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

dc.citation.articleNumbere2201204119en_US
dc.citation.issueNumber35en_US
dc.citation.journalTitleProceedings of the National Academy of Sciencesen_US
dc.citation.volumeNumber119en_US
dc.contributor.authorButcher, Ryan J.en_US
dc.contributor.authorTabor, Jeffrey J.en_US
dc.date.accessioned2022-09-08T14:40:04Zen_US
dc.date.available2022-09-08T14:40:04Zen_US
dc.date.issued2022en_US
dc.description.abstractBacteria 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.en_US
dc.identifier.citationButcher, Ryan J. and Tabor, Jeffrey J.. "Real-time detection of response regulator phosphorylation dynamics in live bacteria." <i>Proceedings of the National Academy of Sciences,</i> 119, no. 35 (2022) National Academy of Sciences: https://doi.org/10.1073/pnas.2201204119.en_US
dc.identifier.digitalpnas-2201204119en_US
dc.identifier.doihttps://doi.org/10.1073/pnas.2201204119en_US
dc.identifier.urihttps://hdl.handle.net/1911/113197en_US
dc.language.isoengen_US
dc.publisherNational Academy of Sciencesen_US
dc.rightsThis open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).en_US
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/en_US
dc.titleReal-time detection of response regulator phosphorylation dynamics in live bacteriaen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
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