# Two-Component System Design Principles

> **NIH NIH R35** · RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL · 2020 · $437,252

## Abstract

Bacteria play important roles in human health. Bacterial communities are important components of normal
physiology, as revealed by studies of human microbiota. In contrast, pathogenic bacteria cause morbidity and
mortality. Whether impacting health or disease, interactions of bacteria with hosts share many common
features. To survive and thrive, bacteria must monitor their intracellular and extracellular environments and
elicit appropriate adaptive responses to changing conditions. "Two-component system” (TCS) phosphotransfer
pathways involving a sensor histidine protein kinase and a phosphorylation-activated response regulator that
generates the output response comprise a versatile regulatory scheme that occurs in hundreds of thousands of
regulatory systems. While structure and function of core elements are conserved, TCSs display enormous
diversity. Data acquired from numerous studies of individual systems as well as global analyses have revealed
differences in the magnitudes of enzyme activities, affinities of macromolecular interactions, levels of signaling
proteins and system architecture, all of which presumably contribute to tuning response behavior to the needs
of individual systems. The overarching goal of this research is to understand design principles of TCSs to the
extent that system behavior can be predicted, or at least rationalized, with knowledge of system parameters.
Protein concentrations are known to be critical parameters that influence reaction kinetics and outcomes in
vitro, yet they are commonly overlooked in cellular studies. Histidine kinase and response regulator
concentrations and stoichiometry are known to differ greatly among TCSs, but the effects of these variations
on system behavior, other than robustness, are largely unstudied. This project will fill this gap by exploring how
histidine kinase and response regulator concentrations impact system design and behavior. Investigations will
be performed using a set 19 Escherichia coli TCSs. Approaches will utilize reporter gene assays and
measurement of intracellular phosphorylation of response regulators to quantitate response output, mass
spectrometry to quantitate levels of two-component proteins in cells under un-induced and activated
conditions, mathematical modeling with experimental data to determine kinetics parameters and predict system
behavior, and competition assays in continuous cultures to assess fitness. Studies will address four broad
questions. Does the size of a TCS regulon place a requirement on the level of response regulator in a TCS?
How does the stoichiometry of histidine kinases and response regulators impact response output in an
activated TCS? How are system parameters configured to accommodate differences in histidine kinase and
response regulator concentrations? Does non-specific phosphorylation of response regulators place a
requirement on the phosphatase activity of histidine kinases? These investigations will identify core design
principle...

## Key facts

- **NIH application ID:** 9922317
- **Project number:** 5R35GM131727-02
- **Recipient organization:** RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
- **Principal Investigator:** ANN M. STOCK
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $437,252
- **Award type:** 5
- **Project period:** 2019-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9922317

## Citation

> US National Institutes of Health, RePORTER application 9922317, Two-Component System Design Principles (5R35GM131727-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9922317. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*