Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

The marine bacterium, V. fischeri actively engages in an exclusive partnership in the light organ of its squid host, Euprymna scolopes. A critical step in this colonization process is the formation of a bacterial aggregate, or biofilm, which is a community of bacteria embedded within a protective extracellular matrix. While V. fischeri readily forms biofilms in nature, genetic overexpression must be utilized to achieve biofilm formation in the laboratory. Recent work investigating media composition led myself and others to evaluate how seawater salts impacted growth and biofilm formation in a number of mutant backgrounds, leading to the identification of calcium as a strong biofilm promoting signal. We identified that in our WT strain, ES114, calcium could induce formation of a biofilm ring adherent to the sides of the test tube. Calcium was also permissive to allow formation of adherent rings and cohesive cellular clumps, in a biofilm competent rscS++ strain, and in strains lacking the novel regulator BinK. These rings and clumps were dependent on cellulose and SYP, respectively. The ability to induce biofilm formation in the absence of genetic overexpression allowed us to probe the roles of known SYP regulators, SypF, SypG, and RscS. This led to the identification of HahK and HnoX, two biofilm regulators whose input had previously been masked due to overexpression conditions. To further examine how calcium is able to promote biofilm phenotypes, I assessed the contribution of the signaling molecule, c-di-GMP, determining that calcium induces c-di-GMP dependent on a specific diguanylate cyclase, CasA. CasA is directly activated by calcium, dependent on residues in an N-terminal sensory domain, and synthesizes c-di-GMP through an enzymatic C-terminal domain. CasA regulates cellulose polysaccharide at the level of transcription, dependent on the transcription factor VpsR, and provides insight into potential function of the V. cholerae homolog, CdgK. Together, these results firmly establish calcium as a key signal for biofilm formation by V. fischeri and provide one mechanism for how calcium works to impact these phenotypes.

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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