Occurrence and expression of glutathione-S-transferase-encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl-utilizing bacteria

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Abstract
The gene bphK of Burkholderia sp. strain LB400 has previously been shown to be located within the bph locus, which specifies the degradation of biphenyl (BP) and chlorobiphenyls, and to encode a glutathione S-transferase (GST) which accepts 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The specific physiological role of this gene is not known. It is now shown that the gene is expressed in the parental organism and that GST activity is induced more than 20-fold by growth of the strain on BP relative to succinate when these compounds serve as sole carbon source. Approximately the same induction factor was observed for 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, which is encoded by the 5′-adjacent bphC gene. This suggests that the expression of bphK is coregulated with the expression of genes responsible for the catabolism of BP. A bphK probe detected only a single copy of the gene in strain LB400. A spontaneous BP− mutant of the organism neither gave a signal with the bphK probe nor showed CDNB-accepting GST activity, suggesting that this activity is solely encoded by bphK. Complementation of the mutant with a bph gene cluster devoid of bphK restored the ability to grow on BP, indicating that bphK is not essential for utilization of this carbon source. BphK activity proved to be almost unaffected by up to 100-fold differences in proton concentration or ionic strength. The enzyme showed a narrow range with respect to a variety of widely used electrophilic GST substrates, accepting only CDNB. A number of established laboratory strains as well as novel isolates able to grow on BP as sole carbon and energy source were examined for BphK activity and the presence of a bphK analogue. CDNB assays, probe hybridizations and PCR showed that several, but not all, BP degraders possess this type of GST activity and/or a closely related gene. In all bacteria showing BphK activity, this was induced by growth on BP as sole carbon source, although activity levels differed by up to 10-fold after growth on BP and by up to 60-fold after growth on succinate. This resulted in a variation of induction factors between 2 and 30. In the majority of bphK + bacteria examined, the gene appeared to be part of LB400-like bph gene clusters. DNA sequencing revealed almost complete identity of bphK genes from five different bph gene clusters. These results suggest that bphK genes, although not essential, fulfil a strain-specific function related to the utilization of BPs by their host organisms. The usefulness of BphK as a reporter enzyme for monitoring the expression of catabolic pathways is discussed.