Award Date
5-15-2026
Degree Type
Thesis
Degree Name
Master of Science (MS)
Department
Life Sciences
First Committee Member
Helen Wing
Second Committee Member
Boo Shan Tseng
Third Committee Member
Christy Strong
Fourth Committee Member
Ronald Gary
Number of Pages
141
Abstract
Bacterial gene regulation is a detailed, meticulous process that is largely shaped by nucleoid-associated proteins (NAPs) and the genetic context in which a gene lies. NAPs are small DNA-binding proteins that compact the genome, affecting DNA topology and transcriptional accessibility of genes. Genetic context refers to the position and orientation of genes and DNA binding sites on a genetic element, playing an often-underappreciated role in shaping how certain genes are expressed. The effect of NAPs and genetic context on gene regulation are evident in the bacterial pathogen, Shigella. Many virulence genes necessary for Shigella pathogenesis are encoded on a large 220 kb plasmid known as pINV. These genes are silenced by the histone-like nucleoid structuring protein (H-NS), a well-conserved NAP in Gram-negative bacteria. However, when Shigella is ingested, the bacterium experiences a shift to 37° C. This temperature triggers the thermosensitive virulence regulatory cascade and leads to the production of the transcriptional anti-silencer, VirB. VirB antagonizes H-NS silencing by binding to DNA and spreading along the double helix. VirB spread introduces a local loss of negative DNA supercoiling, which is sufficient to disrupt DNA engagement by H-NS and makes VirB-regulated promoters transcriptionally accessible. Curiously, the VirB binding site lies 1137 bp upstream from the transcription start site of icsP, a well-characterized VirB-regulated gene. This represents a case of distal transcription regulation, which is rare in bacteria. Investigating non-canonical mechanisms of gene regulation in Shigella is the overlying theme of my work. Here, I take a two-pronged approach to investigate non-canonical mechanisms of bacterial gene regulation. In the first approach, I investigate the capacity for VirB to silence genes that converge on a nearby VirB site. I show that a reporter plasmid featuring a selection marker oriented towards a VirB site cannot effectively be transformed into VirB-producing Shigella and that inhibiting VirB production resolves this issue. I also show that the growth of Shigella transformed with this plasmid is associated with mutation acquisition. In the second approach, I investigate if two NAPs play previously unrecognized roles in Shigella virulence gene regulation. Namely, I investigate the possibility that IHF directs the spread of VirB towards VirB-dependent promoters and show that repositioning an IHF binding site between the VirB site and icsP promoter (PicsP) does not affect PicsP activity. I also investigate if proposed interactions between H-NS and the bacterial messenger molecule, c-di-GMP, upregulate genes silenced by H NS. I show that overexpression of a diguanylate cyclase, an enzyme that produces c-di-GMP, does not affect the activity of PicsP, which is silenced by H-NS. These collective findings highlight the role of genetic context in gene regulation and add to our understanding of how NAPs convert cellular signals into transcriptomic changes.
Keywords
Genetic context; H-NS; IHF; Nucleoid-associated proteins; Shigella; VirB
Disciplines
Bacteriology | Life Sciences | Microbiology
File Format
File Size
2500 KB
Degree Grantor
University of Nevada, Las Vegas
Language
English
Repository Citation
Netherton, Landon, "Genetic Context and NAP Interactions: Non-Canonical Mechanisms of Gene Regulation" (2026). UNLV Theses, Dissertations, Professional Papers, and Capstones. 5594.
https://oasis.library.unlv.edu/thesesdissertations/5594
Rights
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