The LC-MS analysis was carried out as described previously (Moran et al., 2009). Briefly, a 20-μL culture supernatant was injected onto an Atlantis T3, 3-μm C18 column (100 mm × 2.1 mm i.d.) using a Waters Alliance 2695 HPLC system (Waters, Milford, MA) interfaced to a mass spectrometer. Chromatographic separation of analyte was achieved in 60% aqueous acetonitrile containing 0.1% formic acid. Electrospray mass spectra, in the positive and negative ionization mode, were acquired on a Quattro Micro™ mass spectrometer (Waters Corporation, Micromass MS Technologies, Manchester, UK). Electrospray MS (ESMS) and tandem MS (MS/MS) experiments were performed on a quadrupole time-of-flight

instrument (Q-Tof Premier, Waters Corporation, Micromass

MS Technologies). A sample in 65% aqueous acetonitrile (1 : 1) was introduced into the mass spectrometer from a 250-μL gastight syringe Selleckchem HDAC inhibitor at 8 μL min−1. The ESMS data were recorded on a negative Tyrosine Kinase Inhibitor Library order ionization mode for m/z 100 to m/z 2000. The MS/MS data from the selected precursor ions (m/z 1034.7) were obtained for m/z 100 to m/z 1200. A collision energy of 18 eV was used to induce fragmentation of the precursor ions. Our initial interest in Bacillus sp. CS93 was the biosynthesis of chlorotetaine; thus, the strain was cultured using conditions similar to those described previously (Phister et al., 2004), and upon bioassay, visible zones of clearing were apparent on plates of E. coli (8 mm, well plate method), S. aureus (19 mm, disc plate method) and S. cerevisiae (17 mm, disc plate method). There were no zones of clearing in the control experiments. The LC-MS analysis of culture supernatant detected ions characteristics for iturin A (m/z 1043.5 and m/z Stem Cells inhibitor 1057.6), which accounted for the

antifungal activity, and was consistent with the previous work with this strain (Moran et al., 2009). However, the ions expected for bacilysin (m/z 271.1) and chlorotetaine (m/z 289.1, plus an isotopic signal at m/z 291.1) were not observed, despite the culture supernatant exhibiting antibacterial activity. The absence of these secondary metabolites might be a result of very minor changes in the culture conditions between this study and the previous work by Phister and colleagues, since it is well established that small changes in culture conditions can have major impacts on the production of antibiotics (Bode et al., 2002). Alternatively the concentrations of these metabolites were too low in the crude supernatant to be detected by LC-MS. The bac gene cluster was amplified as described in Materials and methods using primers designed from the bacA and bacE gene sequences of B. subtilis A1/3 and Bacillus amyloliquefaciens ATCC15841. Because it is most likely that bacilysin is the immediate precursor of chlorotetaine, it was thought that the bac gene cluster in Bacillus sp. CS93 might contain a gene coding for a halogenase. The PCR experiment yielded a single band at 4.