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Authors: Bartlett, Thomas McCabe
Advisors: Gitai, Zemer
Contributors: Molecular Biology Department
Keywords: cell shape
Subjects: Cellular biology
Issue Date: 2017
Publisher: Princeton, NJ : Princeton University
Abstract: Pathogenic Vibrio cholerae causes the epidemic diarrheal disease known as cholera, which remains a major human health concern. V. cholerae cells exhibit a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism underlying cell curvature, as well as the function of this shape, were previously unknown. This thesis describes how we identified and characterized CrvA, the first curvature determinant identified in V. cholerae. Functional fusion CrvA-msfGFP forms a clear polymer at the inner face of the cell, similar to previously identified bacterial cytoskeletons. However, CrvA has a periplasmic localization sequence, and CrvA-msfGFP is packaged into outer membrane vesicles. This suggests that unlike known cytoskeletons – which are limited to the cytoplasm – CrvA localizes to the periplasm as a periskeleton (periplasmic skeleton). To understand how CrvA generates curvature, we developed the assay QuASAR (Quantitative Analysis of Sacculus Architecture Remodeling), a quantitative method for measuring subcellular peptidoglycan insertion and removal. QuASAR demonstrated that CrvA asymmetrically patterns peptidoglycan insertion, causing proportionally more material insertion into the outer face than the inner face. Furthermore, CrvA-dependent curvature increases at high cell density, crvA is downregulated at low cell density, and quorum-sensing regulatory mutants have altered curvature, suggesting that V. cholerae curvature is quorum regulated. We also demonstrated that curvature promotes motility in dense hydrogel matrices, and that straight CrvA mutants are defective at both colonization and pathogenesis in animal infection models, suggesting that curvature is an important gel motility factor that helps it to colonize and cause disease. We also identified a second curvature determinant, CrvB, and this thesis contains a preliminary description of that protein as well.
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Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Molecular Biology

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