The collected fractions were analyzed by thin layer chromatography (TLC) that was developed with CHCl3/CH3OH/ 2M NH4OH (40:10:1 v/v), and 17-AAG research buy the spots were visualized with iodine and by spraying them with orcinol/H2SO4. The methanol fraction containing the partially purified lipopeptide was then analyzed by ESI-MS in the positive and negative ionization modes. Gas chromatography–mass spectrometry (GC-MS) of fatty acids The lipids (1 mg) were methanolyzed in 0.5 ml of 1 M HCl-MeOH for 4 h at 100°C. The product containing the fatty acid methyl esters (FAMEs) was partitioned by adding H2O (0.5 ml) and extracting with 1 ml of n-hexane [30]. The MeOH/H2O phase was dried

under N2 stream and was acetylated in pyridine-MeOH-Ac2O (1:1:4, v/v) with heating at 100°C for 60 min [31]. The samples were then analyzed using a GC-MS-ion trap detector (Varian, Saturn-2000R) with a capillary column DB-1-MS (J&W) that was 30 m x 0.25 mm x 0.25 μm in size. The chromatograph temperature was programmed to increase from 50 to 280°C at 20°C/min and was then held constant for 30 min. FAMEs were identified on the basis of their relative retention time in comparison with the standard of 3-hydroxy-hexadecanoate methyl ester (Sigma-Aldrich, SP, Brazil) and by their MS-fragmentation profile at electron ionization (EI – 70 eV). Electrospray ionization-mass spectrometry (ESI-MS) The approximately 300 μg/ml RG-7388 in vivo suspension of lipids in MeOH–H2O (3:1, v/v) containing

HCl at 1 mmol/l was submitted to positive and negative mass spectrometry at atmospheric pressure ionization and recorded on a triple quadrupole, Quattro LC (Waters)

with N2 as the nebulization and desolvation gas. Offline SPTLC1 analyses were performed with an infusion pump at a flow rate of 10 μl/min. The energies were set at 3.5 kV on the capillary and 100 V on the cone (negative mode) or at 3.5 kV and 90 V (positive mode). Tandem-MS was obtained by collision-induced dissociation-mass spectrometry (CID-MS) using argon as collision gas and a collision energy of 40 eV. Bioautography In order to confirm the antimicrobial activity of the partial purified lipopeptide fraction, approximately 100 μl of the extract were applied to two thin layer chromatography (TLC) plates (10 cm × 20 cm) and developed with CHCl3/CH3OH/ 2M NH4OH (40:10:1 v/v). One plate was used as the reference chromatogram, and the spots were visualized with iodine and by spraying them with orcinol/H2SO4. The other one was used for bioautography in a Petri dish. A suspension (15 ml) containing 105 cells/ml of D. alaskensis NCIMB 13491 was poured over the TLC plate. After solidification of the medium, the TLC plate was incubated for 7 days at 30°C in an anaerobic chamber. Clear growth inhibition zones were observed against a blackish background. Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) To determine the minimum concentration that the lipopeptide inhibits D.