| Summary: | We report the entire process underlying the NicR2 regulatory mechanism from association between free NicR2 and two promoters to dissociation of the NicR2-promoter complex. NicR2 can bind to another promoter, Pspm, which controls expression of nicotine-degrading genes that are not controlled by the Phsp promoter. We identified specific nucleotides of the Pspm promoter responsible for NicR2 binding. HSP was further demonstrated as an antagonist, which prevents the binding of NicR2 to the Pspm and Phsp promoters, by locking NicR2 in the derepression conformation. The competition between NicR2 and RNA polymerase is essential to initiate transcription of nicotine-degrading genes. This study extends our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has studied, in depth, the pyrrolidine pathway of nicotine degradation in Pseudomonas putida S16. However, little is known regarding whole mechanism(s) regulating transcription of the nicotine degradation pathway gene cluster. In the present study, we comprehensively elucidate an overall view of the NicR2-mediated two-step mechanism regulating 3-succinoyl-pyridine (SP) biotransformation, which involves the association of free NicR2 with two promoters and the dissociation of NicR2 from the NicR2-promoter complex. NicR2 can bind to another promoter, Pspm, and regulate expression of the nicotine-degrading genes in the middle of nic2 gene cluster, which are not controlled by the previously reported Phsp promoter. We identified the function of the inverted repeat bases on the two promoters responsible for NicR2 binding and found out that the –35/–10 motif for RNA polymerase is overlapped by the NicR2 binding site. We clarify the exact role of 6-hydroxy-3-succinoyl-pyridine (HSP), which acts as an antagonist and may prevent binding of free NicR2 to the promoters but cannot release NicR2 from the promoters. Finally, a regulatory model is proposed, which consists of three parts: the interaction between NicR2 and two promoters (Pspm and Phsp), the interaction between NicR2 and two effectors (HSP and SP), and the interaction between NicR2 and RNA polymerase.
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