Quantitative Kinetic Analyses of Shutting Off a Two-Component System.

Rong Gao, Ann M Stock
Author Information
  1. Rong Gao: Department of Biochemistry and Molecular Biology, Center for Advanced Biotechnology and Medicine, Rutgers University-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
  2. Ann M Stock: Department of Biochemistry and Molecular Biology, Center for Advanced Biotechnology and Medicine, Rutgers University-Robert Wood Johnson Medical School, Piscataway, New Jersey, USA stock@cabm.rutgers.edu. ORCID

Abstract

Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype PhoR-PhoB TCS pathway using both transcription reporter assays and phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity , which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression. Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.

Keywords

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Grants

  1. R01 GM047958/NIGMS NIH HHS

MeSH Term

Bacterial Proteins
Escherichia coli
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Histidine Kinase
Kinetics
Phosphates
Phosphorylation
Signal Transduction
Systems Biology

Chemicals

Bacterial Proteins
Escherichia coli Proteins
Phosphates
PhoB protein, Bacteria
PhoR protein, Bacteria
Histidine Kinase

Word Cloud

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