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ItemEmergent collective phenomena in V. Cholerae biofilms, mixed human-autonomous fleets, and nascently multicellular bodies(Georgia Institute of Technology, 2019-11-07) Yanni, DavidHere I focus on the physics of the evolution of multicellularity and division of labor, the cyberphysical risks of hacked internet connected vehicles, and diffusive motion that arises from cell reproduction and lysis (cell death) in biofilms.
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ItemGenetics of type VI secretion and natural transformation in Vibrio cholerae(Georgia Institute of Technology, 2017-01-13) Watve, Samit S.The facultative waterborne pathogen Vibrio cholerae transitions between its human host and the environment where it colonizes chitinous surfaces in aquatic settings. Growth on chitin coordinates the induction of sets of genes for 1) chitin utilization; 2) a type VI secretion system that allows contact-dependent killing of neighboring bacteria; and 3) DNA uptake by natural transformation, which is a mechanism for horizontal gene transfer. This thesis describes the regulatory network controlling these behaviors in V. cholerae and the consequences of their coordinate regulation. Results from high-throughput RNA sequencing (RNA-seq) show that transcription factor CytR is one of four positive regulators comprising the chitin-induced regulatory network. A combination of genetic and phenotypic assays reveal the four regulators TfoX, HapR, QstR and CytR control each behavior in a distinct manner in a commonly used clinical reference strain of V. cholerae. Whole genome sequencing and bioinformatics analyses of a set of strains isolated from diverse sources reveal novel type VI secretion system components present in environmental, but not clinical isolates. Finally, I show that chitin-induced natural transformation can facilitate horizontal gene transfer of distinct type VI secretion system genes between strains. Horizontally acquired effector-immunity proteins are functional in the new genetic background and can be employed in antibacterial antagonism against parental cells and simultaneously protect against attacks by the donor cells. This thesis sheds light on diverse behavioral adaptations that allow this important human pathogen to spread and persist in the environment.
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ItemNatural competence and Type VI secretion in Vibrio cholerae(Georgia Institute of Technology, 2016-07-22) Bernardy, ErynThe waterborne bacterium Vibrio cholerae, responsible for epidemics of cholera diarrhea, associates with the human gut and with chitinous surfaces in aquatic reservoirs. Prior studies of two clinical V. cholerae isolates revealed that natural competence for genetic transformation, a horizontal gene transfer mechanism, requires the chitin-induced TfoX regulator, and quorum sensing transcription factor HapR made at high cell density. To further understand this regulation, I helped identify, in a genetic screen, CytR, a new positive regulator required for competence gene expression and natural transformation. Recently, this complex regulatory network in V. cholerae was shown to also control a type VI secretion system (T6SS) that allows contact-dependent killing of other bacteria by injecting toxic proteins. I characterized a diverse set of sequenced V. cholerae isolates, revealing that transformation was rare in all isolates, while constitutive type VI killing was common among environmental but not clinical isolates. These latter results were consistent with a “pathoadaptive” model that tight regulation is beneficial in a host, while constitutive killing is advantageous in the environment. We hypothesized that two sequenced V. cholerae isolates with distinct T6SSs could generate structured populations from initially well-mixed conditions by killing competitors, but not kin. Indeed, when both isolates were rendered T6SS-, a well-mixed population was observed via fluorescence microscopy. In contrast, mutual killing generated clonal patches with each isolate segregating into distinct groups. Structural dynamics were recapitulated with three mathematical models and a cooperation model developed supports that this assortment promotes cooperation among kin. My work in V. cholerae has helped elucidate a complex regulatory network controlling multiple important phenotypes, diversity of these phenotypes among species members, and ecological consequences of antagonistic microbial interactions in the environment.
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ItemNon-coding small RNAs regulate multiple mRNA targets to control the Vibrio cholerae quorum sensing response(Georgia Institute of Technology, 2013-04-09) Zhao, XiaonanThe waterborne bacterial pathogen Vibrio cholerae uses a process of cell-to-cell communication called quorum sensing (QS) to coordinate transcription of four sRNAs (Qrr1-4; quorum regulatory RNAs) in response to changes in extracellular QS signals that accumulate with cell density. The Qrr sRNAs are predicted to negatively control translation of several mRNAs, including hapR, which encodes the master QS transcription factor that controls genes for virulence factors, biofilm formation, protease production, and DNA uptake. The Qrr sRNAs are also predicted to positively control vca0939, which encodes a GGDEF family protein that promote biofilm formation by elevating intracellular levels of the second messenger molecule c-di-GMP. Using complementary in vivo, in vitro, and bioinformatic approaches, I showed that Qrr sRNAs base-pair with and repress translation of the mRNA encoding HapR. A single nucleotide mutation in Qrr RNA abolishes hapR pairing and thus prevents cholera toxin production and biofilm formation that are important in disease, and also alters expression of competence genes required for uptake of DNA in marine settings. I also demonstrated that base-pairing of the Qrr sRNAs with vca0939 disrupts an inhibitory structure in the 5' UTR of the mRNA. Qrr-activated translation of vca0939 was sufficient to promote synthesis of c-di-GMP and early biofilm formation in a HapR-independent manner. Thus, these studies define the non-coding Qrr sRNAs as a critical component allowing V. cholerae to sense and respond to environmental cues to regulate important developmental processes such as biofilm formation.
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ItemThe regulatory network controlling natural competence for DNA uptake in Vibrio cholerae(Georgia Institute of Technology, 2013-04-02) Antonova, Elena S.The bacterial pathogen Vibrio cholerae is responsible for ongoing cholera outbreaks in Haiti and elsewhere. Association of V. cholerae with the human host is responsible for fatal disease, but the bacteria also reside as natural inhabitants of aquatic environments, commonly attaching as biofilms to chitinous surfaces of copepods and crabs. Prior studies in V. cholerae demonstrated that competence for genetic transformation, a mechanism of horizontal gene transfer (HGT), requires the TfoX regulator protein that is triggered by chitin, and the HapR transcription factor that is made in response to quorum sensing (QS) signals produced by V. cholerae and Vibrios. To define regulatory components connecting extracellular signals to natural competence, I first demonstrated that QS molecules produced by Vibrios within multi-species chitinous biofilms are required for DNA uptake by V. cholerae, confirming the critical role of QS signals in HGT. Second, I identified by transposon-mutagenesis a new positive regulator of competence, CytR (cytidine repressor), only studied prior in E. coli as a regulator of nucleoside scavenging. Specific mutations in V. cholerae CytR impaired expression of competence genes and halted DNA uptake; and the addition of exogenous cytidine had similar affects as predicted in E. coli. V. cholerae and other competent Vibrios encode TfoX, HapR, and CytR, although none of these regulators directly controls genes coding for the DNA uptake apparatus. Thus, these results have uncovered a regulatory network, likely used by many Vibrios, that contains additional factors linking several extracellular chemical molecules (cytidine, chitin, and QS signals) to DNA uptake. My study has begun to define a molecular mechanism by which both environment and genetics contribute to genome evolution for this important marine pathogen.