3 (B1) and 1203.9 (B2). They were attributed to two variants of polymyxin B differing in their fatty acid component, which is either an iso-octanoyl (C8H15O) or a 6-methyloctanoyl (anteisononanoyl, C9H17O) residue [21, 32]. By comparison with polymyxin B and other members of the polymyxin family, we conclude that polymyxin P1 and P2 from strain M-1 contain the same fatty acid Stattic cell line residues consistent with the data reported by Kimura et al. for polymyxin P [14]. The anti-Erwinia activity of polymyxin P produced by P.

polymyxa M-1 In order to identify the compounds which suppress the growth of E. TPCA-1 in vitro amylovora Ea273 and E. carotovora in M-1 GSC culture, the supernatant was subjected to thin layer chromatography (TLC) in combination with bioautography [39] (Figure 4). One spot exhibiting antibacterial activity was observed at R f 0.36 (Figure 4A) which was identical with

that of polymyxin P [14]. It was scraped off from the thin layer plate. The silica gel powder obtained was extracted with methanol, and the extract was analyzed by MALDI-TOF-MS. The obtained mass spectrum ranging from m/z = 850 to 1350 (Figure 4B) indicates the same mass peaks at m/z = 1199.9, m/z = 1213.9, m/z = 1239.9, m/z = 1253.9 and m/z = 1268.0 as previously been detected for series 2 in Figure 2. From these results we conclude, that polymyxin P1 and P2 represent the active compounds inhibiting growth of the Erwinia test strains. There were no mass signals pointing to fusaricidines (m/z = 850 Small molecule library in vitro Casein kinase 1 – 1000) or other metabolites showing antibacterial activity (Figure 4B). Thus, polymyxin P was proven to be an anti-Erwinia metabolite which was produced by M-1. Figure 4 Detection of the anti- Erwinia metabolite produced by P.

polymyxa M-1. (A) Detection of the antibacterially acting metabolite by bioautography. Supernatants prepared from strain M-1 grown in GSC medium for 36 h were separated by TLC and sandwiched with indicator strain E. carotovora. The inhibiting band at R f 0.36 was circled. (B) MALDI-TOF-MS analysis of the antibacterial compounds detected by bioautography. To corroborate these results, a GSC culture supernatant of M-1 was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) (Figure 5A). Fifteen fractions were obtained. The fraction appearing at a retention time of 2 displayed antagonistic effects against the growth of the two phytopathogenic Erwinia indicator strains (Figure 5B). This fraction was analyzed by high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS). Two peaks were detected at m/z = 1191.8 and m/z = 1177.9, which also correspond to the two isomers of polymyxin P [14] (Figure 5C). Figure 5 RP-HPLC analysis and antibacterial activity test of fractions. (A) RP-HPLC (HPLC type: Agilent 1100) analysis of M-1 GSC culture supernatant using a Luna C18 column (100 Å 150 × 4.6 mm, Phenomenex, Aschaffenburg, Germany).