期刊
MBIO
卷 8, 期 5, 页码 -出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/mBio.01089-17
关键词
FRET; PBP5; PBP6a; PBP6b; antibiotics; mCherry; mNeonGreen; periplasm; protein interactions
类别
资金
- Netherlands Organization for Scientific Research (NWO) Middelgroot investment grant [834.09.003]
- NWO ALW open program [822.02.019]
- DIVINOCELL project of the European Commission [FP7-Health-2007-B-223431]
One of the mechanisms of beta-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. 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.
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