Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada.
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
J Bacteriol. 2020 Mar 26;202(8). doi: 10.1128/JB.00684-19.
The Pel polysaccharide is a structural component of the extracellular matrix of biofilms. Recent analyses suggest that Pel production proceeds via a synthase-dependent polysaccharide secretion pathway, which in Gram-negative bacteria is defined by an outer membrane β-barrel porin, a periplasmic tetratricopeptide repeat-containing scaffold protein, and an inner membrane-embedded synthase. Polymerization is catalyzed by the glycosyltransferase domain of the synthase component of these systems, which is allosterically regulated by cyclic 3',5'-dimeric GMP (c-di-GMP). However, while the outer membrane and periplasmic components of the Pel system have been characterized, the inner membrane complex required for Pel polymerization has yet to be defined. To address this, we examined over 500 gene clusters from diverse species of This analysis identified an invariant set of four syntenic genes, three of which, , , and , are predicted to reside within the inner membrane, while the fourth, , encodes a glycosyltransferase domain. Using a combination of gene deletion analysis, subcellular fractionation, coimmunoprecipitation, and bacterial two-hybrid assays, we provide evidence for the existence of an inner membrane complex of PelD, PelE, and PelG. Furthermore, we show that this complex interacts with PelF in order to facilitate its localization to the inner membrane. Mutations that abolish c-di-GMP binding to the known receptor domain of PelD had no effect on complex formation, suggesting that c-di-GMP binding stimulates Pel production through quaternary structural rearrangements. Together, these data provide the first experimental evidence of an inner membrane complex involved in Pel polysaccharide production. The exopolysaccharide Pel plays an important role in bacterial cell-cell interactions, surface adhesion, and protection against certain antibiotics. We identified invariant gene clusters in over 500 diverse proteobacterial species. Using , we demonstrate that PelD, PelE, PelF, and PelG form a complex at the inner membrane and propose that this complex represents the previously unidentified Pel polysaccharide synthase, which is responsible for Pel polymerization and transport across the cytoplasmic membrane. We show that the formation of this complex is independent of cyclic 3',5'-dimeric GMP (c-di-GMP) binding to the receptor PelD. Collectively, these data establish the widespread Pel apparatus as a member of the synthase-dependent pathway of polysaccharide biosynthetic systems and broaden the architectural diversity of already-established bacterial polysaccharide synthases.
Pel 多糖是生物膜细胞外基质的结构成分。最近的分析表明，Pel 的产生是通过依赖于合成酶的多糖分泌途径进行的，在革兰氏阴性菌中，该途径由外膜 β-桶孔道蛋白、周质四肽重复支架蛋白和内膜嵌入的合成酶定义。聚合由这些系统的合成酶成分的糖基转移酶结构域催化，该结构域由环 3'，5'-二核苷酸 GMP（c-di-GMP）变构调节。然而，虽然已经描述了 Pel 系统的外膜和周质成分，但用于 Pel 聚合的内膜复合物尚未确定。为了解决这个问题，我们检查了来自不同种属的 500 多个基因簇。这项分析确定了一组不变的四个基因，其中三个，PelD、PelE 和 PelG，预计位于内膜内，而第四个，PelF，编码糖基转移酶结构域。我们使用基因缺失分析、亚细胞分级分离、共免疫沉淀和细菌双杂交测定的组合，提供了 PelD、PelE 和 PelG 内膜复合物存在的证据。此外，我们表明，该复合物与 PelF 相互作用以促进其定位于内膜。使 c-di-GMP 无法与 PelD 已知受体结构域结合的突变对复合物形成没有影响，这表明 c-di-GMP 结合通过四级结构重排刺激 Pel 产生。总之，这些数据提供了参与 Pel 多糖产生的内膜复合物的第一个实验证据。外多糖 Pel 在细菌细胞间相互作用、表面粘附和保护免受某些抗生素方面发挥着重要作用。我们在 500 多种不同的变形杆菌物种中鉴定出不变的 基因簇。使用 ，我们证明 PelD、PelE、PelF 和 PelG 在内膜形成复合物，并提出该复合物代表以前未识别的 Pel 多糖合成酶，该酶负责 Pel 聚合和穿过细胞质膜的运输。我们表明，该复合物的形成独立于 c-di-GMP 与受体 PelD 的结合。总的来说，这些数据将广泛的 Pel 装置确立为多糖生物合成系统中依赖于合成酶途径的成员，并拓宽了已建立的细菌多糖合成酶的结构多样性。
