Award Date

5-15-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Life Sciences

First Committee Member

Helen J. Wing

Second Committee Member

Eduardo A. Robleto

Third Committee Member

Boo Shan Tseng

Fourth Committee Member

Erin R. Murphy

Fifth Committee Member

Ernesto Abel-Santos

Number of Pages

249

Abstract

Transcriptional silencing and anti-silencing affect many aspects of bacterial physiology, including virulence in bacterial pathogens. In Shigella species, a group of gram-negative pathogens that cause bacillary dysentery in humans, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences virulence genes found on the large virulence plasmid while VirB anti-silences these genes. However, the mechanistic details of their interplay are not fully understood. To elucidate their regulatory mechanisms, I use the icsP virulence locus, which shares a long intergenic region with the divergently transcribed ospZ gene (1535 bp from TSS to TSS). Prior to this work, two discrete H-NS binding regions had been identified, suggesting H-NS-mediated bridging of these two regions as the mechanism of silencing. However, I show that changes to the spacing and helical phasing designed to disrupt the potential bridging were tolerated, suggesting an alternate mechanism of silencing is at play. In addition to H-NS, two other H-NS homologs found in S. flexneri, StpA and Sfh, can also silence the icsP promoter. Interestingly, VirB counters transcriptional silencing mediated by these other H-NS homologs. The site required for VirB-dependent anti-silencing of the icsP promoter is located over 1 kb upstream of the TSS, and nearly 500 bp upstream of the ospZ promoter, but exactly how VirB accomplishes this long-range regulation is not known. I show that VirB docks to this recognition site in vitro and has a high specificity for this site in vivo. Using a combination of 1D and 2D chloroquine-based agarose gel electrophoresis, I demonstrate that, upon docking to its recognition site, VirB triggers a loss of negative supercoiling of our VirB-dependent PicsP-lacZ reporter; importantly, this phenomenon occurs with native VirB levels in S. flexneri. Because H-NS is sensitive to DNA topology at some promoters, it is tantalizing to envision that VirBmediated changes in supercoiling alleviate H-NS-mediated silencing of virulence genes in Shigella. Although anti-silencing proteins in other bacteria, including related pathogens, bear little sequence homology to VirB, the possibility that changes to DNA supercoiling mechanistically unite this group of proteins requires further consideration when studying transcriptional silencing and anti-silencing processes in bacteria.

Keywords

nucleoid structuring proteins; Shigella; supercoiling; transcriptional anti-silencing; transcriptional silencing; virulence

Disciplines

Microbiology | Molecular Biology

Language

English


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