BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.Polysaccharides are essential biopolymers performing diverse biological functions, ranging from energy storage to osmoregulation and cell wall formation. Extracellular polysaccharides, including cellulose, chitin, and alginate, are synthesized inside the cell from nucleotide-activated sugars and are transported across the cell membrane during their synthesis. This remarkable task is performed by membrane-integrated glycosyltransferases (GTs) that couple polymer elongation with translocation (1, 2).Cellulose is a linear polymer of glucose molecules linked via β-1,4 glycosidic linkages (3, 4) and is primarily formed by vascular plants, but also by some algae, protists, and bacteria (4–6). Cellulose is synthesized by cellulose synthase (CesA), a family 2 member of GTs (7) that processively polymerizes UDP-activated glucose via an evolutionarily conserved mechanism (2). CesAs contain eight predicted transmembrane (TM) segments and at least one extended intracellular domain adopting a GT-A fold (2, 8). The intracellular GT-A domain is responsible for donor and acceptor sugar binding, as well as for catalyzing the GT reaction, and the membrane-embedded part forms a TM pore in close juxtaposition with the catalytic site, thereby allowing translocation of the nascent polysaccharide (2).Although most eukaryotic CesAs are believed to form supramolecular complexes that organize the secreted glucans into cable-like structures, i.e., the cellulose microfibrils (9), many Gram-negative bacteria synthesize cellulose as a biofilm component (10, 11). Biofilm formation is stimulated by the bacterial messenger cyclic-di-GMP (c-di-GMP) (12), which affects a diverse group of enzymes via interaction with either covalently or noncovalently attached c-di-GMP-binding domains, such as PilZ (13–15).Bacterial cellulose synthase (Bcs) is a multicomponent protein complex encoded in an operon containing at least three genes, bcsA, B, and C (16, 17). BcsA is the catalytic subunit that synthesizes cellulose and forms the TM pore across the inner membrane and BcsB is a large periplasmic protein that is anchored to the inner membrane via a single C-terminal TM helix. BcsB may guide the polymer across the periplasm toward the outer membrane via two carbohydrate-binding domains (CBDs) (2). BcsA and BcsB are fused into a single polypeptide chain in some species (18). BcsC is predicted to form a β-barrel in the outer membrane, preceded by a large periplasmic domain containing tetratricopeptide repeats likely involved in complex assembly (16). Most cellulose synthase operons also code for a periplasmic cellulase, BcsZ, whose biological function is unknown, yet it appears to enhance cellulose production in vivo (19, 20). Although most biofilm-forming bacteria likely produce amorphous cellulose that is embedded in a 3D matrix of polysaccharides, proteinaceous fibers, and nucleic acids (21), some bacteria produce cellulose microfibrils resembling those synthesized by eukaryotic cells (22). In such bacteria, CesA complexes are linearly arranged along the cell axis, and the CesA operons encode at least one additional subunit, BcsD, that might facilitate the linear organization of the synthases (18).Despite the numerous studies available on a large number of pro- and eukaryotic model systems, revealing the mechanism of cellulose synthesis and translocation has been hampered by difficulties in reconstituting functional CesAs in a purified system, either from eukaryotic or prokaryotic enzymes (23–26). To date, cellulose biosynthetic activities have only been recovered from detergent extracts of native membranes (24–26).To overcome these challenges, we reconstituted an active cellulose synthetic system in vitro from a purified Rhodobacter sphaeroides BcsA-B complex (27). The purified complex efficiently synthesizes amorphous, high-molecular-weight (HMW) cellulose on incubation with UDP-glucose (UDP-Glc) and c-di-GMP, both in a detergent-solubilized state and after reconstitution into proteoliposomes (PLs). We show that cellulose elongation occurs directly from UDP-Glc without lipid-linked intermediates, reveal that c-di-GMP activates the synthase, and demonstrate the strict substrate specificity of BcsA for UDP-Glc. Furthermore, we demonstrate that BcsB is crucial for the catalytic activity of BcsA and localize the region required for cellulose synthesis within BcsB’s C-terminal, membrane-associated domain that packs against the TM region of BcsA.