Date of Award

2022

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

Abstract

Vibrio fischeri bacteria form biofilms that facilitate symbiotic colonization of the Hawaiian bobtail squid, Euprymna scolopes. These host-associated biofilms require production of the SYP polysaccharide. However, it has been difficult to observe robust biofilm formation in vitro in a laboratory setting using wild-type strain ES114; instead, most work investigating syp-dependent biofilm formation has relied on genetically altered strains. Guided by recent findings that increasing calcium induces c-di-GMP (Tischler et al., 2021), a molecule known in other systems to promote biofilm formation, the question was posed as to whether high levels of calcium alone could promote syp-dependent biofilm formation by ES114. Using shaking biofilm, pellicle, and spot plate assays, we observed that ES114 could readily form a biofilm when exposed to high levels of CaCl2. Within 24 hours, shaking cultures formed a ring indicative of cellulose biofilm production, as well as sticky branching structures associated with activation of SYP production. Pellicle assays revealed that ES114 could form cohesive (sticky) syp-dependent pellicles within 24 hours of exposure to high levels of calcium. Although the results were less consistent, ES114 typically produced sticky spots on plates within 5-8 days. Each assay required a different type of media for most efficient biofilm production, an interesting result that could result in further studies. In this research, a variety of gene deletion and overexpression strains were used to determine if previously identified regulators are required for biofilm formation under conditions of high calcium. Overall, this condition presents a new way to reliably induce biofilms in wild type strain ES114, quickly compare the effects of gene deletion or over-expression on biofilm production in vitro, and to investigate mechanisms underlying calcium-stimulated biofilm formation.

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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