Cloning and nucleotide sequence of carbazole catabolic genes from Pseudomonas stutzeri strain OM1, isolated from activated sludge

Abstract

A new carbazole (CAR)-degrading bacterium, called strain OM1, was isolated from activated sludge obtained from sewage disposal plants in Fukuoka Prefecture, and it was identified as Pseudomonas stutzeri. Anthranilic acid (AN), 2'-aminobiphenyl-2,3-diol and its meta-cleavage product, 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid, were identified as metabolic intermediates of CAR in the ethyl acetate extract of the culture broth. Therefore, the CAR catabolic pathway to AN in strain OM1 was indicated to be identical to those found in the Pseudomonas sp, strains 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 EcoRI fragment and a 3-kb Pstl-Sphl fragment were isolated from colonies, forming a clear zone of CAR and a yellow ring-cleavage product from CAT, respectively. Recombinant E. coli carrying the 6.9-kb fragment degraded CAR in the L-broth and produced AN. Cell-free extract from the clone carrying a 3-kb Pstl-Sphl fragment had high meta-ring-cleavage dioxygenase activity for CAT. The nucleotide sequences of these fragments were determined. The 6.9-kb fragment showed a very high degree of homology with the CAR catabolic genes of strain CA10. The amino acid and nucleotide sequences of the 3-kb fragment were found to exhibit significant homology with the genes for the CAT-catabolic enzymes of TOL plasmid pWWO, plasmid NAH7, and plasmid pV1150.