Structural investigation of the multi-membrane spanning bacterial cellulose synthase (Bcs) supramolecular complex

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Abstract Bacteria have evolved several mechanisms to survive harsh environmental factors such as antimicrobial agents produced by competing organisms or in the clinical setting, host defenses, and other external forces. In a still poorly understood response to these environmental factors, quorum sensing planktonic bacteria begin to produce a 3-dimension gel-like extracellular matrix formed by secretion of polysaccharides, lipids, proteins, and nucleic acids known as a biofilm. Formation of the biofilm allows sessile macro-colonies to survive likely by shielding the core inhabitants from dehydration, or diffusion of antimicrobial agents that would normally be effective to a planktonic bacterium. The latter has become an extremely important in the clinical setting with an estimated 60-70% of nosocomial infections are caused by biofilm producing bacteria attached to medical insertion devices. Opportunistic bacteria including Pseudomonas aeruginosa, pathogenic Escherichia coli, and many Staphylococcal species represent the most common organisms, and can be found in patients suffering cystic fibrosis, urinary tract infections and many nosocomial infections. Adhesion is a crucial for biofilm formation, and typically begins with polysaccharide secretion. Cellulose, a linear polymer of b(1®4) D-glucose units, is one of the most abundant terrestrial biopolymers, and a frequent constituent of biofilms likely due to its strength and recalcitrance to degradation. Cellulose synthase operons are complex and vary greatly among bacterial species. In the case of pathogenic E. coli, cellulose is produced using a minimum of three proteins, a 99 kDa inner-membrane synthase (BcsA) responsible for both synthesis and translocation across the inner- membrane, a single pass 80 kDa periplasmic protein (BcsB) with obligate interactions with BcsA, and a 125 kDa outer-membrane porin (BcsC) with a large periplasmic domain. It is likely that the periplasmic domains of BcsB and BcsC both interact with the cellulose polymer and each other, however this has yet to be demonstrated. The goals of this research are to investigate complex formation in the bacterial epoxosaccharide secretion system cellulose synthase from E. coli BcsA-B-C, and to determine the 3- dimensional structure of the periplasmic/outermembrane porin protein BcsC. State-of-the-art structural analysis will be performed by in house cryogenic electron microscopy (cryo-EM) and/or use of synchrotron radiation for X-ray diffraction available from the Advance Photon Source at Argonne National Lab or NSLS-II at Brookhaven Nation Lab. Completion of these goals will result in unprecedented information of uncharted bacterial secretion systems, and give a molecular description of how polysaccharides or related virulence factors are exported into the environment. Furthermore, structural information may reveal attractive targets for designing much needed new therapeutics to ameliorate biofilm formation.