Substrate specificity and expression of three 2,3-dihydroxybiphenyl 1,2-dioxygenases from Rhodococcus globerulus strain P6

Abstract

Rhodococcus globerulus strain P6 contains at least three genes, bphC1, bphC2, and bphC3, coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases; the latter two specify enzymes of the family of one-domain extradiol dioxygenases. In order to assess the importance of these different isoenzymes for the broad catabolic activity of this organism towards the degradation of polychlorinated biphenyls (PCBs), the capacities of recombinant enzymes expressed in Escherichia coli to transform different chlorosubstituted dihydroxybiphenyls formed by the action of R. globerulus P6 biphenyl dioxygenase and biphenyl 2,3-dihydrodiol dehydrogenase were determined. Whereas both BphC2 and BphC3 showed similar activities for 2,3-dihydroxybiphenyl and all monochlorinated 2,3-dihydroxybiphenyls, BphC1 exhibited only weak activity for 2'-chloro-2,3-dihydroxybiphenyl. More highly chlorinated 2'-chlorosubstituted 2,3-dihydroxybiphenyls were also transformed at high rates by BphC2 and BphC3 but not BphC1. In R. globerulus P6, BphC2 was constitutively expressed, BphC1 expression was induced during growth on biphenyl, and BphC3 was not expressed at significant levels under the experimental conditions. Although we cannot rule out the expression of BphC3 under certain environmental conditions, it seems that the contrasting substrate specificities of BphC1 and BphC2 contribute significantly to the versatile PCB-degrading phenotype of R. globerulus P6.

FIG. 1.

Western blot analysis of the expression of BphC proteins. Three monoclonal antibodies specific for BphC1 (AkXIII) (A), BphC2 (AkXIV) (B), and BphC3 (AkXV) (C) were used, and cell extracts of R. globerulus P6 grown on either complex medium (LB medium) or biphenyl or of E. coli cells expressing BphC1 (C1), BphC2 (C2) or BphC3 (C3) (always corresponding to 20 μg of protein) were analyzed. The antibodies were not cross-reactive, since for each antibody, only control cell extracts of E. coli expressing each of the antibody's cognate protein gave positive bands (lanes C1, C2, and C3). M, molecular mass marker.

FIG. 2.

BIAcore analysis of the expression of 2,3-dihydroxybiphenyl dioxygenases BphC1 (dotted bars) and BphC2 (hatched bars) during growth of R. globerulus P6 on biphenyl (A) or succinate (B) or in LB broth (C). The BphC content was analyzed in cell extracts (corresponding to 0.3 mg of protein per ml) during different phases of growth (early exponential growth phase, when cultures had reached less than 20% of maximal optical density; mid-exponential growth phase, when cultures had reached 50% ± 10% of maximal optical density; late exponential growth phase, when cultures had reached 80% ± 10% of maximal optical density; early stationary phase, less than 2 h after cultures had reached maximal optical density; late stationary phase, more than 2 h after cultures had reached maximal optical density). The BphC2 content in panel A was calculated assuming 40% of the signal intensity to be due to cross-reaction with a 42-kDa protein. The data are means of two independent experiments, and variation between respective data points of the experiments was always less than 15%.