Quantitative Kinetic Analyses of Shutting Off a Two-Component System

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, phosphata...

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Autores principales:	Rong Gao, Ann M. Stock
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	PhoBR autoregulation computer modeling histidine kinase phosphatases systems biology Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/fdd73206e3674653b99c87c55f9184d1
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spelling	oai:doaj.org-article:fdd73206e3674653b99c87c55f9184d12021-11-15T15:51:30ZQuantitative Kinetic Analyses of Shutting Off a Two-Component System10.1128/mBio.00412-172150-7511https://doaj.org/article/fdd73206e3674653b99c87c55f9184d12017-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00412-17https://doaj.org/toc/2150-7511ABSTRACT 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 Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo 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 in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo, which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro. 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. IMPORTANCE 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.Rong GaoAnn M. StockAmerican Society for MicrobiologyarticlePhoBRautoregulationcomputer modelinghistidine kinasephosphatasessystems biologyMicrobiologyQR1-502ENmBio, Vol 8, Iss 3 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	PhoBR autoregulation computer modeling histidine kinase phosphatases systems biology Microbiology QR1-502
spellingShingle	PhoBR autoregulation computer modeling histidine kinase phosphatases systems biology Microbiology QR1-502 Rong Gao Ann M. Stock Quantitative Kinetic Analyses of Shutting Off a Two-Component System
description	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 Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo 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 in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo, which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro. 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. IMPORTANCE 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.
format	article
author	Rong Gao Ann M. Stock
author_facet	Rong Gao Ann M. Stock
author_sort	Rong Gao
title	Quantitative Kinetic Analyses of Shutting Off a Two-Component System
title_short	Quantitative Kinetic Analyses of Shutting Off a Two-Component System
title_full	Quantitative Kinetic Analyses of Shutting Off a Two-Component System
title_fullStr	Quantitative Kinetic Analyses of Shutting Off a Two-Component System
title_full_unstemmed	Quantitative Kinetic Analyses of Shutting Off a Two-Component System
title_sort	quantitative kinetic analyses of shutting off a two-component system
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/fdd73206e3674653b99c87c55f9184d1
work_keys_str_mv	AT ronggao quantitativekineticanalysesofshuttingoffatwocomponentsystem AT annmstock quantitativekineticanalysesofshuttingoffatwocomponentsystem
_version_	1718427376889626624

Quantitative Kinetic Analyses of Shutting Off a Two-Component System

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