Research

Functional characterization of two-component signaling systems (and signals)

Whether residing in a freshwater stream or an animal host, the survival of bacterial cells depends on reliable detection and adaptation to changes in their physical and chemical microenvironment. A primary means of such regulation involves the coordinate action of sensor histidine kinases and their cognate response regulators. Together, these proteins form two-component signal transduction systems (TCS), which typically regulate gene expression in response to the presence (or absence) of some signal. While thousands of TCS proteins have been annotated in sequenced bacterial genomes, very little is known about the nature of signals that regulate TCS function. To identify such signals, we have utililized a structure-based bioinformatic method to predict cofactor binding histidine kinases. Working in Caulobacter crescentus, this method provided an entry point to signal identification and has enabled, for the first time, a functional definition of TCS signals in this important model bacterium. We are currently using a range of biochemical and genetic approaches to identify additional functional TCS signals in Caulobacter and in other bacterial species.

Defining the topology and input/output behavior of two-component signaling networks

Once specific functional signals have been identified, we utilize a combination of genetic and genomic tools to define the molecular topology of two-component regulated genetic networks. In the past we have used this approach to define the topology of the oxygen-sensing FixL/FixJ two-component system in C. crescentus. Mathematical modeling is used to simulate the input/output behavior of our experimental network topologies. Such computational studies are aimed at comparing the predicted and observed I/O behavior of a two-component network and assessing the adequacy of our experimental topological models.

Exploring the structural and dynamical basis of two-component signal detection and transduction

Genetic and genomic tools can be used to define the function and topology of two-component signal transduction networks. However, these techniques cannot address the molecular/structural mechanism of signal detection and transduction. We have identified a soluble photosensory histidine kinase in C. crescentus, named LovK, which provides an excellent model to probe how detection of an environmental signal (light) translates into a biomolecular signal. LovK binds a flavin cofactor, and exhibits a reversible photocycle dependent on the absorption of blue photons. In addition to ongoing genetic and physiological studies in which we are working to characterize the function of LovK, we are using solution spectroscopy and X-ray crystallography to define how photon absorption affects the structure, dynamics, and biochemical activity of LovK.