恶臭假单胞菌类脂A次级脂肪酸链转移酶的基因鉴定

发布时间：2018-05-23 21:06

本文选题：恶臭假单胞菌 + 类脂A　；参考：《江南大学》2017年硕士论文

【摘要】：革兰氏阴性细菌细胞外膜外层含有大量类脂A,在细菌细胞生存和环境适应等方面起着重要作用。恶臭假单胞菌(Pseudomonas putida)是自然界广泛存在的革兰氏阴性菌,在环境污染修复和生物塑料聚羟基脂肪酸酯合成方面具有应用价值。类脂A次级脂肪酸链在细胞膜表面疏水性和抗生素耐药性方面起关键作用,但目前P.putida类脂A次级脂肪酸链转移酶还没有报道。本研究以P.putida KT2442为研究对象,通过序列比对发现了两个编码类脂A次级脂肪酸链转移酶的基因,并通过基因敲除和过表达等手段实验确认了两个转移酶在P.putida类脂A合成过程中的具体作用。主要研究结论如下:(1)通过与E.coli LpxL、P.aeruginosa PA0011、P.aeruginosa PA3242、A.baumannii LpxM等不同来源的类脂A次级脂肪酸转移酶进行同源比对,发现P.putida KT2442存在两个类脂A次级脂肪酸转移酶,其编码基因分别为PP_0063和PP_1735。(2)利用同源重组对两个基因PP_0063和PP_1735分别进行敲除,构建了突变菌株KWZ001和KWZ002。分别从突变菌株中提取类脂A,并采用ESI/MS和TLC分析其化学结构,发现PP_0063基因的缺失导致KWZ001菌合成的类脂A缺少一个羟基脂肪酸链,而PP_1735基因的缺失导致KWZ002菌合成的类脂A缺少一个脂肪酸链;证明P.putida PP0063和PP1735为类脂A次级脂肪酸链转移酶。(3)将两个基因PP_0063和PP_1735分别在突变菌株KWZ001和KWZ002中进行表达。从表达菌株KWZ001/pB-0063、KWZ001/pB-1735、KWZ002/pB-0063和KWZ002/pB-1735中提取类脂A,并采用ESI/MS和TLC分析其化学结构,发现PP_0063不能回补PP_1735缺失菌株,而PP_1735也不能回补PP_0063缺失菌株;证明类脂A次级脂肪酸链转移酶PP0063和PP1735具有底物特异性。(4)为进一步确定PP0063和PP1735在类脂A中的作用及底物特异性,将E.coli MG1655中具有底物特异性的两个次级脂肪酸转移酶的编码基因lpxL和pagP分别在突变菌株KWZ001和KWZ002中进行表达。从菌株KWZ001/pB-lpxL、KWZ001/pB-pagP、KWZ002/pB-lpxL和KWZ002/pB-pagP中提取类脂A,并采用ESI/MS和TLC分析其化学结构,明确了P.putida PP0063和PP1735分别在类脂A分子的2位和2’位上添加十二烷基脂肪酸链。(5)通过研究两个敲除突变株KWZ001和KWZ002在不同环境中的生长状况,发现P.putida类脂A次级脂肪酸链的缺失会抑制菌体在酸性环境及低温环境的生长;通过RT-PCR发现酸性条件会激活类脂A分子结构修饰酶Ept A的表达而低温条件会降低修饰酶PagL的活性。进一步研究表明P.putida类脂A次级脂肪酸链的缺失影响了细胞膜的功能,提高了细胞外膜渗透性,降低了细胞自凝集能力,并且在一定程度上影响了菌体对阳离子抗菌肽和部分抗生素的耐药性。
[Abstract]:The outer membrane of Gram-negative bacteria contains a large amount of lipids, which plays an important role in cell survival and environmental adaptation. Pseudomonas putida (Pseudomonas putida) is a kind of gram-negative bacteria, which is widely existed in nature. It has application value in environmental pollution remediation and biosynthesis of polyhydroxyfatty acid esters of biological plastics. Lipid A secondary fatty acid chain plays a key role in cell membrane hydrophobicity and antibiotic resistance, but P.putida lipid A secondary fatty acid chain transferase has not been reported. In this study, two genes encoding lipid-A secondary fatty acid chain transferases were found by sequence alignment with P.putida KT2442. Through gene knockout and overexpression, the specific roles of two transferases in the synthesis of P.putida lipids were confirmed. The main results are as follows: (1) by homologous comparison with E.coli LpxL P.aeruginosa PA0011, P.aeruginosa PA3242 and A. baumannii LpxM, it was found that there were two lipid A secondary fatty acid transferases in P.putida KT2442. The two genes PP_0063 and PP_1735 were knocked out by homologous recombination and the mutant strains KWZ001 and KWZ002 were constructed. Lipoid A was extracted from mutant strains and its chemical structure was analyzed by ESI/MS and TLC. It was found that the absence of PP_0063 gene resulted in the absence of a hydroxyl fatty acid chain in the lipoid A synthesized by KWZ001. The absence of PP_1735 gene resulted in the absence of a fatty acid chain in the lipoid A synthesized by KWZ002 strain, which proved that P.putida PP0063 and PP1735 were the secondary fatty acid chain transferase of lipids A, and the two genes PP_0063 and PP_1735 were expressed in the mutant strains KWZ001 and KWZ002, respectively. Lipoid A was extracted from KWZ001 / pB-0063 / KWZ001 / pB-1735 / KWZ002 / pB-0063 and KWZ002/pB-1735, and its chemical structure was analyzed by ESI/MS and TLC. It was found that PP_0063 could not compensate PP_1735 deficient strain and PP_1735 could not compensate PP_0063 deficient strain. In order to further determine the role of PP0063 and PP1735 in lipid A and substrate specificity, it was proved that PP0063 and PP1735 had substrate specificity. Two substrate-specific encoding genes of fatty acid transferase in E.coli MG1655, lpxL and pagP, were expressed in the mutant KWZ001 and KWZ002, respectively. Lipoid A was extracted from strain KWZ001 / pB-lpxLN KWZ001 / pB-pagPpage KWZ002 / pB-lpxL and KWZ002/pB-pagP, and its chemical structure was analyzed by ESI/MS and TLC. The growth status of two knockout mutants, KWZ001 and KWZ002, was studied by adding dodecyl fatty acid chain to 2 and 2 'sites of lipoid A molecule by P.putida PP0063 and PP1735, respectively. It was found that the deletion of secondary fatty acid chain of P.putida lipids A inhibited the growth of bacteria in acidic and low temperature environments, and that acid conditions activated the expression of Ept A by RT-PCR, while the activity of PagL decreased under low temperature conditions. Further studies showed that the loss of secondary fatty acid chain of P.putida lipid A affected the function of cell membrane, improved the permeability of extracellular membrane, and reduced the self-agglutination ability of cells. To some extent, the resistance of bacteria to cationic antimicrobial peptides and some antibiotics was affected.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

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