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

> **NIH NIH R15** · COLLEGE OF THE HOLY CROSS · 2022 · $155,832

## Abstract

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.

## Key facts

- **NIH application ID:** 11103796
- **Project number:** 7R15GM148908-02
- **Recipient organization:** COLLEGE OF THE HOLY CROSS
- **Principal Investigator:** Benjamin J Stein
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $155,832
- **Award type:** 7
- **Project period:** 2022-09-20 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11103796

## Citation

> US National Institutes of Health, RePORTER application 11103796, Characterization of non-canonical regulatory pathways in the Caulobacter NtrYX signaling system (7R15GM148908-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/11103796. Licensed CC0.

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