Pyrene metabolism in cells of Pseudomonas putida strain KBM-1.

摘要

Pyrene is a well-known mutagen and suspected carcinogen, which causes serious health risks to human beings due to its wide presence in the environment. Using bacteria to decompose pyrene is an ideal procedure due to its non-invasive, non-disruptive and cost-effective features. Pseudomonas putida strain KBM-1 was able to metabolize pyrene in the presence of low amounts of naphthalene or salicylate. The molecular biology of pyrene degradation needs to be better understood in order to utilize strain KBM-1 for bioremediation. This research focused on the identification and cloning of the gene(s) encoding pyrene dioxygenase (Pdo), the first enzyme involved in the pyrene degradation pathway. Southern hybridization results revealed that pdo resides on a megaplasmid at two separate loci. When this megaplasmid was transformed into cells of E. coli, PAH utilization was observed by the transformed cells. Partial sequences of the KBM-1 pdo genes were determined and they were found to be similar to those in Mycobacterium spp. However, the translated peptide sequences suggest that extra amino acids are present in the Pdo of KBM-1. Sau3AI and HindIII genomic libraries were constructed for the sequencing of the complete megaplasmid, which will allow the elucidation of the roles that the megaplasmid plays in pyrene transport and degradation in strain KBM-1. This research will further the understanding of plasmid controlled polycyclic aromatic hydrocarbon (PAH) metabolism in microorganisms and aid in the development of more effective bioremediation strategies for pyrene and other PAH's using genetically engineered bacterial strains.