Pel polysaccharide biosynthesis requires an inner membrane complex comprised of PelD, PelE, PelF and PelG

The Pel polysaccharide is a structural component of the extracellular matrix of Pseudomonas aeruginosa biofilms. Recent analyses suggest 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 guanosine monophosphate (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 pel gene clusters from diverse species of Proteobacteria. This analysis identified an invariant set of four syntenic genes, three of which, PelD, PelE and PelG, are predicted to reside within the inner-membrane while the fourth, PelF, contains a glycosyltransferase domain. Using a combination of gene deletion analysis, sub-cellular fractionation, co-immunoprecipitation, 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. Importance The exopolysaccharide Pel plays an important role in bacterial cell-cell interactions, surface adhesion, and protection against certain antibiotics. We identified invariant pelDEFG gene clusters in over 500 diverse Proteobacterial species. Using P. aeruginosa, 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, responsible for Pel polymerization and transport across the cytoplasmic membrane. We show that the formation of this complex is independent of 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 broadens the architectural diversity of already established bacterial polysaccharide synthases.