Date of Award

2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

Abstract

Biofilms are surface-associated microbial communities surrounded by an extracellular matrix. Through biofilm formation, many pathogens and symbionts are able to colonize, survive, and persist in their host. A model system used to study biofilm formation is the symbiotic bacterium Vibrio fischeri, which colonizes its host, the squid Euprymna scolopes. Complex signaling between the squid and the bacteria is essential for the proper regulation of biofilm formation as well as for persistent colonization.

The signal(s) that promote host-relevant biofilm formation are as-yet unknown, but recently it was discovered that the sugar, L-arabinose, serves as a unique signal to promote biofilm formation in V. fischeri. Wild type V. fischeri cells grown in the presence of L-arabinose form a pellicle at the air-liquid interface of static cultures that does not occur in the absence of this sugar. However, although arabinose can induce biofilm formation in vitro, it inhibited host colonization in vivo. For my thesis, I sought to identify genes required for the cellular response to arabinose. I performed a random transposon mutagenesis, screened for mutants that were unable to form the biofilm in response to arabinose, and identified the transposon location within the genome. Through this approach, I was able to identify various genes that are involved in the production of the arabinose-induced biofilm.

In addition to the arabinose-induced biofilm, V. fischeri produces a second distinct biofilm that is critical for host colonization. The formation of this biofilm

requires the 18-gene symbiosis polysaccharide (syp) locus. Previous results have indicated that one of the regulatory proteins, SypA, is critical for biofilm formation. Deletion of sypA prevents biofilm formation in vitro and colonization in vivo. Current evidence suggests that SypA controls biofilm formation at some unknown level downstream of syp transcription. However, the mechanism by which SypA contributes to biofilm formation and host colonization remains unknown. For my thesis, I investigated mechanisms by which SypA may be contributing to the syp-dependent biofilm formation. I attempted to identify downstream protein targets of SypA that are involved in the syp-dependent biofilm formation through a random transposon mutagenesis screen. In a second approach, I used sigma factor over-expression assays to determine if SypA acted as an anti-sigma factor antagonist as predicted by its STAS (sulfate transporter and anti-sigma factor antagonist) domain. To determine whether SypA interacts with membrane bound Syp Proteins, I attempted to establish an inner membrane isolation protocol that could be used for co-Immunoprecipitation assays. Finally, I identified critical amino acid residues in SypA that are required for syp-dependent biofilm formation. These various approaches led us to develop various tools that will be used for further analysis of SypA’s role in syp-dependent biofilm formation.

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

Available for download on Friday, June 23, 2017

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