高密度二氧化碳诱变的大肠杆菌突变菌株脂肪酸及蛋白质组分析

Fatty Acid and Proteomic Analysis of Escherichia coli Mutant Induced by Dense Phase Carbon Dioxide

采用高密度二氧化碳（DPCD）在8~16 MPa范围,以2 MPa的梯度逐步加压,每梯度压强下胁迫驯化8~10轮,筛选到1株残存率提高5.83个数量级的耐DPCD大肠杆菌突变菌株M8_5。采用气相色谱分析突变菌株与原始菌株细胞中脂肪酸的差异,发现突变菌株的棕榈一烯酸（C16：1）、十七烷酸（C17：0）、油酸（C18：1）和亚麻酸（C18：3）含量显著增加,其总脂肪酸含量也显著升高。通过双向电泳比较发现,突变菌株蛋白质组中含量差异3.5倍以上的蛋白质点有6个,其中DNA结合蛋白H-NS、锰超氧化物歧化酶（Mn SOD）、外膜蛋白F（Omp F）、30S核糖体蛋白S2、琥珀酰辅酶A合成酶含量上调,延胡索酸还原酶铁-硫蛋白簇含量下调。突变菌株十七烷酸含量增加与其DPCD耐受性提高有关,H-NS,Mn SOD和Omp F蛋白含量上调有利于突变菌株DPCD耐受性的提高。

The dense phase carbon dioxide-resistant mutant of Escherichia coli named M8_5 was obtained from Escherichia coli MG1655 after 8-10 cycles of exposure to dense phase carbon dioxide（DPCD） when the pressure was gradually increased from 8 MPa to 16 MPa with 2 MPa intervals. The survival rate of the mutant raised 5.83 magnitudes.The fatty acids of mutant strain and parent strain were analyzed by gas chromatographic. A significant increase of palmitic acid（C16：1）, cis-9, 10-methylenehexadecanoic acid（C17：0）, oleic acid（C18：1） and linolenic acid content were observed, and the total fatty acid content was also significantly increased. Comparison of proteomes of parent and mutant bacteria of MG1655 and M8_5 showed that six proteins were found in the two-dimensional electrophoresis gel with differential expression over 3.5 times. Among these proteins, the DNA-binding protein H-NS, manganese superoxide dismutase（Mn SOD）, outer membrane protein F（Omp F）, 30 S ribosomal protein S2, succinyl coenzyme A synthetase were upregulation, fumarate reductase iron- sulfur subunit was down-regulation. These results showed that the DPCD resistance of mutant strain led to the increased content of cis-9, 10-methylenehexadecanoic acid, and the up-regulation of H-NS,Mn SOD and Omp F was conducive to the improvement of the tolerance to DPCD.