Sequential translocation of an Escherchia coli two-partner secretion pathway exoprotein across the inner and outer membranes
Sequential translocation of an Escherchia coli two-partner secretion pathway exoprotein across the inner and outer membranes
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DOI：
10.1111/j.1365-2958.2009.06993.x
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发表时间：
2010-01-01
影响因子：
3.6
通讯作者：
Bernstein, Harris D.
中科院分区：
文献类型：
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作者：
Choi, Peter S.;Bernstein, Harris D.
在革兰氏阴性菌中，多种高分子量的“外蛋白”通过与专用转运蛋白相互作用经由双伴侣分泌（TPS）途径跨外膜（OM）易位。然而，目前尚不清楚外蛋白跨OM的易位是否与它们跨内膜（IM）的易位相耦合。为了解决这个问题，我们分开生产的大肠杆菌O 157：H7外蛋白（OtpA）和它的转运蛋白（OtpB）暂时通过放置otpA和otpB的控制下，不同的可调控的启动子。我们发现，当全长和截断形式的OtpA表达前OtpB，分泌的外蛋白的一个显着的分数。结果表明，OtpA可以易位到周质，并短暂地保持分泌能力。此外，通过将半胱氨酸残基工程化到OtpA中并使用二硫键形成作为周质定位的报告者，我们获得了另外的证据，即OtpA的C-末端在N-末端易位穿过OM之前进入周质，即使当OtpA和OtpB同时表达时。两者合计，我们的研究结果表明，跨OM的TPS外蛋白的易位可以独立于其跨IM的易位发生。
In Gram-negative bacteria, a variety of high molecular weight 'exoproteins' are translocated across the outer membrane (OM) via the two-partner secretion (TPS) pathway by interacting with a dedicated transporter. It is unclear, however, whether the translocation of exoproteins across the OM is coupled to their translocation across the inner membrane (IM). To address this question, we separated the production of an Escherichia coli O157:H7 exoprotein (OtpA) and its transporter (OtpB) temporally by placing otpA and otpB under the control of distinct regulatable promoters. We found that when both full-length and truncated forms of OtpA were expressed prior to OtpB, a significant fraction of the exoprotein was secreted. The results indicate that OtpA can be translocated into the periplasm and briefly remain secretion-competent. Furthermore, by engineering cysteine residues into OtpA and using disulphide bond formation as a reporter of periplasmic localization, we obtained additional evidence that the C-terminus of OtpA enters the periplasm before the N-terminus is translocated across the OM even when OtpA and OtpB are expressed simultaneously. Taken together, our results demonstrate that the translocation of a TPS exoprotein across the OM can occur independently from its translocation across the IM.