Objective: We investigated differences in morphology, physiology, periplasmic proteins and phagocytosis by macrophage among E. coli strains AD93 (PE- PC-), AD93/ptac67 (PE- PC+), Top10/ptac66(PE+ PC+) and the wild types in order to understand if phosphatidylcholine (PC) can substitute phosphatidylethanolamine (PE) in vivo.
Methods: Bacterial cells were observed under microscope after staining with Gram-staining kit or by electron microscope. Bacterial growth under different conditions was monitored by measuring the absorbance at the wavelength of 600 nm. Periplasmic proteins were analyzed using SDS-PAGE and 2-D electrophoresis. Bacterial adherence and phagocytosis by macrophage were also examined by using murine RAW264.7 macrophage.
Results: 100% bacterial cells in AD93/pDD72 were bar-shaped but 25% AD93/ptac67 cells came out as long filaments. Different from AD93/ pDD72, AD93/ptac67 and AD93 required Mg2+ or Ca2+ for growth. Moreover, AD93/ptac67 displayed a different pattern of periplasmic proteins on a 2-D gel and a low relatively phagocytic efficiency in the phagocytosis test when compared to AD93/pDD72 and AD93. Both Top10/ptac66 and the wild-type Top10/ptac85 cells were bar-shaped under microscope, but the former showed noticeably difference in the outer-layer structure of cell wall, and its stress resistance and periplasmic protein composition were also different from those of the latter.
Conclusion: Substitution of phosphatidylethanolamine with phosphatidylcholine in E. coli cells is unable to restore the phonotype of PE- mutant to the wild type. Biological functions of PE and PC are different, and phosphatidylcholine cannot substitute phosphatidylethanolamine in vivo.