E. coli Phosphonate Bioassay

E. coli WM6242 is an E. coli strain that expresses the phosphonate uptake system phnCDE under the control of a P_tac promoter (21 (link)). Inducing the expression of phnCDE by addition of IPTG increases sensitivity to most phosphonate antibiotics. The phosphonate specific bioassay, which uses E. coli WM6242, was previously described (21 (link)). Briefly, WM6252 was grown to an OD₆₀₀ of 0.8 in LB at 37°C. 6 mL of LB top agar, with and without 1 mM IPTG, was inoculated with 50 μL of the culture and then added to the corresponding LB plates with and without 1 mM IPTG. Test solutions (9 μL) were added to filter discs (6 mm, Becton Dickinson Company), and the discs were placed on the surface of the solidified plates. Plates were incubated for 12 h, and zones of inhibition were measured. When activity is greater on plates containing IPTG, it indicates the presence of a phosphonate antibiotic. Synthetic fosmidomycin (100 μM) was used as a positive control.