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. Author manuscript; available in PMC: 2019 Aug 25.
Published in final edited form as: Nat Chem. 2019 Feb 25;11(4):335–341. doi: 10.1038/s41557-019-0217-x

Figure 4. RfDAAs facilitate real-time in vitro transpeptidation assays.

Figure 4.

a) Scheme of assay procedure. Rf470DL and the synthetic substrate were mixed in a 96-well plate. The fluorescence intensity of Rf470DL was then measured over time upon the addition of recombinant S. aureus PBP4. b) Real-time monitoring of Rf470DL intensity. An intensity increase was observed in the assay; while adding PBP inhibitors blocked the reaction. c) HPLC analysis of the assay products. A shifted retention time was observed when the Rf470DL-substrate mixture was treated with PBP4. d) High-resolution MS analysis of the products from the assay confirmed the formation of the cross-linked product. e) Screening of antibiotic effect on S aureus PBP4 activity. A 1:10 ratio of antibiotics to the substrate was used in the experiments. Kanamycin and Chloramphenicol (ribosome inhibitors) did not inhibit the Rf470DL incorporation. Piperacillin, Ampicillin, Penicillin G, Carbenicillin and Cefoxitin (β-lactam antibiotics) showed different level of inhibition. f) β-lactamase activity of S. aureus PBP4. A competition assay using Nitrocefin was performed to study PBP4 β-lactamase activity toward the β-lactam antibiotics used in the assay experiments. In this panel, low activity stands for a strong inhibition effect of the antibiotics toward PBP4, and vice versa. Values are normalized to the “no antibiotic” sample. Error bars: Mean value and standard deviation.