Gram-negative bacteria employ secretion systems to translocate proteinaceous effectors from the cytoplasm to the extracellular milieu, thus interacting with the surrounding environment or micro-niche. It is known that bacteria can benefit from type VI secretion system (T6SS) by transporting ions to combat reactive oxygen species (ROS). Here, we report that T6SS activities conferred bacterial tolerance to nicotine-induced oxidative stress in Pseudomonas sp. strain JY-Q, a highly active nicotine degradation strain isolated from tobacco waste extract. AA098_13375 was identified to encode a dual-functional effector with anti-microbial and anti-ROS activities. Wild type strain JY-Q grew better than AA098_13375 deletion mutant in nicotine-containing medium by antagonizing increased intracellular ROS levels. It was, therefore, tentatively designated as TseN (Type VI Secretion system Effector for Nicotine tolerance), of which homologs were observed to be broadly ubiquitous in Pseudomonas species. TseN was identified as a Tse6-like bacteriostatic toxin via monitoring intracellular NAD+. TseN presented potential antagonism against ROS to fine tune the heavy traffic of nicotine metabolism in strain JY-Q. It is feasible that the dynamic tuning of NAD+ driven by TseN could satisfy demands from nicotine degradation with less cytotoxicity. In this scenario, T6SS involves a fascinating accommodation cascade that prompts constitutive biotransformation of N-heterocyclic aromatics by improving bacterial robustness/growth. In summary, T6SS in JY-Q mediated resistance to oxidative stress and promoted bacterial fitness via a contact-independent growth competitive advantage besides the well-studied T6SS-dependent antimicrobial activities. IMPORTANCE Mixtures of various pollutants and co-existence of numerous species of organisms are usually found in the adverse environments. Scientific community concerning biodegradation of nitrogen-heterocyclic contaminants commonly focused on screening functional enzymes transforming pollutants into intermediates of attenuated toxicity or for primary metabolism. Here we identified dual roles of T6SS effector TseN in Pseudomonas sp. JY-Q capable of degrading nicotine. T6SS could deliver TseN to kill competitors, and provide growth advantage by contact-independent pattern. TseN could monitor intracellular NAD+ level by its hydrolase activity, conferring cytotoxicity on competitive rivals but metabolic homeostasis on JY-Q. Moreover, JY-Q could be protected from TseN toxicity by its immunity protein TsiN. In conclusion, we found that TseN with cytotoxicity to bacterial competitors facilitated nicotine tolerance of JY-Q. We therefore revealed a working model between T6SS and nicotine metabolism. This finding indicates that multiple diversified weapons have been evolved by bacteria for their growth and robustness.

Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the co-occurrence of these two pathways is only observed in Pseudomonas sp. JY-Q. Nicotine pyrrolidine catabolism pathway of strain JY-Q consists of function modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenases (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm (namely Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 both were in-frame deleted, the mutant still grew well in the basic salt medium supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains re-organized domains similar to that of Spm. When JY-Q_nicAB gene was introduced into another Pseudomonas strain, which is unable to degrade NA, the resultant recombinant strain exhibited the ability transforming SP to HSP except the ability NA metabolism. Here, we ccan conclude that NicAB in strain JY-Q exhibits an additional role of SP transforming. The other genes in NA cluster, NicXDFE (Nic2 homolog), then also exhibit the role of HSP subsequent metabolism for energy yield. This finding also suggests that the co-occurrence of nicotine and NA degradation genes in strain JY-Q represents a contribution for JY-Q making it more effective and flexible for the degradation of nicotine. Importance 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) both are valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant kept the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.

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