Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Forster Resonance Energy Transfer Assay in Escherichia coli

• Main Authors: Nils Y. Meiresonne, René van der Ploeg, Mark A. Hink, Tanneke den Blaauwen, Lotte Søgaard-Andersen
• Format: Article
• Language: English
• Published: American Society for Microbiology 2017-09-01
• Series: mBio
• Online Access: http://mbio.asm.org/cgi/content/full/8/5/e01089-17

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 Forster 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.