AN INTERMEDIATE STEP IN TRANSLOCATION OF LIPOPOLYSACCHARIDE TO THE OUTER-MEMBRANE OF SALMONELLA-TYPHIMURIUM
AN INTERMEDIATE STEP IN TRANSLOCATION OF LIPOPOLYSACCHARIDE TO THE OUTER-MEMBRANE OF SALMONELLA-TYPHIMURIUM
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DOI：
10.1073/pnas.80.5.1159
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
OSBORN, MJ
中科院分区：
文献类型：
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作者：
MULFORD, CA;OSBORN, MJ
新合成的脂多糖在内膜周质面的瞬时本地化的证据是通过免疫电镜技术获得的。S.使用O-抗原合成依赖于添加外源半乳糖的鼠伤寒glE突变体;通过用溶菌酶/EDTA处理制备的原生质球的抗O-抗原IgG间接铁蛋白标记来检查脉冲合成的O抗原的分布和命运。O-反应性脂多糖迅速出现在暴露的周质面的内膜添加半乳糖后，并迅速耗尽后终止的脉冲。对照实验表明，次级再分配的脂多糖从外膜没有发生的条件下采用的原生质球形成和免疫标记，和脉冲追逐动力学与预期的中间体易位的脂多糖的外膜。在半乳糖添加到galE深粗糙双突变体后30秒内，在内膜的周质面可检测到十一烯醇连接的O抗原，并且其在该位置稳定积累。深粗糙菌株中脂多糖核心合成的突变阻止了O-抗原链从磷酸十一异戊烯醇转移到脂多糖。O抗原与脂多糖的附着可能发生在内膜的胞质外侧，脂多糖从内膜的周质面而不是胞质面移位到外膜。
Evidence for transient localization of newly synthesized lipopolysaccharide at the periplasmic face of the inner membrane was obtained by immunoelectron microscopic techniques. S. typhimurium glE mutants in which O-antigen synthesis is dependent on addition of exogenous galactose were employed; the distribution and fate of pulse-synthesized O antigen was examined by indirect ferritin labeling with anti-O-antigen IgG of spheroplasts prepared by treatment with lysozyme/EDTA. O-reactive lipopolysaccharide appeared rapidly at the exposed periplasmic face of the inner membrane after addition of galactose and was rapidly depleted upon termination of the pulse. Control experiments showed that secondary redistribution of lipopolysaccharide from outer membrane did not occur under the conditions employed for spheroplast formation and immunolabeling, and the pulse-chase kinetics were consistent with those expected for an intermediate in translocation of lipopolysaccharide to the outer membrane. Undecaprenol-linked O antigen was detectable at the periplasmic face of the inner membrane within 30 s after addition of galactose to a galE deep rough double mutant and it accumulated stably in that location. The mutation in synthesis of the lipopolysaccharide core in the deep rough strain prevents transfer of O-antigen chains from undecaprenol phosphate to lipopolysaccharide. Attachment of O antigen to lipopolysaccharide may occur on the extracytoplasmic side of the inner membrane and lipopolysaccharide is translocated to the outer membrane from the periplasmic, rather than the cytoplasmic, face of the inner membrane.