Results Characterization of M-1 Culture supernatants of M-1 suppressed growth of several bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa

(Table 1). Remarkably, growth of phytopathogenic E. amylovora Ea 273 and E. carotovora was strongly inhibited (Figure 1). M-1 was identified as P. polymyxa by its 16S rDNA sequence (gb accession: FR727737) and by physiological and biochemical features. The motile, rod-shaped and spore-forming bacterium was facultative anaerobic, was positive in the Voges-Proskauer reaction (acetylmethylcarbinol), able to hydrolyze starch and to utilize glucose, xylose, glycerol, and mannitol, but did not grow at sodium chloride concentrations exceeding 5%. The whole genome sequence of M-1 (gb accession: HE577054.1) displayed close similarity to the sequences of plant-associated P. polymyxa strains SC2 [36] and E681 [3], respectively. Table 1 Antibacterial activity of Paenibacillus polymyxa EGFR inhibitor M-1 culture supernant determined in agar diffusion test Indicator strains Diameter of the inhibition zone (mm) Erwinia amylovora Ea 273 21.5 Erwinia carotovora 20 Escherichia coli K12 18 Pseudomonas aeruginosa 23 Streptococcus faecalis 7 Micrococcus luteus 22.5 Bacillus megaterium 14.5 Bacillus subtilis 168 7.5 Bacillus amyloliquefaciens FZB42 6 Figure 1 In vitro antagonistic effect of P. X-396 purchase polymyxa M-1 against E. amylovora Ea273 and E. carotovora. (A) Inhibiting

effect of M-1 culture supernatant (CS) against E. amylovora Ea273. (B) Inhibiting effect of M-1 culture supernatant against E. carotovora. “M-1CS” represents M-1 GSC culture supernatant. GSC medium was used as a negative control. M-1 cells were also spotted onto lawns of E. amylovora Ea273 and E. carotovora. E. coli DH5α cells were used as a negative control. Detection and structural characterization of polymyxin P The metabolites produced by P. polymyxa M-1,

possessing antagonistic activities against E. amylovora Ea273 and E. carotovora were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in combination with bioautography. Antibacterial activities were detected in both cell-surface extracts 6-phosphogluconolactonase and a GSC culture supernatant of M-1. Cell surface extracts were prepared by extraction of cells picked from agar plates with 70% acetonitrile/0.1% trifluoroacetic acid [37]. By MALDI-TOF-MS, two prominent series of mass peaks were detected, ranging from m/z = 883.1 to 983.5 (series 1) and from m/z = 1177.9 to 1267.9 (series 2) (Figure 2A), respectively. Members of series 1 were attributed to the well-known fusaricidins (unpublished data), a family of lipodepsipeptides exhibiting potent antifungal activities [38]. The compounds of series 2 (Figure 2B) were investigated by MALDI-TOF-MS in more detail. Two metabolites were detected, of which the protonated forms showed masses of m/z = 1191.9 and m/z = 1177.9.