Single-molecule investigations of single-chain cellulose biosynthesis

Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg²⁺, and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.

Cellulose is an integral structural component utilized by several kingdoms of life for its high mechanical strength and chemical stability (1, 2). Lately, cellulose’s contribution to cell walls and microbial mats has garnered great interest as cellulosic biofuels become increasingly competitive (3) and as cellulose-stabilized bacterial biofilms are shown to play significant roles in pathogenesis (4–6). Cellulose is a polysaccharide composed of repeating glucosyl units linked by β (1–4) glycosidic bonds. Investigations of its crystalline fibrillar form show that strands are linearly arranged and flat (7). In gram-negative bacteria, cellulose is manufactured through a multisubunit transenvelope bacterial cellulose synthase (Bcs) complex containing the evolutionarily conserved (8) catalytic BcsA subunit and an inner membrane–anchored domain known as BcsB (9). The membrane-embedded BcsAB complex likely interacts with BcsC in the outer membrane to form a continuous transmembrane conduit for cellulose secretion. In vitro functional and structural studies on the purified Rhodobacter sphaeroides BcsAB complex revealed that it alone is sufficient for cellulose synthesis and secretion across the inner bacterial membrane (9). BcsA is allosterically activated by cyclic diguanosine monophosphate (c-d-GMP), enabling its glycosyltransferase domain to bind the Mg²⁺-coordinated uridine diphosphate glucose (UDP-glc) substrate (10, 11). UDP-glc reacts with and elongates the nonreducing terminal end of the cellulose chain one glucose unit at a time, releasing UDP by-product afterward (12). Subsequently, the polymer translocates through a transmembrane channel formed by BcsA and is likely guided into the periplasmic space by BcsB (13). Surprisingly, the degree of processive polymerization from cellulose synthases of different origins ranges from hundreds to thousands of glucose units (14, 15).The cellulose polymer produced by BcsAB is a main component of biofilm matrices that encase sessile bacterial colonies, particularly among enterobacteria (6). Adherent bacterial populations besiege industrial systems by plugging filters, corroding metal surfaces, and fouling pipes (16). In healthcare settings, robust biofilms are responsible for ∼65% of nosocomial infections and are considerably resistant to antimicrobial treatments (5, 17). Inhibiting the production of extracellular polymeric substances, such as polysaccharides, is a strong potential antibiofilm strategy (18). Thus, a molecular understanding of bacterial cellulose synthesis is paramount for the development of powerful antibacterial agents.BcsAB has been well described by crystallographic snapshots and in vitro analyses; however, these methods lack details of biosynthesis at the molecular level (13, 19). Extensive work has been done to characterize cellulose synthesis and the properties of cellulose (1, 2, 9, 10, 13, 19–21). Cellulose, as an abundant wall polymer of vascular plants, has been described substantially in its amorphous and crystalline forms using X-ray diffraction (22), molecular dynamics simulations (22, 23), and atomic force microscopy (24), among other methods (20, 25, 26). In all cases, studies included cellulose aggregates or atomistic models. While it is known that BcsAB produces high-molecular-weight amorphous cellulose (8), the physical and dynamic properties of single cellulose chain synthesis leading to this structure have not been characterized.A real-time, molecular-scale analysis of cellulose synthesis and single-chain cellulose offers essential insight into the formation and structural qualities of this abundant biopolymer. Biosynthesis requires multiple elements, including activated glucose, c-d-GMP, and Mg²⁺. Furthermore, product transport and product microstructure may also impact biosynthesis. Cellulose production may be impacted by mechanical force, as seen in other molecular machines (27–29). Here, we use optical tweezers to directly probe mechanical and catalytic activity of single BcsAB molecules and their single-strand cellulose polymer products.