探索大肠杆菌细胞膜合成过程中脂肪酸的掺入模式

Fatty acid insertion mechanism during membrane synthesis in Escherichia coli

摘要:

[目的] 探索大肠杆菌生长分裂过程中，脂肪酸作为底物在细胞膜合成过程中的掺入模式。[方法] 本研究解析了以乙酰CoA为底物，合成中间产物长链脂酰-ACP，随后合成磷脂酰乙醇胺(phosphatidylethanolamine，PE)的途径，并将合成途径中的10个关键酶与绿色荧光蛋白(enhanced green fluorescent protein，EGFP)或红色荧光蛋白(monmer Cherry，mCherry)进行融合，在大肠杆菌内表达这些融合蛋白，用激光共聚焦荧光显微镜成像的方式来获得这些融合蛋白的定位信息。[结果] 宽场荧光显微镜成像结果显示，磷脂酰乙醇胺合成途径中的10个酶在不同表达水平下出现不同的定位模式。在大肠杆菌中高水平表达融合蛋白EGFP-FabA、EGFP-FabB、EGFP-FabI、EGFP-FabG、EGFP-PlsB和EGFP-PssA时，细胞两极和中部有大量蛋白聚集的现象。EGFP-FabD、EGFP-FabF、EGFP-CdsA、EGFP-PSD在不同表达水平下，均匀分散在细胞质或细胞膜上。缩时影像(Time-lapse)结果显示，合成途径中的一个关键蛋白EGFP-PlsB在细胞分裂前随着细胞膜的内陷聚集到细胞隔膜，随着细胞分裂，母细胞的隔膜成为新细胞的两极。[结论] 本研究通过获取磷脂酰乙醇胺合成相关蛋白酶在大肠杆菌中的定位结果，推测脂肪酸分子是在细胞分裂隔膜和两极掺入，被催化合成PE后被运送到细胞膜其他位置。

Abstract:

[Objective] This study aimed to explore the mechanism of fatty acid insertion during membrane synthesis when cells grow and divide in Escherichia coli. [Methods] In the phosphatidylethanolamine (PE) synthesis pathway, acetyl-CoA was used as the substrate to synthesize long-chain acyl-ACP, followed by the synthesis of PE. The ten enzymes involved were each fused with a fluorescent protein such as enhanced green fluorescent protein (EGFP) or monmer Cherry (mCherry), and the fusion proteins were expressed in E. coli. The localizations of the fusion proteins were detected by laser scanning confocal fluorescence microscopy. [Results] Fluorescent microscope images showed that the proteins EGFP-FabA, EGFP-FabB, EGFP-FabI, EGFP-FabG, EGFP-PlsB, and EGFP-PssA accumulated in the polar and septal regions when expressed at high levels. Therefore, the ten enzymes displayed different localization patterns at different expression levels. Time-lapse imaging showed that EGFP-PlsB accumulated in the septum before cell division, then these regions of division became poles of the new cells. [Conclusion] This indicates that fatty acid inserted at the septum for PE synthesis and then PE was transported to all the other membrane regions.