Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system
Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system
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DOI：
10.1371/journal.pgen.1008059
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发表时间：
2019-04-01
期刊：
影响因子：
4.5
通讯作者：
Hengge, Regine
中科院分区：
文献类型：
--
作者：
Pfiffer, Vanessa;Sarenko, Olga;Hengge, Regine
普遍存在的第二信使c-di-GMP通过在潜在的调控网络中发挥不同的作用来促进细菌生物膜的形成。这反映在大多数细菌物种中分别合成和降解c-di-GMP的二鸟苷酸环化酶（DGC）和磷酸二酯酶（PDE）的多样性中。大肠杆菌的12个DGC之一，DgcE，作为大菌落生物膜形成过程中细胞外基质产生的顶级触发因子。它的多结构域的结构-一个N-末端膜插入MASE1域，其次是三个PAS，GGDEF和一个简并的EAL域-建议复杂的信号整合和传输通过DgcE。DgcE的遗传解剖揭示了MASE 1结构域和二聚化活性PAS（3）区域的激活作用，而抑制性EAL（deg）结构域抵消了DgcE寡聚体的形成。MASE 1结构域直接被GTdR RdcA（YjdA）靶向，GTdR RdcA是一种二聚体或寡聚体，与其伴侣蛋白RdcB（YjcZ）一起激活DgcE，可能是通过对齐和促进PAS（3）和GGDEF结构域的二聚化。这种激活和RdcA/DgcE的相互作用依赖于GTP水解RdcA，这表明GTP作为抑制剂和显着减少的细胞GTP池在进入稳定期，这与DgcE依赖性激活基质生产，作为一个可能的输入信号RdcA感测。此外，DgcE表现出快速，连续和进行性的蛋白水解周转，这也取决于相对无序的跨膜MASE 1结构域。总的来说，我们的研究揭示了一种新的GTP/C-二-GMP连接信号通路通过多结构域DGC DgcE具有双重作用的以前未表征的MASE1信号domain.Author摘要生物膜代表了一种多细胞的生命形式的细菌，其中大量的细胞生活在社区包围和保护的自我产生的细胞外聚合物基质。由于生物膜耐受抗生素和宿主免疫系统，它们与慢性感染有因果关系。生物膜的形成通常由普遍存在的细菌第二信使c-di-GMP促进。DgcE是大肠杆菌中产生c-di-GMP的12种二鸟苷酸环化酶之一。大肠杆菌，以前被证明是专门作为一个顶级触发器的调控网络，驱动生物膜基质生产在这种细菌。然而，信号输入到DgcE本身，这是一个大的六域蛋白，仍然是未知的。在这里，我们表明，DgcE活性是由一种新型的动力蛋白样GTdR，直接与N-末端膜固有MASE1结构域的DgcE相互作用。我们发现这个MASE1结构域的双重功能，这是必不可少的激活和连续的蛋白水解的DgcE，是这个广泛的细菌信号结构域的第一个表征。通过动力蛋白样GTP酶系统的信号输入表明，DgcE产生的c-di-GMP可能受到进入稳定期期间细胞GTP水平降低的刺激，这正是生物膜基质产生开启的时间。
The ubiquitous second messenger c-di-GMP promotes bacterial biofilm formation by playing diverse roles in the underlying regulatory networks. This is reflected in the multiplicity of diguanylate cyclases (DGC) and phosphodiesterases (PDE) that synthesize and degrade c-di-GMP, respectively, in most bacterial species. One of the 12 DGCs of Escherichia coli, DgcE, serves as the top-level trigger for extracellular matrix production during macrocolony biofilm formation. Its multi-domain architecture-a N-terminal membrane-inserted MASE1 domain followed by three PAS, a GGDEF and a degenerate EAL domain-suggested complex signal integration and transmission through DgcE. Genetic dissection of DgcE revealed activating roles for the MASE1 domain and the dimerization-proficient PAS(3) region, whereas the inhibitory EAL(deg) domain counteracts the formation of DgcE oligomers. The MASE1 domain is directly targeted by the GTPase RdcA (YjdA), a dimer or oligomer that together with its partner protein RdcB (YjcZ) activates DgcE, probably by aligning and promoting dimerization of the PAS(3) and GGDEF domains. This activation and RdcA/DgcE interaction depend on GTP hydrolysis by RdcA, suggesting GTP as an inhibitor and the pronounced decrease of the cellular GTP pool during entry into stationary phase, which correlates with DgcE-dependent activation of matrix production, as a possible input signal sensed by RdcA. Furthermore, DgcE exhibits rapid, continuous and processive proteolytic turnover that also depends on the relatively disordered transmembrane MASE1 domain. Overall, our study reveals a novel GTP/c-di-GMP-connecting signaling pathway through the multi-domain DGC DgcE with a dual role for the previously uncharacterized MASE1 signaling domain.Author summary Biofilms represent a multicellular life form of bacteria, in which large numbers of cells live in communities surrounded and protected by a self-generated extracellular polymeric matrix. As biofilms tolerate antibiotics and host immune systems, they are causally associated with chronic infections. Biofilm formation is generally promoted by the ubiquitous bacterial second messenger c-di-GMP. DgcE, one of the 12 diguanylate cyclases that produce c-di-GMP in E. coli, was previously shown to specifically act as a top level trigger in the regulatory network that drives biofilm matrix production in this bacterium. However, signal input into DgcE itself, which is a large six-domain protein, had remained unknown. Here we demonstrate that DgcE activity is controlled by a novel type of dynamin-like GTPase that directly interacts with the N-terminal membrane-intrinsic MASE1 domain of DgcE. Our finding of a dual function of this MASE1 domain, which is essential for both activation and continuous proteolysis of DgcE, is the first characterization of this widespread bacterial signaling domain. Signal input via the dynamin-like GTPase system suggests that c-di-GMP production by DgcE might be stimulated by the decreasing cellular GTP level during entry into stationary phase, which is precisely the time when biofilm matrix production is turned on.