McNulty Clodagh, Thompson James, Barrett Brendan, Lord Liz, Andersen Christian, Roberts Ian S
Faculty of Life Sciences, 1.800 Stopford Building, The University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Mol Microbiol. 2006 Feb;59(3):907-22. doi: 10.1111/j.1365-2958.2005.05010.x.
The export of large negatively charged capsular polysaccharides across the outer membrane represents a significant challenge to Gram negative bacteria. In the case of Escherichia coli group 2 capsular polysaccharides, the mechanism of export across the outer membrane was unknown, with no identified candidate outer membrane proteins. In this paper we demonstrate that the KpsD protein, previously believed to be a periplasmic protein, is an outer membrane protein involved in the export of group 2 capsular polysaccharides across the outer membrane. We demonstrate that KpsD and KpsE are located at the poles of the cell and that polysaccharide biosynthesis and export occurs at these polar sites. By in vivo chemical cross-linking and MALDI-TOF-MS analysis we demonstrate the presence of a multi-protein biosynthetic/export complex in which cytoplasmic proteins involved in polysaccharide biosynthesis could be cross-linked to proteins involved in export across the inner and outer membranes. In addition, we show that the RhsA protein, of previously unknown function, could be cross-linked to the complex and that a rhsA mutation reduces K5 biosynthesis suggesting a role for RhsA in coupling biosynthesis and export.
带大量负电荷的荚膜多糖跨外膜输出对革兰氏阴性菌而言是一项重大挑战。就大肠杆菌2型荚膜多糖而言，其跨外膜输出机制尚不清楚，也未鉴定出候选外膜蛋白。在本文中，我们证明了此前被认为是周质蛋白的KpsD蛋白是一种参与2型荚膜多糖跨外膜输出的外膜蛋白。我们证明KpsD和KpsE位于细胞两极，多糖生物合成和输出发生在这些极性位点。通过体内化学交联和基质辅助激光解吸电离飞行时间质谱分析，我们证明存在一种多蛋白生物合成/输出复合体，其中参与多糖生物合成的细胞质蛋白可与参与跨内膜和外膜输出的蛋白交联。此外，我们表明功能此前未知的RhsA蛋白可与该复合体交联，并且rhsA突变会降低K5生物合成，这表明RhsA在耦合生物合成和输出中发挥作用。
