Primer extension analyses of total RNA from VPD54 or VPD102 to determine the transcription start sites of peuR (A) and peuA (B and C).

Total RNAs were isolated from VPD54 (vctA- and irgA-deficient mutant derived from VPD5) and VPD102 (peuRS-deficient mutant derived from VPD54) grown at pH 7.0 and 8.0 in LB-Tris, LB-Tris/+EDDA, and LB-Tris/+EDDA/+Ent media. The amounts of total RNA and primers used for reverse transcription were as follows: (A) 10 µg VppeuR-PE, (B) 10 µg VppeuA-PE, and (C) 150 µg VppeuA-PE. The same primers used for primer extension analysis were used to generate the sequence ladders (A, C, G, T). The transcription start sites and putative Fur boxes are indicated at the top of panels A and B (also see Figure 3A).