Analysis of changes in congener selectivity during PCB degradation by Burkholderia sp. strain TSN101 with increasing concentrations of PCB and characterization of the bphBCD genes and gene products.

We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 microg Kaneclor 300/ml, a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls in medium containing 25 microg Kaneclor 300/ml. Using PCB concentrations of 50-150 microg of Kaneclor 300/ml, the congener selectivity pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 microg Kaneclor 300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2'- and 4'-positions. A 5-kb DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated 99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2, 3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the cloned bph gene in biphenyl metabolism.