ΦBP DNA isolated from the phage particles served as a template for positive control reaction. Chromosomal DNA from B. subtilis CCM 2722 (amy+), B. Epigenetics Compound Library flavum CCM 251 and C. glutamicum RM3 were used as templates for negative controls. Amplification was performed using DNA thermal cycler Biometra T-gradient (Whatman) under the following PCR conditions: 95 °C for 5 min; 10 × (95 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s); 20 × (95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min); and 72 °C for 5 min. The amplified DNA fragments were analyzed using agarose gel electrophoresis and DNA sequencing. The
presence of ΦBP sequences on the chromosome of P. polymyxa CCM 7400 was tested by Southern blot analysis. For Southern hybridization, the chromosomal DNA from three isolates of P. polymyxa Palbociclib order CCM 7400 was digested with EcoRI, separated by electrophoresis in 0.8% w/v agarose gel in TAE (40 mM Tris-acetic acid, pH 8.0; 1 mM EDTA) and transferred on Hybond N (Amersham) according to Ausubel et al. (1995). ΦBP DNA isolated from the phage particles was used as positive control. The membranes were hybridized with random-primed digoxigenin-labeled DNA probes at 44 °C in 50% v/v formamide and a signal was detected by DIG detection kit using NBT/BCIP (Roche Applied Science, Germany) according to the manufacturer’s instructions.
Three probes were used for hybridization experiments: a 600-bp Bsp1407I–Bsp1407I DNA sequence from the 2503-bp EcoRI fragment of ΦBP DNA, a 1013 bp EcoRI–EcoRV DNA sequence from the 1662-bp EcoRI fragment of ΦBP DNA and the whole 1213-bp EcoRI fragment of ΦBP DNA. Ureohydrolase For the preparation of the probes, the corresponding plasmids containing cloned EcoRI ΦBP DNA fragments
were digested with restriction endonucleases listed above, separated by electrophoresis in 0.8% w/v agarose gel in TAE, and purified using QIAquick gel extraction kit (Qiagen). After several successive rounds of inoculation and cultivation, we observed spontaneous lysis of the growing P. polymyxa CCM 7400 culture. We screened P. polymyxa culture lysate for the presence of phages. We recovered phage particles from cell-free supernatant of spontaneously lysed culture. We observed delayed or no growth against the control after infection of P. polymyxa CCM 7400 with cell-free lysate. This growth alteration was occasionally followed by a cell lysis coupled with decrease in OD600 nm. The cell culture lysis typically occurred in 6–8 h after infection. We recovered phage particles from those lysates. We used the cells of P. polymyxa CCM 7400 from stationary growth phase diluted to an OD600 nm of 0.5 for phage propagation. Infection of P. polymyxa cells with phage lysate was followed by cultivation of the cells and resulted in cell lysis in 6–8 h. Strains of the genus Paenibacillus tested for sensitivity to ΦBP are listed under Materials and methods. With the exception of the primary host P. polymyxa CCM 7400, only the strain P.