Aims Pandoraea pnomenusa MCB032 completely degrades chlorobenzene, whose metabolic pathway is encoded by cbs and clc gene clusters. The putative regulatory factors ClcR and CbsR are predicted to regulate the cbs and clc gene clusters. This research aims to understand the function of ClcR and CbsR. Methods and results RT-PCR analyses demonstrated that the cbsFAaAbAcAdB operon that encodes catabolic pathways for the degradation of chlorobenzene to chlorocatechol is located on an operon. Moreover, the clcABCDE operon is involved in the 3-chlorocatechol pathway. Gene knockout and transcriptional analysis showed that the transcription of the cbsFAaAbAcAdB operon is positively regulated by CbsR, whereas the clcABCDE operon is activated by ClcR. Primer extension analysis was used to locate the transcription start sites of the cbsFAaAbAcAdB and cbsR operons. Electrophoretic mobility shift assay analyses showed that CbsR is bound to the sites in the promoter regions of cbsFAaAbAcAdB and cbsR operons. Conclusion The XylR/NtrC-type regulator CbsR positively regulates the transcription of the cbsFAaAbAcAdB operon encoding the upstream pathway of chlorobenzene catabolism, while the LysR-type regulator ClcR activates the clcABCDE operon encoding the downstream pathway.

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