The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism

ABSTRACT Two-component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing region of CusS, a cytoplasmic phosphotransfer domain of the sensor phosphorylates the response regulator...

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Autores principales:	Lorena Novoa-Aponte, Cheng Xu, Fernando C. Soncini, José M. Argüello
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Pseudomonas aeruginosa copper homeostasis periplasm two-component regulatory systems Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ccbb9e02c5c14a1d97da7f3496cfffcb
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spelling	oai:doaj.org-article:ccbb9e02c5c14a1d97da7f3496cfffcb2021-11-15T15:31:13ZThe Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism10.1128/mSphere.01193-202379-5042https://doaj.org/article/ccbb9e02c5c14a1d97da7f3496cfffcb2020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.01193-20https://doaj.org/toc/2379-5042ABSTRACT Two-component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing region of CusS, a cytoplasmic phosphotransfer domain of the sensor phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two-component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains CopR in a nonphosphorylated state when the periplasmic Cu levels are below the activation threshold of CopS. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain exhibits maximal expression of the CopRS regulon, lower intracellular Cu+ levels, and increased Cu tolerance compared to wild-type cells. The invariant phosphoacceptor residue His235 of CopS was not required for the phosphatase activity itself but was necessary for its Cu dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly supports the different binding stoichiometries in the two systems. Interestingly, CopS binds Cu+/2+ with 3 × 10−14 M affinity, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm. IMPORTANCE Copper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper-sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two-component systems control the periplasmic response to metal overload. This paper shows that the sensor kinase of the copper-sensing two-component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of its cognate response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the system is activated by copper levels compatible with the absence of free copper in the cell periplasm. These observations emphasize the diversity of molecular mechanisms that have evolved in bacteria to manage the copper cellular distribution.Lorena Novoa-AponteCheng XuFernando C. SonciniJosé M. ArgüelloAmerican Society for MicrobiologyarticlePseudomonas aeruginosacopperhomeostasisperiplasmtwo-component regulatory systemsMicrobiologyQR1-502ENmSphere, Vol 5, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Pseudomonas aeruginosa copper homeostasis periplasm two-component regulatory systems Microbiology QR1-502
spellingShingle	Pseudomonas aeruginosa copper homeostasis periplasm two-component regulatory systems Microbiology QR1-502 Lorena Novoa-Aponte Cheng Xu Fernando C. Soncini José M. Argüello The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism
description	ABSTRACT Two-component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing region of CusS, a cytoplasmic phosphotransfer domain of the sensor phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two-component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains CopR in a nonphosphorylated state when the periplasmic Cu levels are below the activation threshold of CopS. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain exhibits maximal expression of the CopRS regulon, lower intracellular Cu+ levels, and increased Cu tolerance compared to wild-type cells. The invariant phosphoacceptor residue His235 of CopS was not required for the phosphatase activity itself but was necessary for its Cu dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly supports the different binding stoichiometries in the two systems. Interestingly, CopS binds Cu+/2+ with 3 × 10−14 M affinity, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm. IMPORTANCE Copper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper-sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two-component systems control the periplasmic response to metal overload. This paper shows that the sensor kinase of the copper-sensing two-component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of its cognate response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the system is activated by copper levels compatible with the absence of free copper in the cell periplasm. These observations emphasize the diversity of molecular mechanisms that have evolved in bacteria to manage the copper cellular distribution.
format	article
author	Lorena Novoa-Aponte Cheng Xu Fernando C. Soncini José M. Argüello
author_facet	Lorena Novoa-Aponte Cheng Xu Fernando C. Soncini José M. Argüello
author_sort	Lorena Novoa-Aponte
title	The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism
title_short	The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism
title_full	The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism
title_fullStr	The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism
title_full_unstemmed	The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism
title_sort	two-component system coprs maintains subfemtomolar levels of free copper in the periplasm of <named-content content-type="genus-species">pseudomonas aeruginosa</named-content> using a phosphatase-based mechanism
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/ccbb9e02c5c14a1d97da7f3496cfffcb
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The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using a Phosphatase-Based Mechanism

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