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Studies On Nicotine Metabolic Pathway As Well As Molecular Biology In Pseudomonas Sp. HF-1

Posted on:2008-10-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:T J ShaoFull Text:PDF
GTID:1100360215959607Subject:Microbiology
Abstract/Summary:PDF Full Text Request
The response of the antioxidant enzymes superoxide dismutase, catalase, ATPase of Escherichia coli, Bacillus subtilis, and nicotine-degrading bacterium Pseudomonas sp. HF-1 exposed to nicotine were investigated in this thesis. Moreover, a mutant lose the ability to degrade nicotine was mutated and isolated. The metabolic pathway of nicotine by strain HF-1 was elucidated and the degradation enzymes were identified by 2-DE and mass. The results will be valuable to build up alert index systems in nicotine-contaminated soil, bioremediation of nicotine-polluted environments, and to illustrate the catabolic pathway of nicotine by strain HF-1 and molecular fundation. The main results of this study are as follows:1. The responses of superoxide dismutase, catalase and ATPase of nicotine-degrading bacterium Pseudomonas sp. HF-1 exposed to nicotine at different concentrations and culturing time, compared with typical bacteria Escherichia coli and Bacillus subtilis, were investigated. The results suggested that the responses of SOD, CAT and ATPase in Pseudomonas sp. HF-1 at different growth stages to nicotine with various concentrations were different. Different bacteria had different responses to oxidative stress from nicotine and grown cells were more resistant to nicotine than growing younger cells. The inhibition of ATPase in matured Pseudomonas sp. HF-1 appeared an enhancement with the increase of nicotine concentration, showing a striking dose-response relationship, implied that nicotine-degrading bacterium has different responses from non-nicotine-degrading bacterium when they encounter the oxidative stress from nicotine and it is feasible that SOD, CAT and ATPase activities to be used as an available bio-indicator for nicotine pollution. 2. A mutant lost the ability of degrading nicotine was screened and isolated via treating the strain HF-1 with SDS and mitomycin C repetitively and was named as strain 6-13. The morphology of the mutant strain was similar to that of wild-type strain and 16S rDNA sequence analysis showed that two strains had a sequence homology similarity of 100%. Furthermore, analysis of soluble whole cell protein patterns by SDS-PAGE showed that two strains also had identical protein expression map. Two strains were sensitive to amikacin, qingdameisu and cephalosporin while resistant to chloramphenicol, erythromycin, clindamycin, benzylpenicillin sodium, penicillin, ampicillin and cephalothin. 19kb plasmid was successfully isolated from both strains, implying that functional genes related to nicotine degradation maybe are located on chromosome rather than plasmid.3. Studies on microbiological degradation of nicotine demonstrated that Pseudomonas sp. HF-1 could degrade 99.6% nicotine with 1.0 g·L-1 within 21h and there was a perfect liner relationship between growth of the strain and nicotine degradation during the exponential phase of growth. Moreover, the pH in culturing medium during the process of nicotine degradation was relatively stable. Pseudomonas sp. HF-1 seems to be a promising strain for the bioremediation of environments contaminated by tobacco waste.The results attained from GC-MS and LC-MS suggested nicotine-degradation pathway of strain HF-1 was different from metabolism processes described in the previous literatures. Some unreported intermediates such as cotinine, myosmine, nicotyrine, and nornicotine were detected, but N-methylmyosmine, the product of nicotine-degradation by reported Pseudomonas species, didn't. At the same time, a kind of viridescent pigment was observed during nicotine degradation, which was reported for the first time. These results provide evidence that strain HF-1 operates a different nicotine-degrading pathway from the known metabolic pathway of Pseudomonas species.Four main peaks of nicotine metabolites by strain HF-1 with the retention times of them were 3.6-5.0, 5.6-6.7, 7.5-8.4 and 10.2-10.9 min, respectively, were separated using preparative HPLC and analyzed using mass spectrometry. The four peak products were concentrated by freeze-dry evaporation and their purities were up to 93.2%, 90.8%, 89.6% and 88.5% respectively. The product with the highest purity of 93.2% was attempted to be confirmed by NMR, however, only the 1H peak of acetone was observed. The other three peaks were analyzed by ESI-MS and their molecular weights were 179Da, 195Da and 487Da, respectively. The fractions with molecular weights of 179Da and 195Da were tentatively identified as 3-succinoylpyridine and 6-hydroxy-3-succinoylpyridine, respectively. The detection of 3-succinolypyridine and 6-hydroxy-3-succinolypyridine through liquid chromatographic and mass analysis revealed that the degradation pathway of nicotine in Pseudomonas sp. HF-1 was analogous to the metabolism of nicotine in other known Pseudomonas from 3-succinoylpyridine.4. To gain insight into the mechanism underlying nicotine toxicity and the nicotine-degrading pathway in bacteria, a two-dimensional protein reference map was generated for identifying variations in protein expression in nicotine-degrading bacterium Pseudomonas sp. HF-1 following exposure to nicotine. Inspection of the two-dimensional gel electrophoresis gels showed that strain HF-1 cell exposed to nicotine would lead to the up-regulation of 65 proteins and the down regulation of 36 proteins. 22 proteins which presented in nicotine-exposed gel but absent in control or amplified at least two folds (CBB staining) were incised and identified by MALDI-TOF/TOF and ESI-MS. Most of these proteins were homologous to the proteins from Pseudomonas putida KT2440, which has often been used as a model for investigating toxicity mechanisms and adaptation to hazardous chemicals in bacteria. Proteins were grouped in 5 different functional families, taking into consideration only one of the protein biological roles. A series of proteins which related to amino acid biosynthesis, protein folding, energy metabolism and detoxification were induced in response to nicotine.The identification of metallo-beta-lactamase family protein and hydrolase which is an amidase related to nicotinamidase indicated that the nicotine-degrading pathway of strain HF-1 was analogous to that of mammalian although norcotinine and 4-oxo-4-(3-pyridyl)-butanamide were not directly detected. It was reported that the properties of the 2,5-dihydroxypyridine-oxidizing enzyme from Ps. putida N9 were similar to those of dioxygenases catalyzing the cleavage of the aromatic ring, and the 2,5-dihydroxypyridine-oxidizing enzyme appeared the highest similarity to mammalian dioxygenase, homogentisate oxygenase, which contains loosely bound iron and is inhibited by metal-chelating agents. The identification of homogentisate 1,2-dioxygenase could verify the existence of 2,5-dihydroxypyridine indirectly. Furthermore, the identification of nitroreductase family protein by 2-D and MS analysis in nicotine-exposure cultures of strain HF-1 suggest that nicotine is degraded through multiple degradation pathways in Pseudomonas sp. HF-1.
Keywords/Search Tags:Nicotine, Pseudomonas, Oxidative stress, Mutangensis, Biodegradation, Metabolic pathway, Two dimensional electrophoresis, Biological mass spectrometry, Degradation enzymes
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