Date of Award
Doctor of Philosophy (PhD)
Microbiology and Immunology
Biofilms are communities of microorganisms that have intrinsically antimicrobial properties and are difficult to treat in the clinical setting. The marine bacterium Vibrio fischeri efficiently colonizes its symbiotic squid host, Euprymna scolopes, by producing a transient biofilm dependent on the symbiosis polysaccharide (SYP), making it the perfect model system to study biofilm dynamics. In vitro, however, wild-type (WT) strain ES114 fails to form SYP – dependent biofilms. Instead, genetically engineered strains, such as those lacking the negative regulator BinK, have been developed to study SYP biofilms. Historically, V. fischeri has been grown using LBS, a complex medium containing tryptone and yeast extract; supplementation with calcium is required to induce biofilm formation by a binK mutant. Here, I discovered that yeast extract inhibits biofilm formation, which allowed us to uncover signals and underlying mechanisms that control V. fischeri biofilm formation.In contrast to its inability to form a biofilm on unsupplemented LBS, a binK mutant formed cohesive, SYP – dependent biofilms when grown in tTBS without supplementation. Further, WT formed SYP- dependent biofilms when both calcium and the vitamin para-aminobenzoic acid (pABA) were present; neither molecule alone was sufficient, indicating that this phenotype relies on coordinating two cues. Cells grown in tTBS with pABA/calcium were enriched in transcripts for biofilm-related genes and predicted diguanylate cyclases, which produce the second messenger cyclic – di – GMP ( c-di-GMP). They also exhibited elevated levels of c-di-GMP, which were required for biofilm formation as phosphodiesterase overproduction abrogated biofilm formation. I also determined that these SYP – dependent biofilms were reliant on the positive syp regulator RscS, as the loss of this sensor kinase abrogated biofilm formation and syp transcription. This result was significant because the loss of RscS exerts little to no effect on biofilm formation under other genetic and media conditions. Deletion or mutations in the sensory domain(s) of RscS also resulted in a loss of biofilm formation, suggesting that RscS might be the primary sensory regulator. Overexpression of rscS on a multi-copy plasmid, but not the corresponding vector control, permitted biofilm formation on media supplemented with pABA alone, supporting the hypothesis that RscS may be responsible for recognizing the pABA signal. Another key sensor kinase in the syp regulatory network is SypF, which acts as the central regulator funneling phosphoryl groups from RscS to phosphorylate the response regulator; SypG. Expression of an RscS-SypF chimera that contains the N-terminal domains of RscS fused to the C-terminal HPT domain of the required sensor kinase SypF was sufficient to promote biofilm formation on media supplemented with only pABA as long as a full-length copy of rscS was also present. Lastly, pABA induced biofilm formation when I introduced rscS into a heterologous system that does not respond positively to pABA. These data suggest that RscS might be responsible for recognizing pABA to induce biofilm formation. This work thus provides insight into signals and regulators that promote biofilm formation by V. fischeri and a role for RscS in recognizing the environment to induce biofilm formation.
Dial, Courtney Noelle, "Identification of Conditions That Promote Biofilm Formation by Vibrio Fischeri and Elucidation of The Underlying Signal Transduction Pathway" (2023). Dissertations. 4014.
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Copyright © 2023 Courtney Noelle Dial