Post-initiation mechanisms, like those regulating the ethanolamine utilization (eut) genes in E. faecalis, are incompletely understood representing a critical gap in knowledge. The long-term goal of this research is to determine how ethanolamine (EA) utilization is regulated in E. faecalis. The objective of this application is to elucidate the post-initiation regulatory mechanisms that control gene expression. The central hypothesis is that the AmiR and NasR Transcriptional Antiterminator Regulators' (ANTARs) RNA substrates are the central regulatory feature of the system and control gene expression by three interrelated mechanisms. The central hypothesis will be tested in three aims.
Aim 1 will elucidate the molecular details of how EutV, the ANTAR in the eut system, interacts with its RNA substrates to control gene expression. There is evidence that EutV binds a dual hairpin RNA structure with specific features. To further understand the structure of this complex, biophysical approaches will be employed, including resolution of three-dimensional structures of the protein-RNA complex by X-ray crystallography.
In Aim 2, the mechanism by which the AdoCbl riboswitch regulates gene expression will be uncovered. The novel, working hypothesis is that a dual hairpin substrate just downstream of the riboswitch binds and sequesters active EutV, preventing induction by EA alone. AdoCbl binding to the riboswitch causes a conformational change that prevents EutV sequestration, allowing for induction when both EA and AdoCbl are present. The model will be assessed by quantifying in vivo levels of protein and RNA, measuring binding constants, and by mutational analysis of the sRNA. Finally, in silico analysis will investigate how broadly this mechanism applies to microbial systems.
Aim 3 will identify how eut gene expression is turned off. Our working hypothesis is that bacterial microcompartment (BMC) formation sequesters one or more of the crucial ingredients for gene expression - EA, AdoCbl and/or EutV/EutW. By characterizing the dynamics of gene expression and BMC formation with fluorescent markers and transmission electron microscopy (TEM), the hypothesis will be tested. The research in this proposal will further the understanding of how the eut genes are regulated, contributing knowledge to the field of prokaryotic gene regulation and to the identification of potential antimicrobial targets. Specifically, the significance of this contribution will be the uncovering of novel mechanisms by which ANTARs, riboswitches, and BMCs control gene expression. The proposed research is innovative because these new mechanisms will challenge the status quo and expand the field's thinking on how RNA structural features and BMCs can operate.

Public Health Relevance

The research proposed in this application will lead to greater understanding of how regulatory RNAs in a human bacterial pathogen control gene expression. Such knowledge is relevant to public health because it will contribute to efforts focused on exploiting these regulatory mechanisms as potential antimicrobial targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI110432-04
Application #
9394761
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Huntley, Clayton C
Project Start
2015-01-15
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77030
Graham, Carrie E; Cruz, Melissa R; Garsin, Danielle A et al. (2017) Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc Natl Acad Sci U S A 114:4507-4512
De Lay, Nicholas R; Garsin, Danielle A (2016) The unmasking of 'junk' RNA reveals novel sRNAs: from processed RNA fragments to marooned riboswitches. Curr Opin Microbiol 30:16-21
Mayo, Sara A; Song, Ye K; Cruz, Melissa R et al. (2016) Indomethacin injury to the rat small intestine is dependent upon biliary secretion and is associated with overgrowth of enterococci. Physiol Rep 4:
van der Hoeven, Ransome; Cruz, Melissa R; Chávez, Violeta et al. (2015) Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans. PLoS One 10:e0124091
DebRoy, Sruti; Gebbie, Margo; Ramesh, Arati et al. (2014) Riboswitches. A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator. Science 345:937-40