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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\n            <jats:italic>in vivo<\/jats:italic>\n            interactions in this compartment, we have developed and validated a F\u00f6rster 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\n            <jats:sc>d,d<\/jats:sc>\n            -CPases PBP5, PBP6a, and PBP6b of\n            <jats:italic>Escherichia coli<\/jats:italic>\n            change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these\n            <jats:sc>d,d<\/jats:sc>\n            -CPases are not redundant but that their balanced activity is required for robust PG synthesis.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            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.\n          <\/jats:p>","DOI":"10.1128\/mbio.01089-17","type":"journal-article","created":{"date-parts":[[2017,9,13]],"date-time":"2017-09-13T00:10:50Z","timestamp":1505261450000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":30,"title":["Activity-Related Conformational Changes in\n            <scp>d,d<\/scp>\n            -Carboxypeptidases Revealed by\n            <i>In Vivo<\/i>\n            Periplasmic F\u00f6rster Resonance Energy Transfer Assay in\n            <i>Escherichia coli<\/i>"],"prefix":"10.1128","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9184-4361","authenticated-orcid":false,"given":"Nils Y.","family":"Meiresonne","sequence":"first","affiliation":[{"name":"Bacterial Cell Biology and Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2490-9262","authenticated-orcid":false,"given":"Ren\u00e9","family":"van der Ploeg","sequence":"additional","affiliation":[{"name":"Bacterial Cell Biology and Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark A.","family":"Hink","sequence":"additional","affiliation":[{"name":"Molecular Cytology and van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5403-5597","authenticated-orcid":false,"given":"Tanneke","family":"den Blaauwen","sequence":"additional","affiliation":[{"name":"Bacterial Cell Biology and Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2015.0031"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2003.03607.x"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.62.4.1079-1093.1998"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.06.012"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6976.2007.00090.x"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.54.4.1133"},{"key":"e_1_3_3_8_2","first-page":"1","article-title":"Tackling drug-resistant infections globally: final report and recommendations","volume":"84","author":"O\u2019Neill J","year":"2016","unstructured":"O\u2019Neill J . 2016. 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