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Real-time detection of response regulator phosphorylation dynamics in live bacteria

Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here,...

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Autores principales: Butcher, Ryan J., Tabor, Jeffrey J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9436347/
https://www.ncbi.nlm.nih.gov/pubmed/35994658
http://dx.doi.org/10.1073/pnas.2201204119
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author Butcher, Ryan J.
Tabor, Jeffrey J.
author_facet Butcher, Ryan J.
Tabor, Jeffrey J.
author_sort Butcher, Ryan J.
collection PubMed
description Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.
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spelling pubmed-94363472022-09-02 Real-time detection of response regulator phosphorylation dynamics in live bacteria Butcher, Ryan J. Tabor, Jeffrey J. Proc Natl Acad Sci U S A Biological Sciences Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct in vivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology. National Academy of Sciences 2022-08-22 2022-08-30 /pmc/articles/PMC9436347/ /pubmed/35994658 http://dx.doi.org/10.1073/pnas.2201204119 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Butcher, Ryan J.
Tabor, Jeffrey J.
Real-time detection of response regulator phosphorylation dynamics in live bacteria
title Real-time detection of response regulator phosphorylation dynamics in live bacteria
title_full Real-time detection of response regulator phosphorylation dynamics in live bacteria
title_fullStr Real-time detection of response regulator phosphorylation dynamics in live bacteria
title_full_unstemmed Real-time detection of response regulator phosphorylation dynamics in live bacteria
title_short Real-time detection of response regulator phosphorylation dynamics in live bacteria
title_sort real-time detection of response regulator phosphorylation dynamics in live bacteria
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9436347/
https://www.ncbi.nlm.nih.gov/pubmed/35994658
http://dx.doi.org/10.1073/pnas.2201204119
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