Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>

ABSTRACT One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their a...

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Autores principales:	Nils Y. Meiresonne, René van der Ploeg, Mark A. Hink, Tanneke den Blaauwen
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	FRET PBP5 PBP6a PBP6b antibiotics mCherry Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/667f6b6d758e4d2bb1bbb84ac97a7682
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spelling	oai:doaj.org-article:667f6b6d758e4d2bb1bbb84ac97a76822021-11-15T15:51:51ZActivity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>10.1128/mBio.01089-172150-7511https://doaj.org/article/667f6b6d758e4d2bb1bbb84ac97a76822017-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01089-17https://doaj.org/toc/2150-7511ABSTRACT One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies.Nils Y. MeiresonneRené van der PloegMark A. HinkTanneke den BlaauwenAmerican Society for MicrobiologyarticleFRETPBP5PBP6aPBP6bantibioticsmCherryMicrobiologyQR1-502ENmBio, Vol 8, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	FRET PBP5 PBP6a PBP6b antibiotics mCherry Microbiology QR1-502
spellingShingle	FRET PBP5 PBP6a PBP6b antibiotics mCherry Microbiology QR1-502 Nils Y. Meiresonne René van der Ploeg Mark A. Hink Tanneke den Blaauwen Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>
description	ABSTRACT One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies.
format	article
author	Nils Y. Meiresonne René van der Ploeg Mark A. Hink Tanneke den Blaauwen
author_facet	Nils Y. Meiresonne René van der Ploeg Mark A. Hink Tanneke den Blaauwen
author_sort	Nils Y. Meiresonne
title	Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>
title_short	Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>
title_full	Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>
title_fullStr	Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>
title_full_unstemmed	Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>
title_sort	activity-related conformational changes in <sc>d,d</sc>-carboxypeptidases revealed by <italic toggle="yes">in vivo</italic> periplasmic förster resonance energy transfer assay in <italic toggle="yes">escherichia coli</italic>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/667f6b6d758e4d2bb1bbb84ac97a7682
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Activity-Related Conformational Changes in <sc>d,d</sc>-Carboxypeptidases Revealed by <italic toggle="yes">In Vivo</italic> Periplasmic Förster Resonance Energy Transfer Assay in <italic toggle="yes">Escherichia coli</italic>

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