FIG 2.

Effect of the amino acid substitutions on PrfA activity. (A) Transposon-free strains expressing PrfA proteins with the wild-type sequence or indicated amino acid substitutions were tested for growth in DM supplemented with 0.2% G-6-P for 72 h. A representative of five independent experiments is shown. (B) Fosfomycin resistance of the indicated strains. Fosfomycin discs were used, and the clearing zone was measured. The radius of the clearing zone is indicated relative to the wild type as an average of three independent experiments. Statistical analysis was used to compare fosfomycin sensitivity of the PrfA* strain with the that of the wild type and the PrfA* and Δhpt strains (Student’s t test [two tailed; *, P < 0.05; **, P < 0.01; ***, P < 0.001]). (C) (Top) Expression of hpt in the indicated strains grown in BHI until the OD₆₀₀ reached 1. RNA was isolated and hpt expression was examined with Northern blotting using radiolabeled probes against hpt and tmRNA (control). A representative of three independent experiments is shown. (Bottom) Quantification of hpt expression from the top panel. Expression is relative to the WT (set to 1). Student’s t test (two tailed; *, P < 0.05; **, P < 0.01; ***, P < 0.001).