Symbiotic relationships involve a life-long interaction between host and bacteria, and there is much we do not understand about how these interactions are developed and maintained. During the horizontal recruitment of beneficial bacteria by hosts, a complex set of molecular signals and communication ensures specificity. On the bacterial side, these signals are used to govern the transition from the environment to a host-associated lifestyle. Two-component signaling (TCS) systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. The study of TCS networks in the establishment of beneficial host-microbe interactions will help us understand how bacteria integrate cues to coordinate complex and precise behavioral responses during the colonization process. To understand the signaling networks bacteria use during the colonization of a host, I utilized a natural reductionist model. The Hawaiian bobtail squid, <i>Euprymna scolopes</i>, forms an exclusive association with the marine bacterium, <i>Vibrio fischeri</i>. In this symbiosis, <i>V. fischeri</i> colonizes the nutrient-rich environment of the squid light organ and the squid manipulates the bioluminescence produced by the bacteria as a means of camouflage. Within the squid-Vibrio symbiosis, TCS phosphorelays are necessary for the induction of biofilm which allows bacteria to aggregate in the mucus field outside the light organ before entering. Aggregation is critical for subsequent colonization of the host. Previously, the TCS signaling protein, BinK, was identified as a strong negative regulator of <i>V. fischeri</i> biofilm regulation. However, we did not yet understand how BinK functioned and was regulated during the colonization process. Here, I explore the function of BinK and use strains that overexpress or are deleted for <i>binK</i> to further explore biofilm regulation on agar and within the process of host colonization. My work has demonstrated that BinK requires the predicted phosphorylation sites for TCS to inhibit biofilm. The positive regulator of biofilm formation, RscS, is necessary for host colonization in a wild-type (WT) background and I found that it is not necessary for colonization in a Δ<i>binK</i> background. I also demonstrated that <i>syp</i> transcription is regulated at later stages of colonization and that BinK has a role inhibiting <i>syp</i> transcription in the crypts. Then, I collaborated with Katherine Zink of Laura Sanchez’s lab to use the Δ<i>binK</i> mutant to study biofilms outside the host context, and we found that the strains that produce biofilms also produce various small molecules. One such molecule is cyclo(ᴅ-His-ʟ-Pro) which affects luminescence and is produced during the colonization process. Finally, forward genetic screens using <i>binK</i>+/- strains identified both positive and negative regulators of biofilm formation, some of which have been previously implicated as colonization or biofilm factors. Overall, this work has led to a greater understanding of how BinK regulates biofilm gene expression throughout the development of the symbiosis, provided insights into aggregate formation at the host interface, and explored how biofilm formation may coordinate with other bacterial behaviors throughout the establishment of a host-microbe symbiosis.

Creator

• Ludvik, Denise Alberta

DOI

• https://doi.org/10.21985/n2-gx3z-d451

Subject

• Microbiology
• Molecular biology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:15608
• etdadmin_upload_825957

Keyword

• Vibrio fischeri
• symbiosis
• biofilm
• two-component signaling systems

Date created

• 2021-01-01

Resource type

• Dissertation

Rights statement

• In Copyright