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Reconstitution of outer membrane protein assembly from purified components.

ABSTRACT: Beta-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled by highly conserved multi-protein complexes. The mechanism by which these molecular machines fold and insert their substrates is poorly understood. It has not been possible to dissect the folding and insertion pathway because the process has not been reproduced in a biochemical system. We purified the components that fold and insert Escherichia coli outer membrane proteins and reconstituted beta-barrel protein assembly in proteoliposomes using the enzymatic activity of a protein substrate to report on its folding state. The assembly of this protein occurred without an energy source but required a soluble chaperone in addition to the multi-protein assembly complex.

SUBMITTER: Hagan CL

PROVIDER: S-EPMC2873164 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Reconstitution of outer membrane protein assembly from purified components.

Hagan Christine L CL Kim Seokhee S Kahne Daniel D

Science (New York, N.Y.) 20100408 5980

Beta-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled by highly conserved multi-protein complexes. The mechanism by which these molecular machines fold and insert their substrates is poorly understood. It has not been possible to dissect the folding and insertion pathway because the process has not been reproduced in a biochemical system. We purified the components that fold and insert Escherichia coli outer membrane proteins and reconstituted beta ...[more]

PMID: 20378773

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Project description:Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

Dataset Information

Reconstitution of outer membrane protein assembly from purified components.

Publications

Reconstitution of outer membrane protein assembly from purified components.

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