Occurrence and expression of glutathiane-S-transferase-encoding bphK genesin Burkholderia sp strain LB400 and other biphenyl-utilizing bacteria

The gene bphK of Burkholderia sp. strain LB400 has previously been shown to be located within the bph locus, which specifies the degradation of biphen yl (BP) and chlorobiphenyls, and to encode a glutathione S-transferase (GST ) which accepts 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The specif ic physiological role of this gene is not known. It is now shown that the g ene is expressed in the parental organism and that CST 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 inducti on factor was observed for 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, which is encoded by the 5'-adjacent bphC gene. This suggests that the expre ssion of bphK is coregulated with the expression of genes responsible for t he catabolism of BP. A bphK probe detected only a single copy of the gene i n strain LB400. A spontaneous BP- mutant of the organism neither gave a sig nal with the bphK probe nor showed CDNB-accepting CST 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 sourc e. BphK activity proved to be almost unaffected by up to 100-fold differenc es in proton concentration or ionic strength. The enzyme showed a narrow ra nge 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 exa mined for BphK activity and the presence of a bphK analogue. CDNB assays, p robe hybridizations and PCR showed that several, but not all, BP degraders possess this type of CST activity and/or a closely related gene. In all bac teria showing BphK activity, this was induced by growth on BP as sole carbo n source, although activity levels differed by up to 10-fold after growth o n BP and by up to 60-fold after growth on succinate. This resulted in a var iation of induction factors between 2 and 30. In the majority of bphK(+) ba cteria examined, the gene appeared to be part of LB400-like bph gene cluste rs. DNA sequencing revealed almost complete identity of bphK genes from fiv e different bph gene clusters. These results suggest that bphK genes, altho ugh not essential, fulfil a strain-specific function related to the utiliza tion of BPs by their host organisms. The usefulness of BphK as a reporter e nzyme for monitoring the expression of catabolic pathways is discussed.