Cellulose is the most abundant biopolymer on earth. It is a linear polymer of glucose molecules primarily formed by vascular plants but also by green algae, bacteria, and even tunicates. Bacterial cellulose is frequently found in biofilms, which are sessile bacterial communities encased in a 3-dimensional matrix of polysaccharides, proteinaceous fibers, and nucleic acids. Biofilm bacteria are less susceptible to anti-microbial treatments and are responsible for about 80% of hospital-derived infections, thereby posing a significant risk to human health. Developing novel therapeutics to treat or prevent biofilm infections requires a detailed mechanistic understanding of how the biofilm constituents, in particular polysaccharides, are synthesized and deposited outside the cell. The proposed research seeks to provide this information. Bacterial cellulose biosynthesis is an ideal model system to study the mechanism and regulation of exo- polysaccharide secretion. Gram-negatives produce and secrete cellulose via a multi-subunit complex consisting of the inner membrane BcsA and BcsB subunits, the periplasmic BcsZ hydrolase, as well as the outer membrane subunit BcsC. Our previous work provided detailed mechanistic insights into how the inner membrane-integrated BcsA-B complex elongates the cellulose chain and translocates the polymer across the plasma membrane. While current data explain how cellulose is extended, we currently have no information on how cellulose biosynthesis initiates. This question will be addressed biochemically in Aim 1a by reconstituting the initiation reaction in vitro from cell-free expressed 'uninitiated' cellulose synthase. BcsA processively elongates cellulose and pushes the polymer into a transmembrane channel formed by its own membrane-spanning region. Structural snapshots of different cellulose synthase states during cellulose synthesis and membrane translocation provide insights into conformational changes during this process. Yet a precise analysis of energetic requirements for and processivity rates of cellulose translocation is currently missing. We will address these questions on a single molecule level using an optically trapped and catalytically active BcsA-B complex in Aim 1b. Past the inner membrane and in Gram-negatives, cellulose must cross the periplasm and the outer membrane before reaching the biofilm matrix. This section of the translocation path is most likely formed by a direct interaction of periplasmic and outer membrane components with the BcsA-B complex at the inner membrane. In Aim 2 we seek to reconstitute outer membrane transport of cellulose from nanodisc and proteoliposome- reconstituted components for detailed kinetic, biochemical, and interaction studies. This info...