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

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.

Superfolder mTurquoise2ox optimized for the bacterial periplasm allows high efficiency in vivo FRET of cell division antibiotic targets

“…mTq2 and sfTq2 fusions were expressed in the periplasm of E. coli growing in a plate reader at a concentration range of the inducer IPTG (Experimental procedures). Cultures expressing either mTq2 or sfTq2 resulted in toxicity correlated with induction concentrations previously reported for other FPs (Meiresonne et al , ). However, sfTq2 could be expressed at higher levels with less toxicity compared to mTq2.…”

“…A technical negative control, consisting of non‐interacting proteins between the IM and OM, yielded −0.3 ± 1.4% energy transfer. The previously described (Meiresonne et al , ) biological homodimerization interaction of the active and inactive D , D ‐carboxypeptidase PBP5 was confirmed with Ef A values of 4.2 ± 2.7% and 8.8 ± 4.2%, respectively (Fig. B and Table ).…”

“…The technical negative control (IM-OM) did not show any energy transfer. The activity-related conformational changes of the PBP5 dimers previously observed with the mNG-mCh FRET pair (Meiresonne et al, 2017) were confirmed. The error bars represent the mean and standard deviation.…”

“…The FRET efficiencies were calculated using the published algorithms (Alexeeva et al , ; Meiresonne et al , ), using the spectral properties of mTq2 and mNG (Goedhart et al , ; Shaner et al , ). Spectral measurements using a multimode plate reader (BIOTEK Synergy MX, BioTek Instruments Inc., Winooski, VT) were performed as described (Meiresonne et al , ; ) with the mTq2 variant donor channel acquisition set at 450 nm and emission scanning from 470 to 650 nm with minimal slit widths of 9 nm.…”

Building peptidoglycan inside eukaryotic cells: A view from symbiotic and pathogenic bacteria

“…Unlike other d , d ‐carboxypeptidases like PBP4, PBP5, PBP6 and PBP7; PBP6b is the only capable of suppressing a cell shape defect at acidic pH (Peters et al, ). Another recent study involving complementation of an E. coli PBP5‐null mutant with other d , d ‐carboxypeptidases reported divergence in function, concluding that the balanced activity of these d , d ‐carboxypeptidases is required to ensure synthesis of a robust mature PG (Meiresonne, Ploeg, Hink, & Blaauwen, ). Similar cases of specialization include the bifunctional enzymes PBP1a and PBP1b of E. coli , with display optimal GT and TP activities at alkaline and acidic pH, respectively (Mueller et al, ).…”