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Use of a novel high-throughput genetic selection to identify regulators of selected Vibrio cholerae late genes; characterization of PhoB as a regulator of xds.

EmilyKate. McDonough Sackler School of Graduate Biomedical Sciences (Tufts University). Molecular Microbiology. 2014

Dissertation Abstracts International 76-03B(E).

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題名:
Use of a novel high-throughput genetic selection to identify regulators of selected Vibrio cholerae late genes; characterization of PhoB as a regulator of xds.
著者: EmilyKate. McDonough
Sackler School of Graduate Biomedical Sciences (Tufts University). Molecular Microbiology.
主題: Microbiology
所屬期刊: Dissertation Abstracts International 76-03B(E).
描述: Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, is a natural member of temperate aquatic environments around the world. V. cholerae undergoes adaptive shifts in gene expression throughout the various stages of its life cycle. Our laboratory identified 57 V. cholerae genes that are expressed specifically at or near the end of the infection cycle in the infant mouse model of infection. Many of these 'late' genes appear to be involved in preparing the bacteria for the shift from the host small intestine to the aquatic environment. In contrast, these same genes are not required for V. cholerae colonization of the mouse small intestine. These data led us to hypothesize that V. cholerae evolved to preinduce such dissemination genes while still in the host intestinal tract in order to optimize their chances of successfully transitioning to the aquatic environment.
To gain support for this hypothesis and to extend our understanding of gene regulation at this critical transition, the goal of my thesis project was to determine what signals V. cholerae senses in the small intestine that alert the organism to the upcoming change in environment. The first half of my thesis describes the implementation and results of a high throughput genetic selection we developed to identify transcriptional regulators of late genes. The selection was designed such that both activators and repressors could be identified, as it is not known if these genes are induced or de-repressed late in infection. We settled on three late genes as query genes for the selection: cdpA (encoding a cytoplasmic cyclic-diguanylate phosphodiesterase), emrD (encoding a efflux pump inner membrane subunit), and xds (encoding a secreted DNA exonuclease). The selection results did not identify any regulators of cdpA. Conversely, pepA (encoding a leucyl amino-peptidase) was identified as a repressor of emrD, and phoB (encoding the phosphate starvation response regulator) was identified as an activator of xds. Additionally, I have provided evidence that while regulation of xds by PhoB is relevant to the host intestinal environment, expression of xds can occur early or late in infection depending on the physiological state of the inoculum used for infection.
In the second half of my thesis I have worked towards a deeper understanding of the biological significance of the regulation of xds by PhoB. As Xds is an exonuclease, we hypothesized that PhoB induces xds under phosphate limiting conditions as part of the upregulation of phosphate acquisition genes. Indeed, V. cholerae is known to be able to survive using DNA as a sole source of phosphate, and Xds has been described as important for this ability. In this work I describe the identification and preliminary characterization of two nucleotidases, UshA and CpdB, that contribute to the ability of V. cholerae to utilize nucleotides as a source of phosphate. I have shown that both of ushA and cpdB are induced by phosphate limiting conditions, further supporting the significance of nucleotides as sources of phosphate.
出版者: Thesis (Ph.D.)--Sackler School of Graduate Biomedical Sciences (Tufts University), 2014.
建立日期: 2014
格式: 301 p..
語言: 英文
識別號: ISBN9781321336542
資源來源: NUTN ALEPH

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Use of a novel high-throughput genetic selection to identify regulators of selected Vibrio cholerae late genes; characterization of PhoB as a regulator of xds.

EmilyKate. McDonough Sackler School of Graduate Biomedical Sciences (Tufts University). Molecular Microbiology. 2014

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