Characterization by arbitrary primer polymerase chain reaction of polychlorinated biphenyl (PCB)-degrading strains of Comamonas testosteroni isolated from PCB-contaminated soil

Abstract

In this study, we isolated and characterized biphenyl (BP) and polychlorinated biphenyl (PCB) degrading bacterial strains found in PCB-contaminated soil from an auto manufacturing plant located in Syracuse, New York. Twenty-one BP and PCB-degrading bacteria were randomly selected to form a representative sample of the bacterial population present at the site. Of the 21 bacteria, 13 were identified as Comamonas testosteroni, constituting about 60% of the bacterial population examined. Other PCB degraders identified were Acidovorax facilis, Alcaligenes xylosoxydans, Bacillus sphericus, Hydrogenophaga pseudoflava, Pseudomonas avanae, and Rhodococcus fascians. Owing to the abundance of C. testosteroni at this site, only these isolates were further characterized for their PCB congener degradation profile, 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and genetic relatedness by polymerase chain reaction (PCR) analysis. The PCB congener degradation pattern revealed a high degree of variability among the C. testosteroni isolates. The majority of the C. testosteroni isolates tested could degrade more than 95% of the PCB congeners up to pentachiorinated biphenyl. Only four isolates could degrade more than 80% of hexachlorobiphenyl. All 12 isolates of C. testosteroni tested were able to attack 2,3,4,5,6,3′,4′-heptachlorobiphenyl, indicating involvement of biphenyl 2,3-dioxygenase, while 2,3,5,6,2′,3′,6′-heptach!orobiphenyl was attacked by 6 strains, suggesting an oxidation reaction mediated by 3,4-dioxygenase. 2,3-Dihydroxybiphenyl 1,2-dioxygenase activity was also found to vary among the C. testosteroni isolates tested in this study. Eleven strains showed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity specific for 2,3-dihydroxybiphenyl, whereas isolate BW169 could metabolize both 2,3-dihydroxybiphenyl and 4-methylcatechol, and isolate BW74 had the ability to metabolize all three substrates (2,3-dihydroxybiphenyl, 4-chlorocatechol, and 4-methylcatechol). Further molecular characterization of these isolates was done by a PCR-based assay, random amplified polymorphic DNA (RAPD) analysis. Amplifications of common DNA fragments of 450 dp using primer OPJ6 and 650 bp using primer OPJ14 were identified in the C. testosteroni isolates, although a distinct RAPD pattern was obtained for all of the isolates using primer OPJ14, and a combination of the two primers. Therefore, the results presented in this study demonstrate that RAPD can be used to fingerprint the C. testosteroni isolates.Key words: comamonas testosteroni, polychlorinated biphenyl, arbitrary primer-PCR.