Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis
- 26 March 2015
- journal article
- Published by Springer Science and Business Media LLC in Applied Microbiology and Biotechnology
- Vol. 99 (15), 6503-6514
- https://doi.org/10.1007/s00253-015-6529-x
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.Keywords
This publication has 48 references indexed in Scilit:
- Iron(II)-dependent dioxygenase and N-formylamide deformylase catalyze the reactions from 5-hydroxy-2-pyridone to maleamateScientific Reports, 2013
- Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradationScientific Reports, 2012
- Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of NicotineApplied and Environmental Microbiology, 2012
- Novel Nicotine Oxidoreductase-Encoding Gene Involved in Nicotine Degradation by Pseudomonas putida Strain S16Applied and Environmental Microbiology, 2009
- Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesisBMC Microbiology, 2009
- IS4 family goes genomicBMC Evolutionary Biology, 2008
- An NAD(P)H-Nicotine Blue Oxidoreductase Is Part of the Nicotine Regulon and May Protect Arthrobacter nicotinovorans from Oxidative Stress during Nicotine CatabolismApplied and Environmental Microbiology, 2007
- Functional Characterization of IS 1999 , an IS 4 Family Element Involved in Mobilization and Expression of β-Lactam Resistance GenesJournal of Bacteriology, 2006
- A Functional mobA Gene for Molybdopterin Cytosine Dinucleotide Cofactor Biosynthesis Is Required for Activity and Holoenzyme Assembly of the Heterotrimeric Nicotine Dehydrogenases of Arthrobacter nicotinovoransApplied and Environmental Microbiology, 2006
- Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicumGene, 1994