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首页> 外文期刊>Journal of bacteriology >Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop.
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Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop.

机译：删除预期门控环内外的FhuA部分后，大肠杆菌外膜中FhuA通道的特性。

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摘要

Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy-dependent process mediated by the FhuA protein. A FhuA deletion derivative lacking residues 322 to 355 (FhuA delta322-355) forms a permanently open channel through which ferrichrome diffused. This finding led to the concept that the FhuA protein forms a closed channel that is opened by input of energy derived from the electrochemical potential across the cytoplasmic membrane, mediated by the Ton system. In this study, we constructed various FhuA derivatives containing deletions inside and outside the gating loop. FhuA delta322-336 bound ferrichrome and displayed a residual Ton-dependent ferrichrome transport activity. FhuA delta335-355 no longer bound ferrichrome but supported ferrichrome diffusion through the outer membrane in the absence of the Ton system. FhuA delta335-355 rendered cells sensitive to sodium dodecyl sulfate and supported diffusion of maltotetraose and maltopentaose in a lamB mutant lacking the maltodextrin-specific channel in the outer membrane. Cells expressing FhuA delta70-223, which has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only weak ferrichrome uptake, suggesting formation of a nonspecific pore through the outer membrane. FhuA delta457-479 supported Ton-dependent uptake of ferrichrome. None of these FhuA deletion derivatives formed pores in black lipid membranes with a stable single-channel conductance. Rather, the conductance displayed a high degree of current noise, indicating a substantial influence of the deletions on the conformation of the FhuA protein. FhuA also supports infection by the phages T1, T5, and phi80 and renders cells sensitive to albomycin and colicin M. Cells expressing FhuA delta322-336 were sensitive to albomycin and colicin M but were only weakly sensitive to T5 and phi480 and insensitive to T1. Cells expressing FhuA delta335-355 were resistant to all FhuA ligands. These results indicate different structural requirements within the gating loop for the various FhuA ligands. Cells expressing FhuA delta457-479 displayed a strongly reduced sensitivity to all FhuA ligands, while cells expressing FhuA delta70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resistant. It is concluded that residues 335 to 355 mainly determine the properties of the gate with regard to FhuA permeability and ligand binding.
机译：大肠杆菌以FhuA蛋白介导的能量依赖过程将Fe3 +作为三价铬络合物转运通过外膜。缺少残基322至355的FhuA缺失衍生物（FhuA delta322-355）形成了永久开放的通道，亚铁色素通过该通道扩散。这一发现导致了以下概念：FhuA蛋白形成一个封闭的通道，该通道通过输入由Ton系统介导的跨细胞质膜的电化学势所产生的能量来打开。在这项研究中，我们构建了各种FhuA衍生物，它们在门控环的内部和外部都有缺失。 FhuA delta322-336结合了铬铁，并显示出残留的依赖吨的铬铁运输活性。在没有Ton系统的情况下，FhuA delta335-355不再结合铁铬铁，但支持铁铬铁通过外膜的扩散。 FhuA delta335-355使细胞对十二烷基硫酸钠敏感，并支持麦芽四糖和麦芽五糖在没有外部麦芽糖糊精特异性通道的lamB突变体中扩散。表达FhuA delta70-223的细胞在门控环外具有较大的缺失，对十二烷基硫酸钠高度敏感，并在麦芽糖糊精上生长，但仅表现出弱的铬铁摄取，表明在外膜上形成了非特异性孔。 FhuA delta457-479支持吨依赖摄取的铬铁。这些FhuA缺失衍生物均未在黑色脂质膜中形成具有稳定单通道电导的孔。而是，电导显示出高度的电流噪声，表明缺失对FhuA蛋白的构象有实质性影响。 FhuA还支持被噬菌体T1，T5和phi80感染，并使细胞对阿波霉素和大肠菌素M敏感。表达FhuA delta322-336的细胞对阿波霉素和大肠菌素M敏感，但仅对T5和phi480敏感，对T1不敏感。表达FhuA delta335-355的细胞对所有FhuA配体均具有抗性。这些结果表明在门控环内对于各种FhuA配体的不同结构要求。表达FhuA delta457-479的细胞显示出对所有FhuA配体的敏感性大大降低，而表达FhuA delta70-223的细胞对除阿尔巴霉素外几乎对它们具有抗性的所有FhuA配体都相当敏感。结论是，残基335至355主要决定了门的FhuA渗透性和配体结合特性。

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