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Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation.

ABSTRACT: Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain that catabolizes nicotine through the pyrrolidine pathway. In our previous study, we used Tn5 transposon mutagenesis to investigate nicotine metabolism-associated genes, and 18 nicotine degradation-deficient mutants were isolated from 16,324 Tn5-transformants. Three of the mutants were Tn5 inserts into the modABC gene cluster that encoded an ABC-type, high-affinity, molybdate transporter. In-frame deletion of the modABC genes abolished the nicotine-degrading ability of strain J5, and complementation with modABC either from P. putida or Arthrobacter oxidans restored the degrading activity of the mutant to wild-type level. Nicotine degradation of J5 was inhibited markedly by addition of tungstate, a specific antagonist of molybdate. Molybdate at a non-physiologically high concentration (100 ?M) fully restored nicotine-degrading activity and recovered growth of the modABC mutant in a nicotine minimal medium. Transcriptional analysis revealed that the expression of modABC was up-regulated at low molybdate concentrations and down-regulated at high moybdate concentrations, which indicated that at least one other system was able to transport molybdate, but with lower affinity. These results suggested that the molybdate transport system was essential to nicotine metabolism in P. putida J5.

SUBMITTER: Xia Z

PROVIDER: S-EPMC6295455 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation.

Xia Zhenyuan Z Lei Liping L Zhang Hong-Yue HY Wei Hai-Lei HL

Frontiers in microbiology 20181210

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain that catabolizes nicotine through the pyrrolidine pathway. In our previous study, we used Tn5 transposon mutagenesis to investigate nicotine metabolism-associated genes, and 18 nicotine degradation-deficient mutants were isolated from 16,324 Tn5-transformants. Three of the mutants were Tn5 inserts into the modABC gene cluster that encoded an ABC-type, high-affinity, molybdate transporter. In-frame dele ...[more]

PMID: 30627117

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Project description: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.IMPORTANCE 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.

Dataset Information

Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation.

Publications

Characterization of the ModABC Molybdate Transport System of <i>Pseudomonas putida</i> in Nicotine Degradation.

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