Molecular Mechanism Of The Degradation Of N-heterocyclic Pollutant Nicotine By Bacteria

As a typical N-heterocyclic pollutant, nicotine was listed in Toxics Release Inventory by United States Environmental Protection Agency since 1994. Nicotine is very addictive and toxic, and will cause various kinds of diseases when ingested by humans. Nicotine-rich tobacco wastes discharged from tobacco industry and nicotine-rich cigarette butts threw by smokers are the main causes of nicotine pollution. China, as the world’s largest producer and consumer of tobacco, produces millions of tons of tobacco wastes yearly. Nicotine pollution significantly threatens environment and health of human, so its elimination is extremely urgent.Microbial remediation gets wide attention because it is high-efficiency, fast, and does not produce new pollutants. Pseudomonas and Arthrobacter are the most predominant members of reported nicotine-degrading microorganisms. Besides the nicotine-degrading capacities, these bacteria also have strong environmental adaptabilities. Revealing their nicotine-degrading pathways and molecular mechanisms, exploring the enzymic and structural properties of nicotine-degrading enzymes, and elucidating the gene bases of their environmental adaptabilities, can guide the genetic modifications of these strains, and have greatly theoretical and application values.According to the predicted and reported pathways of 2,5-dihydroxypyridine(2,5-DHP) degradation, this study first proposed and proved the late steps of nicotine-degrading pyrrolidine pathway in Pseudomonas. The steps are as following: 2,5-DHP generated from nicotine degradation ring cleaves to form N-formylmaleamic acid(NFM), NFM subsequently hydrolyzes to maleamic acid and formic acid, maleamic acid further deaminizes to maleic acid, then maleic acid isomerizes to fumaric acid which finally enters the citric acid cycle to provide energy for the strain.In this study, a gene cluster(nic2, GenBank No. GQ857548) involved in the late steps of nicotine-degrading pyrrolidine pathway was isolated and identified in the nicotine-degrading bacterium Pseudomonas putida S16. Six ORFs in the same direction were predicted in this cluster. The ORFs are as following: 6-hydroxy-3-succinoylpyridine hydroxylase gene hspB, function unknown gene orfx, maleic acid isomerase gene iso, NFM deformylase gene nfo, 2,5-DHP dioxygenase gene hpo and maleamic acid amidase gene ami. Comparative genome and phylogenetic tree analyses suggested that P. putida S16 obtained nic2 cluster via horizontal gene transfer, and the source of the cluster was different from other P. putida strains.Previous studies found there was an isoenzyme of HspB, named HspA, in P. putida S16. This study showed that hspA gene knockout strain S16 dhspA could grow with nicotine as the sole carbon and nitrogen source and its nicotine-degrading ability was almost the same as the wild type strain S16, while hspB gene knockout strain S16 dhspB could not grow with nicotine as the sole carbon and nitrogen source and it only degraded nicotine to HSP, but could not transform HSP further. Besides, this study also showed that although hpo gene knockout strain S16 dhpo could grow with nicotine as the sole carbon and nitrogen source, it grew much slower than the wild type S16, and turned the medium into puce. In addition, the resting cell of S16 dhpo could transform nicotine to 2,5-DHP but could not degrade 2,5-DHP further. All above results indicate that nic2 cluster is crucial for nicotine degradation in P. putida S16.In order to identify the functions of expression products of genes hpo, nfo and ami, this study cloned these genes to expression vector pET28 a respectively, purified C-terminal His6-tagged proteins His-Hpo,His-Nfo and His-Ami. UV detection showed His-Hpo catalyzed 2,5-DHP degradation and the catalytic ability was dependent on Fe2+. The Km,Vmax and kcat values of His-Hpo for 2,5-DHP were 0.168 mM,16.6 U mg-1 and 10.8 s-1, respectively. The deformylase activity of His-Nfo was identified by measuring the formation of formate with a formate dehydrogenase as coupled enzyme. While the deamination activity of His-Ami was confirmed by detecting the formation of NH4+ with a glutamate dehydrogenase as coupled enzyme.Hpo is the second