of thiocyanate by a novel strain of Burkholderia phytofirmans from soil contaminated by gold mine tailings

“…; Coffey et al . , ), but either the 16S rRNA gene sequence or environmental isolation source information is unpublished at this time. The minimal nutrient requirements of this new B. phytofirmans strain, along with the observed rapid thiocyanate and sulfate consumption, will facilitate bioremediation strategies faced with the dual challenge of mitigating both thiocyanate toxicity and acid production.…”

Assessment of the Bacteriocinogenic Potential of Marine Bacteria Reveals Lichenicidin Production by Seaweed-Derived Bacillus spp.

“…In addition, 16S rRNA gene sequencing was performed on each isolate, by amplifying the 16S rRNA gene in a 15 µL reaction volume using primers 63f and 1387r, purifying the PCR products and sequencing them on an AB 310 genetic analyzer (Applied Biosystems, CA, USA), as outlined by Coffey et al [35]. Nucleotide sequences were compared with those in the GenBank database using the blast program [36] through the National Center for Biotechnology Information (NCBI) server [37].…”

“…T7 promoter and M13 forward (−20) primers were used for the sequencing reactions of the cloned PCR products. DNA sequencing was performed as described by Coffey et al [35] and the sequences obtained were analyzed using BLAST software [36] from the Genbank (NCBI) database. Nucleic acid and deduced amino acid sequences were analyzed with DNASTAR software (DNASTAR Inc., Madison, USA).…”

The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870

“…R. jostii RHA1 expresses a nitrile hydratase (an acetonitrile hydratase) and utilises nitriles such as acetonitrile, acrylonitrile, propionitrile and butyronitrile [123], while R. opacus expresses nitrile hydrolysis activity [116]. R. erythropolis PR4 expresses a Fe-type nitrile hydratase [124], and R. erythropolis strains are well known for expressing this enzyme [116,125,126] as part of a nitrile metabolism gene cluster [122]. This enzyme has been repeatedly determined in this species from isolated diverse locations [127], expressing broad substrate profiles, including acetonitrile, propionitrile, acrylonitrile, butyronitrile, succinonitrile, valeronitrile, isovaleronitrile and benzonitrile [116].…”