Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B(12) receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.

译文

数据表明大肠菌素E1 (ColE1) 跨大肠杆菌外膜转运的双受体模型。ColE1最初与维生素b (12) 受体BtuB结合,然后通过TolC通道-隧道转运,大概处于大部分未折叠状态。在这里,我们研究了cole1导入的早期事件。使用体内方法,我们显示了当添加到整个细胞中时,ColE1被切割。这种切割需要受体BtuB和蛋白酶OmpT的存在,而不是TolC的存在。表达OmpT的菌株在N末端易位结构域的K84和K95处裂解了ColE1,导致去除TolQA盒,这对于ColE1的细胞毒性至关重要。在其他体内数据的支持下,这表明OmpT的功能是降解细胞表面的大肠菌素,从而保护敏感的大肠杆菌细胞免受大肠杆菌的感染。还描述了一种分离tolC突变的遗传策略,该策略赋予ColE1抗性,而不影响其他TolC功能。我们通过使用交联,然后通过组氨酸标记的TolC进行共尿化,进一步提供了TolC和ColE1之间多步相互作用的体内证据。首先,ColE1与TolC的二级结合取决于与BtuB的一级结合。其次,TolC通道中残基的改变会干扰ColE1在TolC孔中的转运,而不是干扰ColE1与TolC的结合。相反,在细胞表面暴露的残基上的取代消除了cole1的结合和易位。

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