Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes
Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes
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DOI：
10.1073/pnas.1830264100
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发表时间：
2003-09-02
影响因子：
11.1
通讯作者：
de la Cruz, F
中科院分区：
文献类型：
--
作者：
Llosa, M;Zunzunegui, S;de la Cruz, F
共轭偶联蛋白（CPs）被认为在细菌偶联过程中将松弛体与IV型分泌系统（T4SS）连接起来。在这里，我们提出了生化和遗传证据，表明原型CP TrwB与质粒R388的松弛体和IV型分泌成分相互作用。用亲和树脂固定的TrwB细胞质结构域保留了R388松弛体的组分TrwC和TrwA蛋白。利用细菌双杂交系统，检测到TrwB和TrwE之间存在强相互作用，TrwE是共轭T4SS的核心成分。当TrwB或TrwE的跨膜结构域被删除时，这种相互作用就会消失，从而表明它发生在两种蛋白质的膜或质周部分。我们还分析了与相关IncN质粒pKM101组分的相互作用。它的CP TraJ不与TrwA相互作用，表明与松弛体有高度特异性的相互作用。另一方面，来自三种不同缀合体系的CPs显示与其同源trwe -样组分和异源trwe -样组分相互作用，表明这种相互作用的特异性较低。三个系统之间的交配实验证实，松弛体成分需要同源CP才能转移，而t4ss是可互换的。一般来说，双杂交分析所看到的相互作用强度与传递效率之间存在相关性。
Conjugative coupling proteins (CPs) are proposed to play a role in connecting the relaxosome to a type IV secretion system (T4SS) during bacterial conjugation. Here we present biochemical and genetic evidence indicating that the prototype CP, TrwB, interacts with both relaxosome and type IV secretion components of plasmid R388. The cytoplasmic domain of TrwB immobilized in an affinity resin retained TrwC and TrwA proteins, the components of R388 relaxosome. By using the bacterial two-hybrid system, a strong interaction was detected between TrwB and TrwE, a core component of the conjugative T4SS. This interaction was lost when the transmembrane domains of either TrwB or TrwE were deleted, thus suggesting that it takes place within the membrane or periplasmic portions of both proteins. We have also analyzed the interactions with components of the related IncN plasmid pKM101. Its CP, TraJ, did not interact with TrwA, suggesting a highly specific interaction with the relaxosome. On the other side, CPs from three different conjugation systems were shown to interact with both their cognate TrwE-like component and the heterologous ones, suggesting that this interaction is less specific. Mating experiments among the three systems confirmed that relaxosome components need their cognate CP for transfer, whereas T4SSs are interchangeable. As a general rule, there is a correlation between the strength of the interaction seen by two-hybrid analysis and the efficiency of transfer.