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Molecular Mechanisms Used by Auxiliary Regulator NlpE to Signal the Cpx Envelope Stress Response in Escherichia coli

  • Author / Creator
    Wang, Junshu
  • The Cpx two-component system senses and mediates adaptation to envelope stresses in Escherichia coli. NlpE is an outer membrane lipoprotein that senses surface adhesion and signals the Cpx pathway. This work focused on investigating the molecular mechanisms used by NlpE to signal the Cpx pathway when overexpressed and upon surface adhesion in E. coli. The previously solved crystal structure of NlpE revealed two distinctly formed β-barrel domains connected by an unstructured flexible linker. Mutagenesis studies revealed that both the amino- and carboxyl-terminal β-barrel domains, as well as the flexible linker contribute to signaling the Cpx pathway. The results suggest a signaling model that involves collaboration between all regions of NlpE. The amino-terminus and the flexible linker also stabilize the lipoprotein for its proper function in E. coli. In vitro pull-down, in vivo co-immunoprecipitation and cross-linking assays were carried out to identify proteins that may interact with NlpE in E. coli. The outer membrane adhesin OmpA was identified. OmpA is also required for NlpE to sense surface adhesion and convey this signal to the Cpx response. In summary, the findings of this thesis suggest a model in which outer membrane lipoprotein NlpE senses surface adhesion through a direct interaction with the adhesin OmpA and signals the Cpx pathway in a manner that involves all three structural domains of NlpE. The results provide molecular details of the manner in which auxiliary regulator NlpE communicates a signal generated at the outer membrane to a sensory kinase in the inner membrane and may be useful in the future for the design of tools to manipulate bacterial behavior.

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  • Degree
    Doctor of Philosophy
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    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.