CLONING AND NUCLEOTIDE-SEQUENCE OF CARBAZOLE CATABOLIC GENES FROM PSEUDOMONAS-STUTZERI STRAIN OM1, ISOLATED FROM ACTIVATED-SLUDGE

A new carbazole (CAR)-degrading bacterium, called strain OM1, was isol ated from activated sludge obtained from sewage disposal plants in Fuk uoka Prefecture, and it was identified as Pseudomonas stutzeri. Anthra nilic acid (AN), 2'-aminobiphenyi-2,3-diol and its meta-cleavage produ ct, 2-hydroxy-6sxo-6-(2'-aminophenyl)-hexa-2 acid, were identified as metabolic intermediates of CAR in the ethyl acetate extract of the cul ture broth. Therefore, the CAR catabolic pathway to AN in strain OM1 w as indicated to be identical to those found in the Pseudomonas sp. str ains CA06 and CA10. The strain OM1 degraded catechol (CAT) via a meta- cleavage pathway in contrast to strains CA06 and CA10, which transform catechol into cis, cis-munonic acid. Clones containing a 6.9-kb EcoRl fragment and a 3-kb Pstl-Sphl fragment were isolated from colonies, f orming a clear zone of CAR and a yellow ring-cleavage product from CAT , respectively. Recombinant E. coli carrying the 6.9-kb fragment degra ded CAR in the L-broth and produced AN. Cell-free extract from the clo ne carrying a 3-kb Pstl-Sphl fragment had high meta-ring-cleavage diox ygenase activity for CAT. The nucleotide sequences of these fragments were determined. The 6.9-kb fragment showed a very high degree of homo logy with the CAR catabolic genes of strain CA10. The amino acid and n ucleotide sequences of the 3-kb fragment were found to exhibit signifi cant homology with the genes for the CAT-catabolic enzymes of TOL plas mid pWW0, plasmid NAH7, and plasmid pVl150.