All bacteria respond to changes in environmental conditions using two-component sensory systems. These systems consist of a sensory histidine kinase that phosphorylates the receiver domain of a response regulator, inducing a downstream cellular response. One such signaling module, the FixLJ system, controls transcription of a set of genes in response to changes in oxygen levels in rhizobiales and caulobacterales. Interestingly, this system also controls expression of the receiver domain derived inhibitor FixT, which directly affects FixL kinase activity. In Caulobacter crescentus, preliminary data suggests that FixT activity is further regulated by the association of an iron-sulfur cluster. Thus, C. crescentus FixT represents a novel receiver domain protein that integrates environmental information in dampening the FixLJ signaling cascade. The proposed research program describes a multidisciplinary approach to characterize FixT biochemical function, structural features, and sensory regulation.
The first aim of this research strategy will employ solution assays to examine the kinetic and equilibrium parameters governing FixT inhibition of FixL kinase activity. To complement this biochemical approach, the second aim will use biophysical techniques to obtain atomic level detail of FixT structure, alone and in complex with FixL. Finally, the third aim will take a holistic approach in identifying and characterizing in vivo conditions that control FixT function, and thereby modulate FixLJ signaling. FixT presents an important model for understanding how diverse bacteria carefully tune their response to environmental oxygen, and more generally, how two-component systems may coopt isolated receiver domains to integrate environmental information. Moreover, FixT presents a rare example of a post-translationally regulated histidine- kinase inhibitor, and thus, principles uncovered in this work will aid in the design of antimicrobial treatments that inhibit two-component histidine kinases. Importantly, these experiments will be performed under the guidance of an expert microbiologist and biochemist, and will thus provide excellent training for a future faculty position.
Bacteria employ two-component signaling systems to sense and respond to dynamic environmental conditions, such as fluctuating oxygen levels in plant and animal host tissues. The goal of this work is to characterize the activity and structure of a novel, sensory inhibitor that modulates an oxygen-responsive two-component system in a-proteobacteria. In the future, this research will aid in the development of antimicrobial therapies that modulate signaling systems in pathogenic bacteria.
