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ItemAn investigation into the multiple Type VI Secretion Systems of Enterobacter cloacae(Georgia Institute of Technology, 2021-05) Wilson, AshleyEnterobacter cloacae is a Gram-negative, opportunistic bacterial pathogen that is commonly acquired by patients in hospitals. The Type VI Secretion System (T6SS) is a harpoon-like apparatus that injects toxins into the cell envelop of neighboring bacteria to defend or compete for resources. It’s commonly found in a range of bacteria including Vibrio cholerae, Pseudomonas aeruginosa, and Escherichia coli. Previous research in this lab has shown that E. cloacae kills in a contact dependent manner and bioinformatic analysis found three vask genes, which encode for a protein in the Type VI apparatus. We created three single mutant strains (Δvask1, Δvask2, Δvask3) as well a double mutant (Δvask1Δvask3). These strains were tested in competition assays with target WT E. coli, with the survival of the target being indicative of E. cloacae killing ability. We show here that Δvask2 had no change in killing ability, Δvask1 and Δvask3 had some reduction in killing ability, and Δvask1Δvask3 had a complete reduction in killing ability. This initial result suggests E. cloacae in vitro killing ability is dependent on two T6SSs.
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ItemGlucose protects E. coli from death by the Vibrio cholerae type VI secretion system(Georgia Institute of Technology, 2019-05) Nichols, Holly L.Vibrio cholerae, the causative agent of the intestinal disease cholera, interacts with other bacteria in dense multispecies communities within both host and environmental settings. Using the harpoon-like type VI secretion system (T6SS), V. cholerae delivers toxic effector proteins into neighboring cells, causing cell lysis and death. The T6SS is frequently studied in V. cholerae using a qstR* mutant which constitutively expresses the T6SS. A qstR* V. cholerae strain can effectively kill target species Escherichia coli, Aeromonas veronii., and T6SS-sensistive V. cholerae cells in a standardized lab killing assay, causing a drop in viable cell counts of five orders of magnitude. This study finds that addition of glucose to a standardized killing assay against qstR* V. cholerae restores E. coli survival by three to four orders of magnitude, though the same effect is not found for Aeromonas or T6SS-sensitive V. cholerae. A growth assay revealed that E. coli doubling time does not affect killing by V. cholerae. Additional evidence shows that E. coli does not produce a diffusible molecule that represses the T6SS of V. cholerae. Investigation by fluorescence microscopy revealed that E. coli cells when entirely surrounded by V. cholerae cells survive in the presence but not the absence of glucose, which suggests that glucose causes a relevant physiological change in individual E. coli cells. We propose that further study should focus on the E. coli capsule as a potential mechanism for surviving T6SS attack. This study makes an unprecedented case that attack via the T6SS can be thwarted by sugar metabolism in target cells.
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ItemDeveloping Tools to Monitor Vibrio cholerae Type VI Secretion During Range Expansion on Solid Surfaces(Georgia Institute of Technology, 2015-12) Ng, Siu LungThe waterborne bacterium Vibrio cholerae causes the fatal cholera diarrhea, and thrives in aquatic environments attached to chitinous surfaces with other bacteria. V. cholerae has been used to study natural competence, which promotes DNA uptake through horizontal gene transfer (HGT). A newly described Type VI Secretion System (T6SS) that V. cholerae employ to kill neighboring cells could increase the chance for a competent V. cholerae to take up and utilize the released DNA. The T6SS apparatus, which is similar to a bacteriophage spike, injects toxic effector proteins into prey cells causing lysis that can aid V. cholerae in acquiring DNA from its neighbors. Although previous studies have investigated the regulation and mechanism of T6SS in V. cholerae at a single cell level, the role of T6SS in the dynamics of mixed bacterial populations, such as those found in the environment, remains poorly understood. Fluorescence microscopy was used in this study to determine the effect of T6SS by growing V. cholerae predators and isogenic prey populations on agar surfaces as a model for competitors undergoing spatial expansion. These conditions may mimic conditions encountered on chitinous surfaces in marine settings. In this study, plasmids expressing green and red fluorescent proteins were constructed to visualize the predator and prey populations. Preliminary spatial expansion experiments were ultimately performed using different fluorescent protein alleles that were encoded on the chromosome of competing bacteria. Preliminary results suggest that the presence of a functional T6SS plays an important role in competition. The tools developed for this study are now being used to study interaction between diverse V. cholerae isolates, and as a platform for experimental evolution.
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ItemEffects of biofilm production on horizontal gene transfer to Vibrio cholerae(Georgia Institute of Technology, 2014-05-02) Wilson, SarahThe waterborne bacterial pathogen Vibrio cholerae utilizes a cell-cell communication system called quorum sensing to coordinate group behavior in both a human host and in aquatic environments. Virulence genes like the cholera toxin, biofilm genes for sticky secreted attachment factors, and competence genes for DNA uptake are all regulated by this population density-dependent system. In a human host, both virulence and biofilm genes are repressed at high cell densities that occur late in infection, presumably to promote transmission upon exhausting the host’s resources. However, in the natural environment, regulation is more complex. Namely, at high cell densities, repression of biofilm production is coordinated with activation of competence genes that can promote horizontal gene transfer (HGT). Based on this model, it was proposed that accumulation of biofilm material on bacterial cells could hinder the uptake of extracellular DNA in aquatic settings. In support of this hypothesis, significant decreases were detected in DNA uptake by V. cholerae strains engineered to overproduce biofilm. However, reductions in DNA uptake were also observed in strains that produced no biofilms. These results suggest that proper timing of biofilm formation plays an important role in the capacity of V. cholerae to engage in HGT, one mechanism thought to allow this pathogen to rapidly evolve in changing environments.
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ItemCharacterizing a novel direct target of the quorum-sensing controlled small RNAs in V. cholerae(Georgia Institute of Technology, 2013-12-13) Elsherbini, Joseph Ahmedn/a