Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that rely on conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthases. Polymer structure and modifications, however, depend on the ensemble of synthase modules and accessory subunits, thus defining several types of bacterial cellulose secretion (Bcs) systems. In E. coli, a BcsRQABEFG macrocomplex, encompassing the inner membrane and cytosolic subunits, and an outer membrane porin, BcsC, secure the biogenesis of phosphoethanolamine (pEtN)-modified cellulose. Resolution-limited studies have proposed different macrocomplex stoichiometries and its assembly and regulation have remained elusive. Using cryo-EM, we visualize the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, remains dinucleotide-bound and retains the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activation even in the absence of dramatic intracellular c-di-GMP increase and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where c-di-GMP sensing and/or synthase stabilization are carried out by key co-polymerase subunits.