Studies On The Degradation Of 4-Chloronitrobenzene In Pseudomonas Putida ZWL73

A strain of Pseudomonas putida ZWL73 was isolated from soil contaminated with chloronitrobenzenes and identified by 16S rDNA sequencing. This bacterium released chloride and ammonia into the medium when grown on 4-chloronitrobenzene (4CNB) as sole source of carbon, nitrogen and energy. The 4CNB nitroreductase and 2-amino-5-chlorophenol 1,6-dioxygenase activities were detected from the cell-free extract of ZWL73, which implied presence of a partial reductative pathway for the 4CNB degradation in this strain. A plasmid designated pZWL73 of approximately 100 kb in this strain was also found to be responsible for 4CNB degradation. This was based on the fact that the plasmid-cured strains showed 4CNB- phenotype and the 4CNB~+ phenotype could be conjugally transferred. The cell free extracts of strain ZWL73 exhibited chloronitrobenzene nitroreductase and 2-amino-5-chlorophenol 1, 6-dioxygenase (2A5CPDO) activities, but neither activity was found from that of the plasmid-cured strain.A plasmid DNA library was constructed , from which we have cloned a 4.9-kb EcoRI fragment exhibiting 2A5CPDO activity. Sequencing results revealedβsubunit (cnbCa) andαsubunit (cnbCb) of a meta-cleavage dioxygenase, which were subsequently expressed in E.coli with 2A5CPDO activity. 2A5CPDO also showed activities towards 2-aminophenol(2AP) ,catechol and protocatechuate. The K_m values of...