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.
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 ... read moreinfant 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.
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Andrew Camilli.
Committee: Michael Malamy, Abraham Sonenshein, Ralph Isberg, and Paula Watnick.
Keyword: Microbiology.read less