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-d egrading (mcd) gene in bacterial cells by the polymerase chain reactio n (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 sp ecific primers, was strongly correlated to its detection by DNA hybrid ization with an mcd gene probe. There was substantial homology, as det ermined by Southern hybridization and PCR-RFLP analyses, amongst the P CR-amplified mcd gene products from 24 diverse carbofuran-degrading so il bacteria. Hybridization of plasmid profiles of 57 carbofuran-degrad ing bacteria with plasmid pPDL11 showed that there is extensive homolo gy between this plasmid and other approximately 100 kb plasmids, from 23 geographically, phenotypically and genetically diverse soil bacteri a, 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 degradat ive plasmid and that very similar plasmids are present in bacteria wit h different chromosomal and plasmid backgrounds. (C) 1997 Published by Elsevier Science Ltd.