Quorum Sensing Adaptation in the Bacterial Symbiont Vibrio fischeri
Restricted (Penn State Only)
- Author:
- Provencher, Ed
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 21, 2024
- Committee Members:
- Santhosh Girirajan, Program Head/Chair
Jordan Bisanz, Major Field Member
Istvan Albert, Major Field Member
Timothy Miyashiro, Dissertation Advisor
Timothy Meredith, Chair of Committee
Claude Depamphilis, Outside Unit & Field Member - Keywords:
- quorum sensing
experimental evolution
symbiosis
lipopolysaccharide
bioluminescence production
evolution - Abstract:
- Quorum sensing describes the mechanism by which populations of bacteria exhibit coordinated behaviors. These bacteria synthesize small molecules known as autoinducers. Detection of autoinducer by the cell induces the expression of traits beneficial to the whole population. The concentration of autoinducer is typically correlated with cell density, so detection of autoinducer often serves as an indirect measurement of population size. Traits regulated by quorum sensing are often energetically expensive to express, making their careful regulation imperative to avoid unnecessary resource consumption, thereby increasing the fitness of the cell. Quorum sensing systems are widespread among bacteria and are often associated with bacterial symbionts, which form beneficial, long-term associations known as symbioses with eukaryotic hosts. These bacterial symbionts possess traits that increase the fitness of the host, such as digestion, synthesis of essential nutrients, and protection from invasion by pathogens. In exchange, the host provides a nutrient-rich niche within which the symbiont thrives. Failure of the symbiont to establish symbiosis can have severe negative consequences for the host since the expression of symbiotic traits often facilitates normal host physiology. It is therefore important to increase understanding of the role of quorum sensing in both establishing and maintaining these beneficial associations. The insight gained from studies of quorum sensing in symbiosis has the potential to facilitate the development of new therapeutics that improve host health by precisely controlling interactions with beneficial quorum sensing bacteria. The marine bacterium Vibrio fischeri represents an excellent model organism for the study of quorum sensing in the context of a beneficial symbiosis. V. fischeri utilizes quorum sensing to regulate bioluminescence production during symbiosis with its host, the Hawaiian Bobtail squid Euprymna scolopes. The squid possesses a specialized symbiotic organ within its mantle known as the light organ, where it houses a pure culture of V. fischeri bacteria. The bioluminescence produced by V. fischeri is leveraged by the squid in an anti-predation behavior known as counterillumination, whereby the downward-dwelling light eliminates the squid’s shadow as it forages in the water column at night, allowing it to avoid detection by benthic predators and prey. The squid must acquire its bacterial partner from the environment, so V. fischeri must be capable of surviving independent of the host. Because bioluminescence production is only necessary during symbiosis when cell density is high, quorum sensing regulation prevents unnecessary expression of bioluminescence while outside the host, where autoinducer concentration is unlikely to reach sufficient concentration to activate bioluminescence production. This maximizes the fitness of the cell by preventing excess resource consumption outside of symbiosis. While much is known about the molecular mechanisms of bioluminescence production, the evolutionary trajectories of quorum sensing and the selective pressures driving quorum sensing adaptation over time are less clear. The work presented in this dissertation addresses this knowledge gap by increasing understanding of how quorum sensing regulation in populations of V. fischeri adapts over time. To investigate the potential evolutionary trajectories of V. fischeri forced to produce bioluminescence independent of symbiosis, populations of V. fischeri were maintained in rich medium supplemented with the autoinducer N-3-oxohexanoyl-L-homserine lactone (3OC6), which activates bioluminescence production following detection by the response regulator LuxR. The results of this short-term evolution experiment indicate that populations of V. fischeri maintained in the presence of exogenous 3OC6 adapt rapidly to this environment, leading to the attenuation of bioluminescence production and, by extension, the loss of symbiotic competence. These adaptations conferred increased fitness relative to the ancestral strain, demonstrating that bioluminescence production is energetically costly and that exposure to exogenous 3OC6 exerts selective pressure that drives quorum sensing adaptation. Underlying the observed attenuation of bioluminescence production are mutations in the response regulator and transcription factor, LuxR. These mutations attenuate the ability of LuxR to interact with 3OC6 and activate transcription of the bioluminescence genes. As part of investigations into the functional consequences of these mutations, it was revealed that insertion of the luxR gene elsewhere in the genome of a luxR deletion mutant was not sufficient to restore normal bioluminescence production, which suggests that the molecular mechanisms of LuxR-mediated regulation of bioluminescence production must be revisited. These findings provide insight into the sources of immense strain diversity among the quorum sensing circuits of distinct V. fischeri strains while also increasing our understanding of the importance of quorum sensing regulation in maximizing symbiont fitness while not in symbiosis. One strain isolated from a lineage maintained in the presence of exogenous 3OC6 also exhibited increased biofilm formation, which was determined to be due to a defect in lipopolysaccharide (LPS) biosynthesis. The strain was found to carry a mutation in the glycosyltransferase VF_0133, which is shown through the work presented here to be involved in the assembly of the inner core of V. fischeri LPS. LPS is especially important for V. fischeri to establish symbiosis. V. fischeri possesses sheathed flagella, so disruptions to LPS structure can impact cellular motility, which is necessary for V. fischeri to establish symbiosis with the squid. Additionally, LPS serves as a signaling molecule for the host that triggers morphological development of the light organ during and following colonization by V. fischeri. Disruptions to this structure can therefore negatively impact host development and health by failing to induce important steps in light organ maturation and development. These results highlight the important role of LPS in both host and bacterial physiology. Together, the work presented in this dissertation serves to shed light on the evolutionary plasticity of V. fischeri quorum sensing, as well as to highlight the importance of increasing understanding of the current model of quorum sensing regulation of bioluminescence production in V. fischeri. This work also identifies a previously-unknown component of the LPS biosynthesis pathway in V. fischeri, which has the potential to facilitate future studies of the role of LPS in communication with the host during colonization. The findings presented here increase understanding of how 3OC6 exposure can dictate the evolutionary trajectory of V. fischeri and the effects of adaptation on the ability of a bacterial symbiont to establish symbiosis with its eukaryotic host.