Two-component signaling (TCS) systems are a predominant form of biological sensing of environmental change, with over 45,000 TCS systems found in eubacteria and other organisms. TCS pathways regulate infectivity, virulence and resistance in a wide range of pathogenic bacteria and fungi - but are not found in mammals, making them prime targets for antimicrobial development. As suggested by the name, TCS systems are minimally composed of two types of proteins: histidine kinases (HKs) which convert changes in environmental stimuli into altered levels of catalytic activity, and response regulators (RRs) that are phosphorylated by HKs and subsequently activated to execute biological responses. While several TCS systems have served as paradigms of signal transduction studies, the mechanisms of two fundamental steps are still unclear: 1). how signal detection is relayed through sensory and catalytic domains in HKs and 2). how phosphorylation-induced structural and dynamic changes are propagated between RR receiver and output domains. Here we address these gaps with an integrated and comprehensive research plan to examine such allosteric control in TCS systems from a conserved stress response pathway. Based on physiological importance and experimental tractability, we have focused on light-regulated TCS proteins from Erythrobacter litoralis, concentrating on two HKs (EL346, EL368) and their RR substrates (R1, R5). Taking advantage of light as a known and easily manipulated stimulus, coupled with the excellent suitability of these HK and RR proteins for structural and functional studies, we have a unique opportunity to examine three goals: 1). Testing a model of HK regulation by examining how photosensitive sensor domains control histidine kinase activity;2). Establishing the effects of activation on response regulators immediately downstream from the kinases;3). Determining the in vivo effects of structure-guided mutations in these HK and RR proteins. We will use a combination of biophysical methods - including solution NMR spectroscopy and X-ray crystallography - to characterize the structure, dynamics and interactions of these proteins and complexes. Insights from these structural models will be tested with a mix of in vitro and cell-based functional assays. Outcomes from this research will include information about fundamental regulatory processes employed by these proteins, giving insights that will be broadly applicable for signal transduction studies.

Public Health Relevance

Understanding how bacteria use two-component signaling (TCS) to sense and react to changes in the environment around them is a fundamental problem in the area of biological signal transduction. Among other responses, such TCS systems control the infectivity and virulence of pathogenic bacteria (including E. coli, S. aureus and M. tuberculosis) and fungi, but are not used in humans. As such, our proposed mechanistic studies of the control of TCS systems will provide the foundation for the development of novel targeted antimicrobial strategies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Flicker, Paula F
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University of Texas Sw Medical Center Dallas
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