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Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on 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 BcsE 2 R 2 Q 2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain 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 activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits., (© 2024. The Author(s).)

Most bacteria respond to surfaces by biogenesis of intracellular c-di-GMP, which inhibits motility and induces secretion of biofilm-promoting adherence factors. Bacterial cellulose is a widespread biofilm component whose secretion in Gram-negative species requires an inner membrane, c-di-GMP-dependent synthase tandem (BcsAB), an outer membrane porin (BcsC), and various accessory subunits that regulate synthase assembly and function as well as the exopolysaccharide's chemical composition and mechanical properties. We recently showed that in Escherichia coli , most Bcs proteins form a megadalton-sized secretory nanomachine, but the role and structure of individual regulatory components remained enigmatic. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other's folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner BcsF. Crystallographic and solution-based data show that BcsE's predicted GIL domain is a degenerate receiver-GGDEF domain tandem (BcsE REC * -GGDEF *), where the divergent diguanylate cyclase module binds both dimeric c-di-GMP and BcsQ through mutually independent interfaces. In addition, we reveal that a third N-terminal domain (BcsE NTD ) determines the protein's homooligomerization and targeting of BcsERQ to the membrane as well as previously unreported interactions with transcription antitermination complex components. Together, the data suggest that BcsE acts on multiple levels to fine-tune bacterial cellulose secretion, from the early stages of secretion system assembly to the maintenance of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation. IMPORTANCE Bacterial cellulose is a widespread biofilm component that can modulate microbial fitness and virulence both in the environment and infected hosts. Whereas its secretion generally involves an inner membrane c-di-GMP-dependent synthase tandem (BcsAB) across the bacterial domain of life, enterobacteria feature sophisticated Escherichia coli -like Bcs secretion systems, where multiple additional subunits are either required for secretion or contribute to the maximal production of the polysaccharide in vivo. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other's folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner, BcsF. Crystallographic and functional data reveal that BcsE features unexpected domain architecture and likely acts on multiple levels to fine-tune bacterial cellulose production, from the early stages of secretion system assembly to the maintenence of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation., (Copyright © 2020 Zouhir et al.)