a, Influence of heat and proteases on Agr inhibition. B. subtilis culture filtrate was subjected to heat (95 °C for 20 min) or digestion with proteinase K (50 μg ml−1, 37 °C, 1 h) and the effect on inhibition of Agr activity was measured using the luminescence assay with the USA300 P3–luxABCDE reporter strain (see Fig. 3a). RLU, relative light units. The experiment was performed with n = 2 independent biological samples. Lines connect the means. (The observed additional suppression of Agr activity in the proteinase-K-treated sample at 6 h, compared with the B. subtilis culture filtrate sample, is expected owing to proteolytic inactivation of intrinsic AIP.) b, Preparative RP chromatography of B. subtilis culture filtrate to determine the Agr-inhibiting substance. The peaks labelled 2 and 3 showed substantial Agr-inhibiting activities in the Agr-activity assay and were identified as fengycins using subsequent RP-HPLC/ESI-MS and MS/MS analysis (see c, d). The peaks labelled 1 and 4–6 also contained fengycin species (see e). AU, arbitrary units. The applied gradient (% buffer B) is shown in green. c, Fractions corresponding to Agr-inhibitory peaks 2 and 3 from the preparative RP run (b) were subjected to RP-HPLC/ESI-MS. Top, total ion chromatograms (TICs) of the RP-HPLC/ESI-MS runs; bottom, ESI mass spectrogram of the major peaks. d, MS/MS analysis of the peak 2 and 3 fractions. Peaks that are characteristic of a given fengycin subtype (A or B in this case) are marked in colour. ‘Parent’ refers to the relevant numbered peak in the spectrograms above. e, Analysis of further fengycin-containing fractions. Peaks 1, 4, 5 and 6 from the preparative RP run (b) were also found to contain fengycin species as determined by subsequent RP-HPLC/ESI-MS analysis. Shown are the mass spectrograms of the major peaks of those runs and the tentative characterization for fengycin type. The preparative and analytical chromatography and RP-HPLC/ESI-MS analyses (as shown in b, d) were repeated multiple (more than ten) times for fengycin purification, with similar results. MS/MS analyses were not repeated. f, Analysis of fengycin and surfactin lipopeptide expression by the B. subtilis wild-type strain and its isogenic ΔfenA mutant.