DETECTION OF CHLOROCATECHOL 1,2-DIOXYGENASE GENES IN PROTEOBACTERIA BY PCR AND GENE PROBES

In various bacterial strains belonging to the beta-subdivision of prot eobacteria which are capable of degrading chlorinated monoaromatic com pounds, chlorocatechol 1,2-dioxygenase genes were detected by PCR and Southern hybridization. Using PCR primers derived from the conserved s equence motifs of chlorocatechol 1,2-dioxygenase genes tfdC, clcA and tcbC, PCR products of the expected size were obtained with the test st rains, but not with negative control strains. The specificity of the P CR products was verified by hybridization using an oligonucleotide pro be for an internal sequence motif which is evolutionarily conserved am ong chlorocatechol 1,2-dioxygenases and some other dioxygenases that c atalyze the intradiol aromatic-ring-cleavage. Hybridization with the t fdC PCR product from the 2,4-D degradative plasmid pJP4 under stringen t conditions revealed different extents of homology of the chlorocatec hol 1,2-dioxygenase genes to the canonical tfdC sequence in the variou s strains. These findings were confirmed by the nucleotide sequence an alysis of the tfdC-specific PCR products. From our results, we conclud e that the PCR primer set is more suitable than the hybridization with pJP4-derived gene probes for the detection of diverse chlorocatechol 1,2-dioxygenase genes in proteobacteria.