Abstract

Transcription activation is typically carried out by soluble proteins engaging basal elements of the transcription apparatus - including promoter DNA and RNA polymerase - in the bacterial cytoplasm. In the Gram negative pathogen Vibrio cholerae, virulence gene expression is under control of an unusual set of membrane proteins. We hypothesize that a membrane complex including two activators, ToxR and TcpP, binds to the toxT promoter, recruits RNA polymerase, and activates toxT gene expression leading to activation of ToxT-controlled virulence genes. The mechanism by which membrane proteins can access DNA in the cell and recruit RNA polymerase has not been uncovered with standard genetic and biochemical approaches. Single-molecule imaging methods with nanometer-scale resolution now make it possible to investigate this mechanism in living cells, and these techniques will be applied to the ToxR/TcpP system to test specific hypotheses. This exploratory proposal has the following two specific aims: 1. Construct Vibrio cholerae strains expressing photo-activatable fluorescent fusion proteins of ToxR and TcpP, and mark toxT promoter DNA in the V. cholerae genome using the lacO operator site for binding of a LacI-EYFP fusion protein. 2. Carry out single-molecule super-resolution imaging in live cells to test specific hypotheses about the mechanism and dynamics by which membrane activators bind to toxT promoter DNA for activation of virulence gene expression.

Public Health Relevance

Bacteria control gene expression tightly so as not to waste energy and thereby decrease their fitness. These mechanisms represent good targets for drug discovery to control bacterial pathogens. This work will examine single molecules in living cells using state of the art approaches, thereby uncovering knowledge that is the most suitable for eventual therapeutic discovery research.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI099497-02
Application #
8424204
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Hall, Robert H
Project Start
2012-02-01
Project End
2014-07-31
Budget Start
2013-02-01
Budget End
2014-07-31
Support Year
2
Fiscal Year
2013
Total Cost
$227,688
Indirect Cost
$77,688

  Institution

Name
University of Michigan Ann Arbor
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109