【作者】 李艳霞；

【导师】 王磊；

【作者基本信息】 南开大学 ， 微生物学， 2012， 博士

【摘要】 近年来，石油污染越来越多地受到人们的关注。在石油的复杂成分中，单环烷烃，主要是BTEX化合物（苯、甲苯、乙苯和二甲苯）以及可溶的组分，成为了污染地下水的主要污染物和威胁之一。石油中的芳香族成分经过一系列的微生物的氧化还原等作用后使得污染水域内经常检测到芳香酸类的中间代谢物，如苯甲酸，邻、间、对-甲基苯甲酸等。而且，单环芳香化合物几乎在所有生物体内大量地存在，因为他们是芳香族氨基酸（苯丙氨酸、色氨酸和酪氨酸）转化过程中的重要产物。芳香族化合物是在自然界中除了糖结构外分布最为广泛的化合物，不仅源于高等植物的木质素的解聚过程，而且还因为在制药、化学和石油工业中发生的事故和产生的副产物等释放过程所造成。芳香有机废物因为其苯环的高热稳定性导致的长时期难以被降解的特性，以及他们对人类产生的急性致癌、致诱变和致畸等危害性而引起高度重视。因此，在近二十年里苯环族芳香烃的代谢途径的研究也受到了人们很大程度地关注。嗜热土壤脱氮芽孢杆菌NG80-2是一株分离于中国大港油田地层水中的兼性厌氧、嗜热、可降解长链烷烃的革兰氏阳性菌株。前期的基因组学分析发现，NG80-2可能存在四组不同的苯环化合物代谢基因簇，分别为GTNG₁888-GTNG₁899（苯甲酸降解基因簇）、GTNG₁919-GTNG₁930（苯乙酸降解基因簇）、GTNG₂973-GTNG₂993（4-羟基苯乙酸降解基因簇）、GTNG₃150-GTNG₃164（邻氨基苯甲酸降解基因簇），这暗示着NG80-2可能具备降解丰富苯环化合物的能力。在本研究中，NG80-2编码的四组不同苯环化合物的基因簇通过生物信息学和蛋白组学相结合的方法被验证，它们分别是苯甲酸（苯甲酰辅酶A途径）、苯乙酸（苯乙酰辅酶A途径）、4-羟基苯乙酸（3,4-二羟基苯乙酸途径）和邻氨基苯甲酸（3-羟基邻氨基苯甲酸途径）降解途径。数据表明，这些催化第一步激活过程、环氧化和开环过程的酶往往会被所预测的苯环化合物特异地诱导，而负责下游途径的酶却表现出了较为广泛的底物特异性。另外，NG80-2的这些苯环化合物降解基因簇还编码着一些新颖的降解酶，分别为苯甲酰辅酶A环氧酶、3，4-二羟基苯乙酸-2,3-双加氧酶等。在苯甲酸降解基因簇中存在着一个paaX的同源体，在NG80-2通过苯甲酸培养的过程中起到正调控因子的作用。除了NG80-2这些苯环化合物降解酶被证明外，在NG80-2代谢这些苯环化合物的过程中，糖酵解的过程被下调了，糖异生的途径表现出上调的趋势。此外，在苯乙酸为唯一碳源的条件下，乙醛酸旁路的GTNG₅83（异柠檬酸裂合酶）和GTNG₁384（苹果酸合酶G）均有明显的上调现象，这与苯乙酸代谢过程中产生大量的乙酰辅酶A作为碳源利用密不可分，因为一分子的苯乙酸可产生2分子的乙酰辅酶A和琥珀酰辅酶A。本项目通过蛋白组学方法对NG80-2中多种苯环化合物降解途径进行了验证，也说明该嗜热菌株因其具备降解丰富苯环化合物的能力而存活在复杂的油层环境中。维氏气单胞菌是可以经常在环境、临床和食物中发现的革兰氏阴性、杆形细菌。气单胞菌属中的致病菌可引起免疫缺陷病人一系列包括创伤感染、腹泻和败血病等多种疾病。维氏气单胞菌同时也是引起鱼类出血性败血症的主要病原菌，给鱼类生产造成重大经济损失。维氏气单胞菌B565分离于中国天津的池塘淤泥沉积物中。在前期的研究中发现，该菌可通过产生的几丁质酶来控制真菌或者粘体动物引起的相关疾病。在本研究中，维氏气单胞菌B565的全基因组序列被报道并且将其与两株已报道的气单胞属的嗜水气单胞菌ATCC7966与杀鲑气单胞菌A449菌株的全基因组序列进行对比分析发现了两步进化过程。在维氏气单胞菌B565的全基因组序列中发现了5个几丁质酶基因和一些可能的毒力因子，包括溶血因子、RTX蛋白、黏附因子、鞭毛和甘露糖敏感血凝素（MSHA）等。通过将B565与已报道的两个同属的致病菌的基因组比较分析发现，该属致病菌株的毒力因子是通过两步独立的过程获得的，即一些毒力因子在很早的时间就从他们的同源祖先继承下来，另一些则可能从其他细菌通过横向转移的方式获得。更多还原

