Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis

Abstract

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive whole-genome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5-transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5 insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3-succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5.

The enzymes of microbial nicotine metabolism

“…Thus, the function of NicA1 remains unclear, and NicA2 is likely the true nicotine oxidase. The ndaA gene in P. putida J5, required for degradation of nicotine by that organism, codes for a protein that is 99% identical in sequence to that of NicA2 [52], so that NdaA is also likely to be a nicotine oxidase. The structure of NicA2 was recently determined, showing that the protein is a member of the MAO family with the same overall structure as LHNO (Fig.…”

“…Both are FAD-containing enzymes whose sequences place them in the MAO family of flavoproteins. The sequence of NdaC from P. putida J5 is identical to that of Pnao, although the protein itself has not been characterized, and loss of ndaC eliminates the ability of cells to metabolize pseudooxynicotine [52]. In the case of Pnao the source of the oxygen in the 3-succinoylsemialdehyde pyridine product has been shown to be water [55], establishing the reaction catalyzed by the enzyme as shown in Scheme 9, with the hydrolytic step being nonenzymatic.…”

“…The sequence of the enzyme identifies it as an aldehyde dehydrogenase [56], but the protein itself does not appear to have been characterized. In P. putida S16 and J5, sequence analyses have identified Spad and ndaD, respectively, as the likely 3-succinoylsemialdehyde pyridine dehydrogenases, and ndaD is required for P. putida J5 to convert 3-succinoylsemialdehyde pyridine to 3-succinoylpyridine [48,52]. …”

Identification and Characterization of an Aeromonas hydrophila Oligopeptidase Gene pepF Negatively Related to Biofilm Formation

“…As a powerful approach for functional gene analysis, DNA transposition has been performed in multifarious bacteria, including Listeria monocytogenes ( Chang et al, 2012 ), Riemerella anatipestifer ( Wang X. et al, 2014 ), Pseudomonas putida ( Xia et al, 2015 ) and Salmonella typhimurium ( Sahu et al, 2013 ), to ascertain genetic determinants. In this study, we identified 24 biofilm-associated genes based on a library of 1030 mutant colonies of A. hydrophila , among which 14 genes were shown to enhance biofilm formation and 10 were involved in reducing biofilm formation.…”

Characterization and Genome Analysis of a Nicotine and Nicotinic Acid-Degrading Strain Pseudomonas putida JQ581 Isolated from Marine

“…nic22 [23], Pseudomonas sp. ZJUSKD [24], Pseudomonas putida J5 [25], Pseudomonas putida HF-1 [26], Pseudomonas plecoglossicida TND35 [27], Pseudomonas sp. HZN6 [28,29,30], Pseudomonas geniculata N1 [31], Shinella sp.…”