本文選題：惡臭假單胞菌 + 類脂A�。� 參考：《江南大學(xué)》2017年碩士論文

【摘要】：革蘭氏陰性細(xì)菌細(xì)胞外膜外層含有大量類脂A,在細(xì)菌細(xì)胞生存和環(huán)境適應(yīng)等方面起著重要作用。惡臭假單胞菌(Pseudomonas putida)是自然界廣泛存在的革蘭氏陰性菌,在環(huán)境污染修復(fù)和生物塑料聚羥基脂肪酸酯合成方面具有應(yīng)用價(jià)值。類脂A次級脂肪酸鏈在細(xì)胞膜表面疏水性和抗生素耐藥性方面起關(guān)鍵作用,但目前P.putida類脂A次級脂肪酸鏈轉(zhuǎn)移酶還沒有報(bào)道。本研究以P.putida KT2442為研究對象,通過序列比對發(fā)現(xiàn)了兩個(gè)編碼類脂A次級脂肪酸鏈轉(zhuǎn)移酶的基因,并通過基因敲除和過表達(dá)等手段實(shí)驗(yàn)確認(rèn)了兩個(gè)轉(zhuǎn)移酶在P.putida類脂A合成過程中的具體作用。主要研究結(jié)論如下:(1)通過與E.coli LpxL、P.aeruginosa PA0011、P.aeruginosa PA3242、A.baumannii LpxM等不同來源的類脂A次級脂肪酸轉(zhuǎn)移酶進(jìn)行同源比對,發(fā)現(xiàn)P.putida KT2442存在兩個(gè)類脂A次級脂肪酸轉(zhuǎn)移酶,其編碼基因分別為PP_0063和PP_1735。(2)利用同源重組對兩個(gè)基因PP_0063和PP_1735分別進(jìn)行敲除,構(gòu)建了突變菌株KWZ001和KWZ002。分別從突變菌株中提取類脂A,并采用ESI/MS和TLC分析其化學(xué)結(jié)構(gòu),發(fā)現(xiàn)PP_0063基因的缺失導(dǎo)致KWZ001菌合成的類脂A缺少一個(gè)羥基脂肪酸鏈,而PP_1735基因的缺失導(dǎo)致KWZ002菌合成的類脂A缺少一個(gè)脂肪酸鏈;證明P.putida PP0063和PP1735為類脂A次級脂肪酸鏈轉(zhuǎn)移酶。(3)將兩個(gè)基因PP_0063和PP_1735分別在突變菌株KWZ001和KWZ002中進(jìn)行表達(dá)。從表達(dá)菌株KWZ001/pB-0063、KWZ001/pB-1735、KWZ002/pB-0063和KWZ002/pB-1735中提取類脂A,并采用ESI/MS和TLC分析其化學(xué)結(jié)構(gòu),發(fā)現(xiàn)PP_0063不能回補(bǔ)PP_1735缺失菌株,而PP_1735也不能回補(bǔ)PP_0063缺失菌株;證明類脂A次級脂肪酸鏈轉(zhuǎn)移酶PP0063和PP1735具有底物特異性。(4)為進(jìn)一步確定PP0063和PP1735在類脂A中的作用及底物特異性,將E.coli MG1655中具有底物特異性的兩個(gè)次級脂肪酸轉(zhuǎn)移酶的編碼基因lpxL和pagP分別在突變菌株KWZ001和KWZ002中進(jìn)行表達(dá)。從菌株KWZ001/pB-lpxL、KWZ001/pB-pagP、KWZ002/pB-lpxL和KWZ002/pB-pagP中提取類脂A,并采用ESI/MS和TLC分析其化學(xué)結(jié)構(gòu),明確了P.putida PP0063和PP1735分別在類脂A分子的2位和2’位上添加十二烷基脂肪酸鏈。(5)通過研究兩個(gè)敲除突變株KWZ001和KWZ002在不同環(huán)境中的生長狀況,發(fā)現(xiàn)P.putida類脂A次級脂肪酸鏈的缺失會抑制菌體在酸性環(huán)境及低溫環(huán)境的生長;通過RT-PCR發(fā)現(xiàn)酸性條件會激活類脂A分子結(jié)構(gòu)修飾酶Ept A的表達(dá)而低溫條件會降低修飾酶PagL的活性。進(jìn)一步研究表明P.putida類脂A次級脂肪酸鏈的缺失影響了細(xì)胞膜的功能,提高了細(xì)胞外膜滲透性,降低了細(xì)胞自凝集能力,并且在一定程度上影響了菌體對陽離子抗菌肽和部分抗生素的耐藥性。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q936

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本文編號：1926393