探索大肠杆菌细胞膜合成过程中脂肪酸的掺入模式
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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-Pls B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。
[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-Co A 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-Pls B 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.
[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-Co A 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-Pls B 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.
引文
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[13]Beaty NB,Lane MD.The polymerization of acetyl-Co A carboxylase.The Journal of Biological Chemistry,1983,258(21):13051-13055.
[14]Cabeen MT,Jacobs-Wagner C.Bacterial cell shape.Nature Reviews Microbiology,2005,3(8):601-610.
[15]Gitai Z,Dye N,Shapiro L.An actin-like gene can determine cell polarity in bacteria.Proceedings of the National Academy of Sciences of the United States of America,2004,101(23):8643-8648.
[16]Bean GJ,Amann KJ.Polymerization properties of the Thermotoga maritima actin Mre B:roles of temperature,nucleotides,and ions.Biochemistry,2008,47(2):826-835.
[17]Gowrishankar K,Ghosh S,Saha S,Rumamol C,Mayor S,Rao M.Active remodeling of cortical actin regulates spatiotemporal organization of cell surface molecules.Cell,2012,149(6):1353-1367.
[18]Morgenstein RM,Bratton BP,Nguyen JP,Ouzounov N,Shaevitz JW,Gitai Z.RodZ links MreB to cell wall synthesis to mediate Mre B rotation and robust morphogenesis.Proceedings of the National Academy of Sciences of the United States of America,2015,112(40):12510-12515.
[2]Cronan JE.Bacterial membrane lipids:where do we stand?Annual Review of Microbiology,2003,57:203-224.
[3]Parsons JB,Rock CO.Bacterial lipids:metabolism and membrane homeostasis.Progress in Lipid Research,2013,52(3):249-276.
[4]Nishibori A,Kusaka J,Hara H,Umeda M,Matsumoto K.Phosphatidylethanolamine domains and localization of phospholipid synthases in Bacillus subtilis membranes.Journal of Bacteriology,2005,187(6):2163-2174.
[5]Hughes HV,Huitema E,Pritchard S,Keiler KC,Brun YV,Viollier PH.Protein localization and dynamics within a bacterial organelle.Proceedings of the National Academy of Sciences of the United States of America,2010,107(12):5599-5604.
[6]Johnson AS,van Horck S,Lewis PJ.Dynamic localization of membrane proteins in Bacillus subtilis.Microbiology,2004,150(9):2815-2824.
[7]Huang B,Bates M,Zhuang XW.Super-resolution fluorescence microscopy.Annual Review of Biochemistry,2009,78:993-1016.
[8]Bryksin AV,Matsumura I.Overlap extension PCR cloning:a simple and reliable way to create recombinant plasmids.Biotechniques,2010,48(6):463-465.
[9]You C,Zhang XZ,Zhang YHP.Simple cloning via direct transformation of PCR product(DNA Multimer)to Escherichia coli and Bacillus subtilis.Applied and Environmental Microbiology,2012,78(5):1593-1595.
[10]Yu XY,Liu TG,Zhu FY,Khosla C.In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli.Proceedings of the National Academy of Sciences of the United States of America,2011,108(46):18643-18648.
[11]Gregory JA,Becker EC,Pogliano K.Bacillus subtilis Min C destabilizes Fts Z-rings at new cell poles and contributes to the timing of cell division.Genes&Development,2008,22(24):3475-3488.
[12]Rapp M,Drew D,Daley DO,Nilsson J,Carvalho T,Melén K,De Gier JW,Von Heijne G.Experimentally based topology models for E.coli inner membrane proteins.Protein Science,2004,13(4):937-945.
[13]Beaty NB,Lane MD.The polymerization of acetyl-Co A carboxylase.The Journal of Biological Chemistry,1983,258(21):13051-13055.
[14]Cabeen MT,Jacobs-Wagner C.Bacterial cell shape.Nature Reviews Microbiology,2005,3(8):601-610.
[15]Gitai Z,Dye N,Shapiro L.An actin-like gene can determine cell polarity in bacteria.Proceedings of the National Academy of Sciences of the United States of America,2004,101(23):8643-8648.
[16]Bean GJ,Amann KJ.Polymerization properties of the Thermotoga maritima actin Mre B:roles of temperature,nucleotides,and ions.Biochemistry,2008,47(2):826-835.
[17]Gowrishankar K,Ghosh S,Saha S,Rumamol C,Mayor S,Rao M.Active remodeling of cortical actin regulates spatiotemporal organization of cell surface molecules.Cell,2012,149(6):1353-1367.
[18]Morgenstein RM,Bratton BP,Nguyen JP,Ouzounov N,Shaevitz JW,Gitai Z.RodZ links MreB to cell wall synthesis to mediate Mre B rotation and robust morphogenesis.Proceedings of the National Academy of Sciences of the United States of America,2015,112(40):12510-12515.