The bacterial indicator strains used in this study are listed in Table 1. Bacterial growth was performed in Luria–Bertani (LB) broth at 37 °C. The producer strain B. subtilis B38 was grown in tryptic soy broth (TSB) at 30 °C. The antibacterial activity was assayed using the agar disk diffusion method as described previously (Tabbene et al., 2009a). The titer of antibacterial activity was expressed as activity units (AU) mL−1 and corresponded to the reciprocal of the highest dilution showing growth inhibition of the Pseudomonas aeruginosa ATCC 27853 indicator strain. To purify the S07-2 compound,

B. subtilis B38 was cultured in 1 L TSB as described previously (Tabbene et al., 2009a). The cell-free supernatant was subjected to methanol extraction. After centrifugation, the supernatant learn more was evaporated and the resulting precipitate was dissolved in MilliQ water and fractionated onto a Sep-Pak plus C18 cartridge (Waters, Division of Millipore Corp., Bedford, MA) using a discontinuous gradient of acetonitrile (0%, 20%, 40%, 60%, 80%

and 100%). The active fraction was applied onto a DEAE-Sepharose column (Amersham Pharmacia Biotech). Elution was performed using 10 mM ammonium acetate buffers at different pH (7.5, 6, 5, 4 and 3). The active fraction was applied onto a C18 RP-HPLC column (250 × 4.6 mm). Elution was performed using a linear gradient of acetonitrile from 0% to 100% at a flow rate of 1 mL min−1 for 70 min. All collected fractions were dried under vacuum, dissolved in methanol and tested for their antibacterial activity against P. aeruginosa. The active fraction was chromatographed once more, onto an HS PEG HPLC www.selleckchem.com/products/PLX-4032.html column (250 ×

4.6 mm). Elution was performed using a linear gradient of acetonitrile from 0% to 100% in 10 mM ammonium acetate buffer, pH 6.8, at a flow rate of 0.8 mL min−1 for 40 min. The HPLC-purified fraction was subjected to TLC using n-butanol–methanol–water (39 : 10 : 20, v/v/v) as the mobile phase. The bioassay was performed as described previously (Tabbene et al., 2009a) using P. aeruginosa as the indicator strain. S07-2 compound was detected by UV light at 254 nm or by exposure to iodine and subjected to ninhydrin and 4,4′-bis(dimethylamino)diphenylmethane (TDM) staining methods according to Yu et al., 2002. The iron-binding capacity of the S07-2 compound was determined PIK3C2G using chrome azurol sulfonate (CAS) agar blue solution according to Schwyn & Neilands (1987). The CAS agar solution was poured onto the developed TLC plate. A positive reaction was revealed by a change in the color of the CAS–iron complex from blue to orange. A preliminary detection of the radical-scavenging activity was conducted as described previously (Sreenivasan et al., 2007). The developed TLC plate was sprayed with 0.1% w/v 1-diphenyl-2-picrylhydrazyl (DPPH) methanolic solution. The compound with antiradical activity appeared as a yellow spot against the purple–blue background.