Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Biofilms, or surface-associated microbial communities encased in a matrix, represent a common mode of growth for many bacterial species. Importantly, biofilms often play significant roles in the association of bacteria with their animal hosts, including promoting attachment to and colonization of host tissues. The symbiosis between the bacterium Vibrio fischeri and its animal host Euprymna scolopes provides a natural model system for investigating the impact of biofilms on host infection. In particular, V. fischeri produces a specific biofilm to successfully initiate host colonization.

Biofilm formation by V. fischeri is a tightly regulated process involving a complex network of regulatory genes. These include the response regulator (RR) SypE, which preliminary studies suggest both negatively and positively impacts biofilm formation. Bioinformatic analysis predicts sypE encodes a novel, multi-domain RR consisting of a central regulatory receiver (REC) domain flanked by an N-terminal serine kinase and a C-terminal serine phosphatase domain. The aim of this study was to investigate the role of SypE in regulating biofilms and symbiotic colonization.

To examine the regulatory role of SypE, I performed a genetic analysis and assessed the contribution of the SypE domains to overall protein function. These studies determined that SypE indeed possesses both negative and positive regulatory activities, mediated by the protein's N- and C- terminal effector domains, respectively. Furthermore, the protein's central REC domain regulates these opposing activities. Finally, colonization studies revealed that inactivation of SypE's inhibitory activity is critical to permit efficient host colonization.

To further elucidate the role of SypE, I next assessed the downstream target of SypE's regulatory activities. Co-immunoprecipitation studies revealed that SypE interacts with another putative, regulatory protein, SypA. Using biochemical and genetic approaches, I determined that SypE regulates biofilms through controlling SypA phosphorylation, and that phosphorylation inhibits SypA activity. Furthermore, both biofilms and host colonization required active, unphosphorylated SypA. Together, these studies demonstrate that regulation of SypA activity by SypE is a critical mechanism by which V. fischeri controls biofilms and host colonization.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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