Mechanism of Cellulose Synthesis and Transport Across Biological Membranes

纤维素合成和跨生物膜运输的机制

基本信息

批准号：
10061615
负责人：
Jochen Zimmer
金额：
$ 54.73万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-05 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10061615
关键词：
3-Dimensional Address Anabolism Architecture Bacteria Binding Biochemical Biological Biological Assay Biological Models Biophysics Biopolymers C-terminal Cell membrane Cells Cellulose Chemicals Chromosomal translocation Complex Couples Crystallization Data Deposition Electron Microscopy Enzymes Fiber Glucans Glucose Glycosides Green Algae Health Hospitals Human Hydrolase In Vitro Individual Infection Kinetics Length Lipids Measurement Membrane Microbial Biofilms Modeling Molecular Conformation Nucleic Acids Oligosaccharides Planet Earth Polymers Polysaccharides Polystyrenes Process Property Reaction Regulation Research Resolution Risk Roentgen Rays Side Slide Structure Surface System Testing Transmembrane Transport Urochordata Vascular Plant Vesicle Work X-Ray Crystallography antimicrobial aptamer bacterial community biophysical techniques cell envelope cellulose synthase experimental study genetic analysis genetic linkage analysis in vivo insight microbial community nanodisk novel therapeutics optical traps periplasm prevent proteoliposomes reconstitution single molecule small molecule

3-Dimensional Address Anabolism Architecture Bacteria Binding Biochemical Biological Biological Assay Biological Models Biophysics Biopolymers C-terminal Cell membrane Cells Cellulose Chemicals Chromosomal translocation Complex Couples Crystallization Data Deposition Electron Microscopy Enzymes Fiber Glucans Glucose Glycosides Green Algae Health Hospitals Human Hydrolase In Vitro Individual Infection Kinetics Length Lipids Measurement Membrane Microbial Biofilms Modeling Molecular Conformation Nucleic Acids Oligosaccharides Planet Earth Polymers Polysaccharides Polystyrenes Process Property Reaction Regulation Research Resolution Risk Roentgen Rays Side Slide Structure Surface System Testing Transmembrane Transport Urochordata Vascular Plant Vesicle Work X-Ray Crystallography antimicrobial aptamer bacterial community biophysical techniques cell envelope cellulose synthase experimental study genetic analysis genetic linkage analysis in vivo insight microbial community nanodisk novel therapeutics optical traps periplasm prevent proteoliposomes reconstitution single molecule small molecule

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项目摘要

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 information will support our efforts to determine the structure of an inner and outer membrane-spanning cellulose synthase complex as outlined in Aim 3. We will use X-ray crystallography and/or electron microscopy to determine the structure of individual periplasmic and outer membrane components as well as their complexes with BcsA-B.