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

mBio. 2017 Sep 12;8(5):e01089-17. doi: 10.1128/mBio.01089-17.

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.

Keywords: FRET; PBP5; PBP6a; PBP6b; antibiotics; mCherry; mNeonGreen; periplasm; protein interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carboxypeptidases / chemistry*
  • Carboxypeptidases / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Fluorescence Resonance Energy Transfer
  • Luminescent Proteins
  • Peptidoglycan / chemistry
  • Peptidoglycan / metabolism
  • Periplasm / chemistry
  • Periplasm / metabolism
  • Periplasm / ultrastructure*
  • Protein Conformation
  • Serine-Type D-Ala-D-Ala Carboxypeptidase / chemistry
  • Serine-Type D-Ala-D-Ala Carboxypeptidase / metabolism

Substances

  • Escherichia coli Proteins
  • Luminescent Proteins
  • PBP5 protein, E coli
  • Peptidoglycan
  • red fluorescent protein
  • Carboxypeptidases
  • DacC protein, E coli
  • Serine-Type D-Ala-D-Ala Carboxypeptidase