【Abstract】 IIncreasing attention has been directed towards crude-oil pollution in recent years. Among the complex chemicals in crude oil, mono-aromatic hydrocarbons, mainly the BTEX group （benzene, toluene, ethyl benzene and xylene）, most soluble constituents, tend to be one of the dominant pollutants and serious threats in contaminated groundwater. Aromatic acids, metabolic intermediates such as benzoic acid, o-, m-,/>-toluic acids, frequently detected in the contaminated aquifer indicated a series of active microbial-mediated oxidation/reduction on the aromatic hydrocarbon components. Furthermore, mono-aromatics are produced in large amounts during the conversion of aromatic amino acids in nearly all living organisms, including phenylalanine, tryptophan and tyrosine. Next to glucosyl structures, aromatics are most widely distributed in nature, not simply resulting from lignin depolymerization of higher plants, but the release from the accidents and by-products in pharmaceutical, chemical and petroleum industries. Aromatic organic wastes are of great concern for their long-term persistence due to the high thermodynamic stability of the benzene moiety, and their acute carcinogenic, mutagenic, and teratogenic risks to public health. Therefore, considerable efforts have been devoted to understanding the fate of the aromatic hydrocarbons and their various metabolic pathways in the past two decades.Geobacillus thermodenitrificans NG80-2is a Gram-positive, facultative thermophilic, long-chain alkane-degrading bacterium isolated from an oil reservoir in China. Previous analysis of the NG80-2genome revealed the presence of four distinct aromatic catabolic gene clusters predicted for benzoate （via benzoyl-CoA, GTNG₁888-GTNG₁899）, phenylacetate （via phenylacetyl-CoA, GTNG₁919-GTNG₁930）,4-hydroxyphenylacetate （via3,4-dihydroxyphenylacetate, GTNG2973-GTNG₂993）,and anthranilate （via3-hydroxyanthranilate, GTNG₃150-GTNG₃164）,indicating the ability of NG80-2to utilize diverse aromatic compounds.In this study, these gene clusters and pathways for the degradation of benzoate （via benzoyl-CoA）, phenylacetate （via phenylacetyl-CoA）,4-hydroxyphenylacetate （via3,4-dihydroxyphenylacetate） and anthranilate （via3-hydroxyanthranilate） were confirmed using combined in silico analysis and proteomics approaches. It was found that synthesis of the enzymes responsible for the initial activation, ring oxidation and ring cleavage reactions were generally induced specifically by their respective substrates, while many of the enzymes catalyzing downstream reactions exhibited broader substrate specificities. Novel genes encoding benzoyl-CoA epoxidase and3,4-dihydroxyphenylacetate2,3-dioxygenase, were proposed. A paaX homologue in the benzoate gene cluster serves as a positive regulator of benzoate degradation. Downregulation of the glycolysis pathway, along with upregulation of the gluconeogenesis pathway were detected in association with the utilization of the aromatics. Moreover, the glyoxylate bypass was upregulated in the phenylacetate-cultured NG80-2cells as shown by the increased synthesis of isocitrate lyase （GTNG₅83） and malate synthase G （GTNG₁384）, indicating the utilization of acetyl-CoA as the sole carbon source, which is produced in excess （2copies of acetyl-CoA and1succinyl-CoA per phenylacetate）. This novel proteomics analysis confirmed the presence of multiple metabolic pathways for aromatic compounds in NG80-2, which is highly advantageous to the survival of this thermophilic bacterium under reservoir conditions.ⅡAeromonas veronii is a Gram-negative, rod-shaped bacterium commonly found in the environmental, clinical, and food samples. The pathogens in the genus Aeromonas can cause diseases ranging from wound infections and diarrhoea to septicaemia in immune compromised patients.A. veronii is also the causative agent of bacterial hemorrhagic septicemia in fish, which is a major economic problem in the fish-farming industry. A. veronii strain B565was isolated from aquaculture pond sediment in Tianjin, China. In a separate study, it was found to be able to produce chitinase which can be used to control fungal or Myxozoa-related diseases.In this study, the complete genome sequence of B565was presented here and compared with2published genome sequences of the pathogenic strains in Aeromonas genus, which are A.hydrophila ATCC7966and A.sa/monicida A449.B565encodes5 chitinase genes and some putative virulence factors, such as hemolysins, RTX protein, adhesion factor, flagella, and mannose-sensitive hemagglutinin （MSHA）.The result represents an independent step-wise acquisition of virulence factors of pathogenic strains in this genus:some virulence genes were inherited long time ago from their common ancestors, while most of the other weapons can be acquired quickly from other related species by lateral transfer.更多还原