FIG 4.
Amycobactin inhibits protein secretion through the Sec translocon. (a) The M. smegmatis ′BlaTEM-1 reporter strain was used to monitor the presence of β-lactamase in the culture supernatant (CF) (red curves) and whole-cell lysate (WCL) (black curves), either untreated (UT) (circles) or after treatment with amycobactin (squares). β-Lactamase was monitored by using the cleavage of the chromogenic β-lactamase substrate nitrocefin and monitoring the absorbance at 490 nm and 390 nm every 5 min for 60 min. The data, displayed as the ratio of absorbance at 490 nm (cleaved product) to absorbance at 390 nm (uncleaved nitrocefin), represent the results of three independent experiments. (b) Culture filtrate samples were analyzed for the maximum change in absorbance at 490 nm (Vmax490) over the course of the 60-minute experiment for which results are shown in panel a. (c) Representative Western blot analysis of β-lactamase protein in the CF and WCL of the untreated or amycobactin-treated M. smegmatis ′BlaTEM-1 reporter strain. (d) Densitometry analysis, using ImageJ software, of the Western blots of WCL and CF from untreated and amycobactin-treated cultures. AUC, area under the curve. Error bars display standard errors of the means. Significance was determined by Student’s t test (b) or one-way analysis of variance (d). ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. Data represent the results of three independent experiments.