Characterization of non-canonical regulatory pathways in the Caulobacter NtrYX signaling system

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Project Summary/Abstract All bacteria must sense and respond to metabolic and environmental changes to thrive in diverse habitats. These adaptive responses are often achieved using two-component signaling systems. Two- component signaling systems are composed of two core elements: a sensory histidine kinase and response regulator. The histidine kinase detects a cue and phosphorylates the response regulator. In general, phosphorylation of response regulators leads to changes in gene expression. Although canonical two-component signaling systems are somewhat simple, it has become increasingly apparent that many systems are more complex sensory networks, incorporating atypical features and additional regulatory components. NtrY-NtrX (NtrYX) is an emerging example of a complex sensory system linked to diverse phenotypes in many ⍺- proteobacteria. Prior studies have investigated NtrYX within the framework of canonical two-component signaling. However, recent work reveals that, in the freshwater and soil bacterium Caulobacter crescentus, the NtrYX system is more elaborate, incorporating an unidentified source of phosphorylation and a novel NtrY regulator, NtrZ. In addition, genetic and transcriptional data suggest that phosphorylated and unphosphorylated NtrX have distinct and physiologically relevant activities. The proposed work will investigate integrated regulatory strategies in the C. crescentus NtrYX system at multiple levels. The first aim of this project will determine how NtrX phosphorylation affects its DNA-binding and regulatory activities. To better understand how this phosphorylation is controlled, the second aim will apply biochemical and structural approaches to probe the interaction between NtrY and the NtrZ regulator. Finally, the third aim will explore an additional pathway regulating phosphoryl-flow, employing candidate-driven and unbiased approaches to identify the in vivo source of NtrX phosphorylation. Together, these approaches will establish C. crescentus NtrYX as a powerful model for understanding how intersecting regulatory pathways can control two-component signaling. Our work will also shed light on conserved and important NtrYX systems in diverse ⍺-proteobacteria, including pathogenic and symbiotic organisms like Brucella abortus and Sinorhizobium meliloti. Moreover, this project will reveal novel and generalizable strategies for tuning signal transduction pathways in bacteria.