CONSTRUCTION OF A DNA-PROBE TO DETECT ISOQUINOLINE-DEGRADING BACTERIA

To facilitate the cloning of DNA encoding isoquinoline degradation an assay was developed that allowed the rapid visual scoring of the isoqu inoline degradation phenotype of single colonies. Transposon mutagenes is of one of the isolates, Comamonas acidovorans IQ3, was performed us ing Tn5, and nine Isq(-) mutants deficient in the ability to utilise i soquinoline as the sole nitrogen source were isolated. These mutants w ere also incapable of utilising the first metabolite of the isoquinoli ne degradation pathway, 1-hydroxyisoquinoline, as the sole carbon sour ce. For each Isq(-) mutant, the EcoRI fragment containing the Tn5 inse rtion was cloned into pBR322. Restriction and Southern analyses of the cloned DNA revealed that of the nine Isq(-) mutants, six contained Tn 5 insertions in a common 8.9-kb EcoRI fragment derived from the wild t ype, C. acidovorans IQ3. The cloned DNA thought to be involved in the degradation of isoquinoline proved to be specific when used as a probe in colony hybridization to some bacteria possessing the ability to de grade isoquinoline.