Molecular Mechanisms Of Nicotine Degradation By Pseudomonas Putida S16

Nicotine is not only a natural compound of tobacco products but also an occurring alkaloid found in many plants. As its heterocyclic structure, it is soluble in water and can cross biological membranes and the blood brain barrier easily. In the manufacture and processing of tobacco products, powdery solid wastes, with a high content of nicotine as the main toxic compound can not be recycled. Nicotine can be easily transported into ground water, and it is so toxic that it is very harmful to health. Because of the harmful effects, it has been designated as Toxic Release Inventory (TRI) chemical by the United States Environmental Protection Agency (EPA) since 1994. Therefore, it is important to remove nicotine from tobacco waste, or even convert it to valuable compounds. The CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) put forward the "Disposal of Tobacco Waste", which summarized the methods for the disposal of tobacco waste. Microbial treatment is one of the recommended technologies for the rapid decomposition of tobacco waste. Many microorganisms can use nicotine as sole carbon and nitrogen source from the leaves of tobacco and soils. They can change the contents of nicotine during the manufacture of tobacco. Research on mechanisms of nicotine degradation can reveal chemical biology mechanisms and provide further theoretical foundation.Pseudomonas putida S16 strain was able to use nicotine as sole nitrogen and carbon source, which was isolated in our laboratory previously. The metabolic pathway of nicotine by strain P. putida S16 has been previously proposed based on the results of nuclear magnetic resonance, Fourier-transform Infrared, Ultraviolet (UV) spectroscopy, GC-MS, and high-resolution MS analyses. However, the pathway was not characterized at the genetic level, and this report was focused on the strain S16. We constructed the genomic library, screened for the positive clones, sequenced the clones, cloned two novel genes containing nicA and hsp, and expressed successfully to reveal the molecular mechanisms of this pathway.Two genomic DNA libraries of strain S16 containing more than 20,000 transformants were constructed. Three positive transformants were isolated from the library, named as x-2, 10-52, and GTPF. All of them could use nicotine as the sole source of nitrogen and carbon. According to the results, the nicotine degradation pathways of x-2 and 10-52 were identical, different from the strain S16. The sequences of transformants x-2 and 10-52 were identical based on sequence reports. According to the results of National Center for Biotechnology Information (NCBI) BLAST program, the genes of x-2 and 10-52 showed 99% identity with many dehydrogenases such as mannitol dehydrogenase. On the position 831, it showed 60% identies with Fe-S oxidase family(LTLTELGRNLPTKARTKHNIKRIDRLLGNRHLHKE) and showed 100% identies with the sequence of transposon Tn10, however, the function were never known. It was suggested that it needed to perform experiment to certify the function of this gene. Three important intermediates were identified by ESI-Q-TOF-MS, and it showed that the first compound (C₁₀H₁₃N₂O₂) had a molecular ion peak at m/z 193.09715 ((M+H)⁺), the second compund (C₁₀H₁₅N₂O₂) had a molecular ion peak at m/z 195.11280 ((M+H)⁺), and the third compound (C₁₀H₁₃N₂O) had a molecular ion peak at m/z 177.10224 ((M+H)⁺). None of these compounds were identical to the pyrrolidine pathway, and its chemical structure should be further identified.While, the transformant GTPF could transform nicotine to N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine. It might be poposed that the transformant contained the key genes of pyrrolidine pathway. After determining the nucleotide sequence of the 4,879-bp insert of pUC19, we obtained three large open reading frames (ORF1, ORF2 and ORF3) by computer analysis, which were linked in tandem and considered to be translated in both directions. All these ORFs started with Valine or Methionine, and were greater than 100 amino acids in length. At both ends of the gene cluster, putative transcriptional regulators in the species of P. putida were found by NCBI BLAST program. The Shine-Dalgamo (SD) sequence was found in the upstream region of the putative initiation codon of ORF1. The deduced amino acid sequence of ORF1 showed 40% identity with cytochrome c oxidase subunit I. The deduced amino acid sequence of ORF2 showed 38% identity with the hypothetical protein of Photobacterium profundum SS9. The deduced amino acid sequence of ORF2 showed no identity with the protein of known function according to the results of BLASTx program. Immediately upstream of ORF2 was another open reading frame of 582 bp (ORF3). The deduced amino acid sequence of ORF3 showed 97% identity with the helix-turn-helix of P. putida F1, and 97% identity with the transcriptional regulator AsnC family of P. putida KT2440. However, no significant homology of these three ORFs with other degradative genes was observed. Therefore, subcloning reactions were necessary to identify the function of these catabolic genes.Different DNA fragments, which were amplified from the sequence of GTPF by PCR, were subcloned into pMD18-T vector in both orientations and transformed into E. coli DH5a. Nicotine and HSP was added to the resting cells to detect the degrading ability. We identified, subcloned, and sequenced the nicA gene, a 1,854 bp fragment showed no significant similarity with any known heterocyclic compounds degradative genes, which was the first nicotine degradation gene encoding an enzyme that converted nicotine through pseudooxynicotine to 3-succinoylpyridine. The nicA gene was cloned into pET vectors, and the recombinant proteins NicA was purified and characterized. Nucleotide sequence analysis followed by enzymatic activities assays of the gene products indicated that under the catalysis of NicA, the pyrrolidine ring of nicotine was opened up with the formation of P, and P was further oxidized to SP, which was consistent with previous report. Experiments with ¹⁸O labeling gave direct evidence for the incorporation of oxygen from H₂¹⁸O into the produced pseudooxynicotine.The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directly to DHP was successfully cloned. DNA sequence analysis of this 936 bp fragment reveals that the deduced amino acid shows no similarity with any protein of known function. We scanned for any possible known motifs in this sequence and found it is similar with some motifs. The hsp gene was cloned into pET vectors, and the recombinant proteins HSP was purified and characterized. After cloning of nicA and hsp, we futher cloned SP degrading gene. However, it was a pity that the cloning was unsuccessful.According to the reports, mobile genetic elements, including plasmids and transposons, play important roles in the generation/evolution of novel catabolic pathways, and many nicotine degradation genes were focused on the plasmids in bacteria. Several methods were used to isolate and detect the plasmids in strain S16. Aorrding to the results of sample preparation and pulsed field gel electrophoresis, there might be four megaplasmids in S16 with a size of about 60 kb, 200 kb, 600 kb and 1000 kb, and further research is being done.