Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.
mBio. 2012 Feb 28;3(2). doi: 10.1128/mBio.00251-11. Print 2012.
Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells.
Many proteins localize to specific sites within bacterial cells, and localization to these sites is frequently critical to proper protein function. The mechanisms that underlie protein localization are incompletely understood, in part because of the paucity of methods that allow saturation screening for mutants in which protein localization is altered. We developed a genetic reporter assay that enables screening of bacterial populations for changes in localization of proteins to the bacterial pole, and we demonstrate the utility of the system in identifying factors required for proper localization of the polar Shigella autotransporter protein IcsA. Using this method, we identify the conserved cell division protein FtsQ as being required for positioning of IcsA to the bacterial pole. We demonstrate further that the requirement for FtsQ for polar positioning applies to other autotransporters and that the method can be applied to polar proteins other than autotransporters.
细菌内的空间组织对于许多细胞过程至关重要，尽管将蛋白质定位到细菌内特定位置的基本机制仍未被很好地理解。蛋白质定位的研究受到缺乏允许快速大规模筛选蛋白质定位发生改变的突变体的方法的限制。我们开发了一种用于将蛋白质定位到细菌细胞质内极区的遗传报告系统，该系统允许对蛋白质定位发生改变的大肠杆菌突变体进行饱和筛选。利用该系统，我们确定了 Shigella 自转运蛋白 IcsA 正确定位所需的蛋白质。自转运蛋白是广泛分布的细菌毒力蛋白，在细菌极区分泌。我们表明，保守的细胞分裂蛋白 FtsQ 是 IcsA 和其他自转运蛋白定位到极区所必需的。我们进一步证明，该系统可应用于研究除自转运蛋白以外的在细菌细胞内呈现极定位的蛋白质。
许多蛋白质定位于细菌细胞内的特定位置，并且蛋白质的定位通常对其正确功能至关重要。蛋白质定位的机制尚未完全理解，部分原因是缺乏允许对蛋白质定位发生改变的突变体进行饱和筛选的方法。我们开发了一种遗传报告测定法，该测定法可用于筛选细菌群体中蛋白质向细菌极区定位的变化，并且我们证明了该系统在鉴定正确定位极区 Shigella 自转运蛋白 IcsA 所需的因子方面的实用性。使用该方法，我们确定了保守的细胞分裂蛋白 FtsQ 是将 IcsA 定位到细菌极区所必需的。我们进一步表明，FtsQ 对极区定位的要求适用于其他自转运蛋白，并且该方法可应用于除自转运蛋白以外的极区蛋白。
