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 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 is repressed and ompC is activated. A model arising from genetic studies predicts that phospho- OmpR (OmpR-P) binds with high affinity to activate ompF and with low affinity to repress ompF and activate ompC. Recent work by the PI indicates that this currently accepted hypothesis is not sufficient to account for porin gene regulation. This application contains three aims. The first is to use in vitro footprinting to examine the pattern of binding as a function of OmpR and OmpR-P concentration. If the binding is sufficiently different, it would lead to the rejection of the affinity hypothesis and alternatives would need to be considered.
The aim further examines binding at low and high osmolarity both in vitro and in vivo, to correlate occupancy with osmoregulated expression of ompF and ompC.
The second aim i s to determine whether the linker of OmpR is required for communicating between the amino-terminal phosphorylation domain and the carboxyl-terminal DNA-binding domain and whether it plays an active or passive role in this process. If the linker is required, studies to determine the length and amino acid requirements (if any) will be conducted. The use of site-specific spectroscopic probes will explore the conformational changes that occur during signaling.
The final aim i s to determine the contact sites and to understand the interactions between OmpR and the alpha subunit of RNA polymerase (RpoA) that are important for transcriptional activation. This application begins with DNA-binding of OmpR and OmpR-P to the regulatory regions of ompF and ompC. It then focuses on the response regulator OmpR and examines conformational changes that are important for signaling. It lastly considers the role of OmpR interactions with RNA polymerase in stimulating transcription. Information gained in studying the porin regulon is relevant to systems in animal cells in which transmembrane signaling-dependent changes in kinase activity result in a phosphorylation cascade and ultimately to changes in gene expression. It is also relevant to pathogenic systems that use two-component systems to modulate their virulence properties.
|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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