PCR detection of the mcd gene and evidence of sequence homology between the degradative genes and plasmids from diverse carbofuran-degrading bacteria

We report a method for the specific detection of the methylcarbamate-degrading ( mcd) gene in bacterial cells by the polymerase chain reaction (PCR) and suggest that combined use of the PCR with a specific gene probe would improve sensitivity of detection of this gene in isolated bacteria and directly in soil DNA. PCR detection of this gene using specific primers, was strongly correlated to its detection by DNA hybridization with an mcd gene probe. There was substantial homology, as determined by Southern hybridization and PCR-RFLP analyses, amongst the PCR-amplified mcd gene products from 24 diverse carbofuran-degrading soil bacteria. Hybridization of plasmid profiles of 57 carbofuran-degrading bacteria with plasmid pPDL11 showed that there is extensive homology between this plasmid and other approximately 100 kb plasmids, from 23 geographically, phenotypically and genetically diverse soil bacteria, which all contain sequences homologous to the mcd gene. Restriction fragment length polymorphism (RFLP) patterns using the plasmid probe showed that there are at least five polymorphic types of this degradative plasmid and that very similar plasmids are present in bacteria with different chromosomal and plasmid backgrounds.