Quantitative Kinetic Analyses of Shutting Off a Two-Component System

MBio
Rong Gao, Ann M Stock

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 archetypeEscherichia coliPhoR-PhoB TCS pathway using both transcription reporter assays andin vivophosphorylation 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 assessin vivokinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an ...Continue Reading

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Citations

Jan 10, 2018·Frontiers in Microbiology·M Graciela PucciarelliFrancisco García-Del Portillo
May 19, 2019·Annual Review of Microbiology·Rong GaoAnn M Stock
Oct 17, 2019·Molecular Microbiology·Heidi A CrosbyAlexander R Horswill
Jul 12, 2017·Journal of Bacteriology·Robert B Bourret
Jun 22, 2019·Annual Review of Microbiology·Alejandro Buschiazzo, Felipe Trajtenberg
Oct 21, 2020·Journal of Bacteriology·Katherine J Graham, Lori L Burrows
Sep 15, 2019·EcoSal Plus·Stewart G Gardner, William R McCleary

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Methods Mentioned

BETA
light scattering
phosphotransferase
PCR

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