For the use of four-sectored 100 mL petri plates, volumes were adjusted to 100 μL of overnight culture and 2 mL molten top agar per sector. Phage lysates were either added to top agar prior to pouring onto an LB agar plate or were spotted onto solidified top agar containing signaling pathway bacteria and allowed to dry prior to incubation at 37°C. Phage lysates were diluted in either Phage buffer [PB; 50 mM Tris–HCl

(pH 7.4), 10 mM MgSO4, 2 mM CaCl2, 75 mM NaCl] or SM buffer [50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 8 mM MgSO4, 0.002% gelatin] [19]. Phage isolation and enumeration φX216 was plaque-purified twice from spontaneously formed plaques by released phage on B. pseudomallei E0237 using small scale liquid lysates using B. pseudomallei 2698a as a host strain. Plate lysates were

prepared by flooding inverted plates with 5 mL of PB followed by incubation for either 3 h at 37°C or overnight at 4°C without agitation. The liquid was recovered from plates and bacteria pelleted by centrifugation at 16,000xg for 1 min at room temperature. Supernatants were combined and sterilized AG-881 cell line with a 0.2 μm disposable syringe filter (DISMIC-25AS Life Science Products, Inc., Frederick, CO). To create adapted lysates, plate lysates were used sequentially to infect a host strain followed by lysate recovery and reinfection for two to four cycles. For liquid lysates, 1 mL of a B. mallei ATCC23344 overnight culture, 1 mL phage lysate at approximately 106 pfu/mL, 1 mL 10 mM CaCl2 and 10 mM MgCl2 were combined and incubated without agitation at 37°C for 15 min for initial phage attachment. 1.5 mL each of these mixtures were inoculated into 2 × 250 mL of pre-warmed LB with 2% glycerol in two 1 L disposable fretted Erlenmeyer flasks (Corning, Elmira, NY) and

incubated overnight at 37°C with aeration. After overnight incubation, lysates were PRIMA-1MET sometimes treated with 1% chloroform although better results were obtained when this step was omitted. Lysates were centrifuged at 4,000xg for 20 min at 4°C. Supernatants were combined with 25 mL 1 M Tris–HCl (pH 7.4) to a final concentration of 50 mM Tris–HCl, pre-filtered through a 0.8 μm disposable vacuum filtration unit and then filtered through a 0.2 μm disposable vacuum Baf-A1 mouse filtration unit to achieve sterility (Nalgene, Rochester, NY). Lysates were stored at 4°C in the dark. To determine phage titers, lysates were serially diluted in PB and 10 μL aliquots spotted onto top agar plates with appropriate Burkholderia sp. tester strains. Isolated plaques were counted and titers (pfu/mL) calculated. Burst size determination Phage burst sizes were determined by generation of one-step growth curves as previously described [19]. Briefly, a B. mallei ATCC23344 liquid lysate was inoculated using the same procedure described above for a single 250 mL volume.