Supernatant solubility was tested in different polar solvents: 2 × ethyl acetate, chloroform or butanol were added,

respectively, to a 250-mL flask with 50 mL of the different culture filtrates. Extraction of potentially active fractions was Selleck STA-9090 carried out as described (Rydberg et al., 2004). The organic phase was vaporized to remove the organic solvent using a vacuum at 50 °C followed by the addition of sterile distilled water until the original volume was restored. The aqueous phase and aqueous solution components of the respective organic extracts were then tested for nematicidal activity. The molecular size of the nematicidal components was determined by dialyzing the culture filtrates against a Millipore ultrafiltration membrane (1000 nominal molecular weight limit) using a vacuum PI3K inhibitor rotary evaporator. The culture filtrates in the Buchner flask and the residual portion in the Buchner

funnel were, respectively, diluted to their starting volumes with ddH2O (solutions E and F, which were then tested for nematicidal activity). Genomic DNA was extracted from Bacillus spp. as described (Hoflack et al., 1997). Plasmids from E. coli and Bacillus spp. were both extracted with an AxyPrep™ plasmid miniprep kit (Axygen Scientific Inc., Union City, CA), except that Bacillus spp. were resuspended in solution I and treated with 10 mg mL−1 lysozyme (Sigma, pH 8.0) at 37 °C for 20 min. Escherichia coli DH5α and B. subtilis OKB105 strains were transformed as described (Spizizen, 1958; Sambrook et al., 1989) as was the OKB105 mutant library (Breton et al., 2006). Southern hybridizations were performed using DIG High Prime DNA Labeling and the Detection Starter Kit I as described by the manufacturer (Roche Applied Science, Mannheim, Germany). All enzymes used in this study were purchased from TaKaRa Bio Inc. (Japan). The specific primers used are described in Table 2. For complementation of the

M1 mutant, two types of plasmids were constructed. pMA5-purL (multicopy shuttle expression vector) was constructed as follows. The entire purL gene was amplified from strain OKB105 chromosomal DNA using primers P1/P2. The sequences of Cm were obtained from pDG1661 using primers P3/P4. The two fragments were ligated by overlap PCR using primers P1/P4. The PCR product was purified, Astemizole digested with BamHI and NdeI, and cloned into the E. coli–B. subtilis pMA5 shuttle vector (Dartois et al., 1994) to generate expression vector pMA5-purL. The pMA5-purL was transformed into strain M1, and selected on solid LB agar medium supplemented with 5 μg mL−1 chloramphenicol and 50 μg mL−1 kanamycin. M1∷pMA5-purL was confirmed by PCR and BamHI/NdeI digestion, and the resulting transformant was designated M1-1. M1-1 used the constitutive promoter HpaII for purL expression. pUC18-purL (suicide vector) was constructed as follows.