Degradation of pentachlorophenol by mutant strains of Sphingomonas chlorophenolica ATCC 39723

Abstract

The site-directed insertion mutagenesis of pcpD can be obtained via allelic exchange with the corresponding mutated gene for the study of the role of pcpD gene in pentachlorophenol degradation in Sphingomonas chlorophenolica ATCC 39723. The targeting vector was constructed by cloning the pcpD open reading frame into pUC 19 and interrupting the open reading frame by insertion of kanamycin resistance gene. This targeting vector was transformed into S. chlorophenolica. Homologous recombination between pcpD locus in genome and mutated pcpD fragment in targeting vector could be occurred and pcpD locus in genome could be replaced by mutated pcpD fragment in targeting. Transforments containing mutated pcpD gene could be selected in medium containing kanamycin. In this work, after transformation of this targeting vector into S. chlorophenolica, kanamycin resistance colonies became visible after incubation for three days. Southern blot analysis of genomic DNAs isolated from twenty-seven kanamycin resistance clones showed that all of them gave signal bands with pcpD gene probe but not with kanamycin resistance gene probe. Therefore, S. chlorophenolica did not have kanamycin resistance gene inserted into their genomic DNAs. All of which were mutant strains of S.chlorophenolica which did not contain disrupted pcpD gene. Extraction of plasmid DNA in mutant strains did not give the targeting vector. Analysis of pentachlorophenol degradation ability by mutant strains showed that these mutants could degrade pentachlorophenol 25-99% in two hours as compared to the wild type strain.