Two component regulatory systems have emerged as a paradigm for adaptive responses. The simplest systems consist of a sensor and a response regulator. The two-component system in E. coli that regulates the porin genes responds to changes in osmolarity of the growth medium. EnvZ, the presumed osmosensor is phosphorylated by intracellular ATP and then phosphorylates OmpR. At low osmolarity, the major porin in the outer membrane is OmpF and at higher osmolarity, ompF transcription is repressed and ompC is activated. Two-component systems are intimately involved in the coordinate expression of virulence factors in many different pathogens and OmpR is an important global regulatory protein. In Salmonella, OmpR lies at the first step of a regulatory cascade that turns on a downstream two-component regulatory system and activates expression of a type three secretion system required for systemic infection. In the present application, the hypothesis to be tested is that EnvZ controls the concentration of OmpR approximately P by adjusting its phosphatase activity in response to the osmotic signal. Prior to the previous funding period, we discovered that DNA binding stimulates OmpR phosphorylation. This observation may have important mechanistic implications for signaling and it suggests that response regulators that function as transcription factors may be phosphorylated while bound to the DNA. In the first aim, we will attempt to isolate and characterize such an EnvZ/OmpR/DNA complex. An EnvZ-GFP fusion will be employed to examine signaling and transcription in intact cells and in spheroplasts to determine the role (if any) of the outer membrane. An OmpR approximately P dephosphorylation assay has been developed and will be used to test the role of EnvZ in OmpR-P turnover. In the previous funding period, we discovered that OmpR is capable of binding to DNA in a head-to-head orientation rather than the previously proposed head-to-tail mode. Our new model predicts that the recognition helix is actually helix 2 rather than helix 3. We will determine the role of helices 2 and 3 in DNA recognition and test the hypothesis that OmpR can bind to DNA in more than one orientation.
In aim three, we propose to solve the full-length structure of OmpR, the structures of the isolated N- and C-terminal domains, the phosphorylated N-terminal domain and the C-terminal domain bound to DNA by NMR. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058746-08
Application #
7490379
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Chin, Jean
Project Start
2000-04-01
Project End
2010-08-31
Budget Start
2008-09-01
Budget End
2009-08-31
Support Year
8
Fiscal Year
2008
Total Cost
$282,949
Indirect Cost
Name
University of Illinois at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Wang, Loo Chien; Morgan, Leslie K; Godakumbura, Pahan et al. (2012) The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm. EMBO J 31:2648-59
Naveed, Hammad; Jimenez-Morales, David; Tian, Jun et al. (2012) Engineered oligomerization state of OmpF protein through computational design decouples oligomer dissociation from unfolding. J Mol Biol 419:89-101
Walthers, Don; Li, You; Liu, Yingjie et al. (2011) Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription. J Biol Chem 286:1895-902
Liu, Yingjie; Chen, Hu; Kenney, Linda J et al. (2010) A divalent switch drives H-NS/DNA-binding conformations between stiffening and bridging modes. Genes Dev 24:339-44
Kenney, Linda J (2010) How important is the phosphatase activity of sensor kinases? Curr Opin Microbiol 13:168-76
Gerken, Henri; Charlson, Emily S; Cicirelli, Elisha M et al. (2009) MzrA: a novel modulator of the EnvZ/OmpR two-component regulon. Mol Microbiol 72:1408-22
Lin, Wan-Jung; Walthers, Don; Connelly, James E et al. (2009) Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR. Mol Microbiol 71:1477-95
Osborne, Suzanne E; Walthers, Don; Tomljenovic, Ana M et al. (2009) Pathogenic adaptation of intracellular bacteria by rewiring a cis-regulatory input function. Proc Natl Acad Sci U S A 106:3982-7
Carroll, Ronan K; Liao, Xiubei; Morgan, Leslie K et al. (2009) Structural and functional analysis of the C-terminal DNA binding domain of the Salmonella typhimurium SPI-2 response regulator SsrB. J Biol Chem 284:12008-19
Rhee, Jee Eun; Sheng, Wanyun; Morgan, Leslie K et al. (2008) Amino acids important for DNA recognition by the response regulator OmpR. J Biol Chem 283:8664-77

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