Searching for nitrile hydratase using the Consensus-Degenerate Hybrid Oligonucleotide Primers strategy.
Consensus-Degenerate Oligonucleotide Hybrid Primers (CODEHOP) were designed by using the WWW-implemented strategy, based on multiple alignments of nitrile hydratase (NHase) alpha subunit, available from EMBL database. These primers were successfully tested with known NHase-producing bacterial strains such as Agrobacterium tumefaciens DSM 9674, Rhodococcus erythropolis DSM 9675, R. erythropolis 9685 and R. erythropolis 11397 and also allowed amplification from organisms not previously referenced (Variovorax sp. DSM 11402 and Agrobacterium sp. DSM 11401). Hybrid primer utilisation was evaluated by analysing the incorporated sequences in cloned PCR fragments. Several primers from the oligonucleotide pool seemed to participate in the amplification of the correct fragment. Common garden soil was used as a source for both acetonitrile culture enrichment screening and direct DNA extraction. A R. erythropolis strain (CCMI 1005) was isolated by culture enrichment and allowed the PCR amplification of a DNA sequence (AJ548493) identical to the NHase coding sequences commonly described for that species, while the direct use of soil DNA as template led to the CODEHOP detection of two putative NHase coding sequences (AJ548498 and AJ548499), which were significantly different from any other known sequence. The phylogenetic relationship between all sequences obtained and the published NHase coding sequences was assessed by neighbour-joining analysis. The results demonstrate the use of consensus-degenerate primers in NHase detection from organisms known to express it and in the screening for new NHase family members.
Lourenço
Pedro Miguel Lopes
PM
Departamento de Biotecnologia, Instituto Nacional de Engenharia e Tecnologia Industrial,. Ed. F, 1649-038 Lisboa, Portugal.
Almeida
Tânia
T
Mendonça
Diogo
D
Simões
Fernanda
F
Novo
Carlos
C
D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D042621:Comamonadaceae / Q000201:enzymology / Q000235:genetics; D016384:Consensus Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D021122:Protein Subunits; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D017931:DNA Primers; D004269:DNA, Bacterial; D021122:Protein Subunits; D006836:Hydro-Lyases; C048831:nitrile hydratase