|其他摘要||Nicotine [1-methyl-2-(3-pyridyl-pyrrolidine), C10H14N2] is the main alkaloid component of cigarettes and the main non-recyclable powdery waste in tobacco manufacturing process. Biological treatments with micro-organisms have potential for manipulation of nicotine content in cigarettes production and detoxification of tobacco wastes containing high concentrations of nicotine. In particular, the bacterial community residing in the tobacco rhizosphere has presumably adapted to use nicotine as a growth substrate and has developed biochemical strategies to decompose this organic heterocyclic compound. But the exploitation and application of nicotine degradation with micro-organisms are restricted by the nicotine-degrading microbial resources and their metabolic mechanisms. In this thesis, we fix our attention on two primary contents, diversity of tobacco-associated nicotine-degrading bacteria and identification of genes involved in nicotine degradation in Pseudomonads. The main results of this study are as follows:
A total of 96 nicotine-degrading bacterial strains were isolated with nicotine as the sole carbon source, in which 56 strains from tobacco rhizosphere and 40 from tobacco leaves. The degrading efficiency of 19 endophytic strains (47.5%) and 39 strains (69.6%) from rhizosphere exceeded 90%. The endophytic nicotine-degrading bacterial strains were distributed into 10 genera, in which 62.5% were Pseudomonas spp. and 12.5% were Arthrobacter spp. The nicotine-degrading bacterial strains in tobacco rhizosphere were distributed into 6 genera, in which 60.7% were Arthrobacter spp. and 25.0% were Pseudomonas spp. The species richness, diversity and dominance index of endophytic nicotine-degrading bacterial community were higher than that of rhizosphere. But the evenness index of nicotine-degrading bacteria in tobacco rhizosphere was higher compared with endophytic nicotine-degrading bacteria. In this thesis, we firstly reported nicotine-degrading Ensifer, Sinorhizobium, Sphingomonas, Massilia, Erwinia, Brevundimonas, Paenibacillus, and Cellulosimicrobium.
Strain J5 and N7 were the most effectual nicotine-degrading bacteria isolated from tobacco rhizosphere. The optimum nicotine concentration for the growth of strain J5 and N7 were 2.0 g/l. 3 g of nicotine/l could be fully decomposed being treated with strain J5 for 24 h. And there was no more nicotine detected in the medium containing 2.0 g nicotine/l after N7 growth for 24 h. There were statistically significant linear relationships between nicotine degradation and biomass of strain J5 and N7. Based on morphology, flagella dyeing, physiological characteristics, and 16S rDNA sequence analysis, strain J5 and N7 were identified as Pseudomonas putida and Ensifer sp., respectively. When strain J5 and N7 cell suspensions (108 CFU/ml) were applied to treat tobacco leaves, the nicotine concentration was decreased by 11.7% and 16.0%, respectively. These results suggest that the novel strain J5 and N7 may be useful for nicotine biodegradation.
A mini-Tn5 mutagenesis library of P. putida J5 was constructed, and 28 mutants that failed to grow in the M9 medium with 1.0 g nicotine/l as the sole carbon source were screened from 16324 transformants. The flanking sequences of Tn5 transposon were cloned with shotgun method from the mutant genome. The mutant sites could be divided into six groups as follows: oxidoreductases, proteins and metals transport systems, proteases and peptidases, transcriptional and translational regulators, unknown protein, and interspaces between two genes. The Tn-5 inserted genes of M728, M430 and M9502 were identical to molybdenum transport system (modABC), which regulated the expression of nicotine dehydrogenase in A. nicotinovorans. A homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in A. nicotinovorans, was identified from the mutant M2022. Furthermore, a homolog of ketopantoate hydroxymethyltransferase, the first enzyme for biosynthesis of vitamin pantothenate, was identified from the mutant M10. The further research of these mutants and the Tn5-inserted genes will help to find out the mechanism of transformation and metabolism of nicotine in P. putida J5.
Sequence analysis revealed that the Tn5 cassette of mutant M10 was inserted into a putative open reading frame of about 801 bp and encoding a polypeptide of 266 amino acids with a molecular mass of 27.8 kDa. The deduced amino acid sequence showed 54% identity to ketopantoate hydroxymethyltransferase (PanB) of E. coli K-12, in which PanB initiates the first reaction of pantothenate bosynthesis. The insertion occurred at the site of 185 amino acids of PanB. In-frame deletion of the panB gene abolished the ability to utilize nicotine, pyruvic acid and ketoisovalerate as the sole carbon resources, respectively, while complementation with panB from P. putida J5 and E. coli K-12 restored the mutant to the wild-type level. These results suggest that ketopantoate hydroxymethyltransferase is a crucial enzyme in nicotine metabolism in P. putida J5. In E. coli, Ketopantoate hydroxymethyltransferase is a key enzyme for synthesis of the vitamin pantothenate, a vital and central metabolic compound in all organisms. PanB initiates the first reaction by using ketoisovalerate to generate ketopantoate, which is reduced to D-pantoic acid. In the synthesis of pantothenate, pyruvic acid is the direct precursor of ketoisovalerate in many bacteria such as Escherichia, Salmonella, and Pseudomonas. Alternatively, pyruvic acid could be the end product of nicotine catabolism in Pseudomonas via 2,5-dihydroxypyridine and maleic acid. Given the known reaction mechanism and general chemical considerations, we propose that nicotine, when used as the carbon source by P. putida J5, is catabolized to pyruvic acid via pyrrolidine pathway and then participates in the synthesis of vitamin pantothenate and CoA, which supply the energy for the normal functioning.
Investigation of nicotine-degrading bacterial resources not only has basilic scientific significance, but also lays an experimental foundation for exploitation of new nicotine-degrading bacterial agents and owning the independent intellectual property rights. Identification of nicotine metabolic genes of Pseudomonas will reinforce the research on molecular pathway of nicotine metabolism, and also provide theoretical support for fully and reasonably utilizing the microbial resources.|