member of a new dioxygenase family whose founding member is NicX. This study purified these two 2,5-DHP dioxygenases, characterised and compared their catalytic and enzymic properties, and uncovered the commonnesses and specialities between them. The 2,5-DHP dioxygenases add two oxygen atoms which both come from O2 to 2,5-DHP, transform 1 mol 2,5-DHP consuming 1 mol O2. Both of them bind with Fe2+ at the ratio 1:1. In solution, Hpo is a trimer while Nic X is a hexamer assembled by two trimers. Enzyme kinetics comparison suggested the affinity of His-Hpo for 2,5-DHP was much lower than that of His-NicX, while the catalytic efficiency of His-Hpo was about 3.7 times of that of His-NicX. With structure prediction and site-directed mutagenesis, this study identified a 2-His-1-carboxylate Fe2+-binding motif HX52 HXD in Hpo, which is composed of H257, H310 and D312. This motif is highly conserved among Hpo and NicX but different from those of other non-heme Fe2+-dependent enzymes.It is disputed for more than 40 years which protein catalyze the NFM hydrolysis. Gauthier and Rittenberg reported that 2,5-DHP dioxygenase had both dioxygenase and deformylase activities and catalyzed NFM hydrolysis. However, Behrman showed that both NFM and its trans-isomer, N-formylfumaramic acid(NFF) hydrolyzed rapidly and spontaneously. While Jiménez et al. demonstrated that not 2,5-DHP dioxygenase but a separate deformylase catalyzed the hydrolysis of NFM. Based on syntheses of NFM and NFF, and the results of ESI-TOF-MS analyses, this study concluded that 2,5-DHP dioxygenases could not catalyze the hydrolysis of NFM or NFF; although NFM and NFF did hydrolyze spontaneously, the deformylase Nfo greatly speeded these reactions, and the catalytic efficiency of His-Nfo for NFM was about 1,400 times of that for NFF.Arthrobacter is one of the most predominant members of nicotine-degrading bacteria, but studies of nicotine degradation in Arthrobacter still stay at plasmid level(plasmid pAO1). This study sequenced the genome of strain M2012083, first revealed genome informations of nicotine-degrading Arthrobacter strain, then found moeB and mogA genes which are essential for nicotine degradation in Arthrobacter but are absent in plasmid pAO1. Using synteny analysis, all the homologues of nicotine degradation-related genes of plasmid pAO1 were located on a 68,622-bp DNA segment(nic segment) in M2012083 genome, with 98.1% identity to the 69,252-bp nic segment of plasmid pAO1. However, integrase gene xerD and IS1473 sequence containing resolvase gene are absent in nic segment of M2012083, while transposase gene sequence Tn554 is incomplete in it. Meanwhile, the rest plasmid pAO1 sequences other than the nic segment, which contain all plasmid function genes, have no significant similarity with M2012083 genome. Most of the pAO1 genes involved in plasmid function were not found in M2012083 genome. All of above suggest that the nicotine degradation-related genes of A. aurescens M2012083 locate on chromosome or a plasmid different from plasmid pAO1.Based on the genome comparison among M2012083 and other 6 Arthrobacter strains, this study found 17 σ70 transcription factors supposed to be involved in stress responses and 109 genes involved in environmental adaptability of strain M2012083, including 8 universal stress-related genes, 35 osmotic stress-related genes, 29 oxidative stress-eliminating genes, 21 cold and heat shock responses genes, 4 detoxification genes, and 12 other stress-related genes. This study also illustrated the functions of above genes in detail.In summary, this study focused on the most predominant nicotine-degrading bacteria, Pseudomonas and Arthrobacter, researched on P. putida S16 and A. aurescens M2012083, revealed the molecular mechanism of the late steps of nicotine-degrading pyrrolidine pathway in P. putida S16, uncovered the gene bases of nicotine-degrading capacity and environmental adaptability of A. aurescens M2012083. This study fill the blank of the molecular mechanism research of nicotine catabolism in bacteria and provide theoretical basis for selective modification of nicotine-degrading bacteria. Therefore, this study is significant for the application of nicotine-degrading bacteria in bioremediation of nicotine-contaminated environments.