Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.

中文翻译：

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RetS 通过破坏 GacS 的典型组氨酸激酶二聚化界面来抑制铜绿假单胞菌生物膜形成。

长期以来，细菌信号组氨酸激酶 (HK) 一直被认为仅通过线性信号转导链发挥作用。然而，最近发现一些 HK 可以形成复杂的多激酶网络 (MKN)。最重要的 MKN 涉及酶 RetS 和 GacS，控制着病原菌铜绿假单胞菌运动生活方式和生物膜生活方式之间的转换。GacS 促进生物膜形成，而 RetS 使用三种不同的机制抵消 GacS。两个是去磷酸化机制。第三种是 RetS 和 GacS HK 区域之间的直接结合，阻止 GacS 自磷酸化。针对第三种机制，我们确定了 RetS 的 HK 区域与 GacS 的二聚化和组氨酸磷酸转移 (DHp) 结构域之间的共复合物的晶体结构。这是首次报道两种不同细菌信号 HK 之间的复合物结构。在该复合物中，典型的 HK 同二聚化界面被 RetS 和 GacS 之间惊人相似的异二聚化界面所取代。我们进一步证明了 GacS 反式自磷酸化，从而解释了为什么 RetS-GacS 复合物的形成会抑制 GacS 自磷酸化。将突变分析与细菌双杂交和生物膜测定结合使用，我们不仅证实了观察到的 RetS-GacS 相互作用的生物学作用，而且还确定了对 RetS-GacS 复合物与各自的 RetS 和 GacS 之间的平衡至关重要的残基同型二聚体。总的来说，我们的研究结果表明，在未知信号导致其解离和解除 GacS 抑制以促进生物膜形成之前，RetS 和 GacS 在铜绿假单胞菌的浮游生长阶段形成结构域交换的异源寡聚体。