FIG. 1.

Mapping of the iraD TSSs and correlation of iraD expression with growth phase and RpoS levels. (A) Agarose gel of 5′ RACE PCR products generated from MG1655 cells in either logarithmic or stationary phase as indicated. Sequencing of the PCR products revealed two products of 794 and 514 bp, predicting transcript lengths of ∼800 and 500 nt, respectively. Neither transcript is present in ΔiraD cells (data not shown). (B) Map of the two TSSs identified in the iraD 5′ upstream region based on the sequencing data after the 5′ RACE shown in panel A. TSSs are indicated with their positions relative to the 5′ end of the iraD ORF, and putative −10 and −35 elements are indicated for each. The distal promoter is labeled as P1, and the proximal promoter is labeled as P2. (C) iraD::luxCDABE expression for the full-length promoter, the distal promoter P1, and the proximal promoter P2 throughout growth. Full-length reporter is a fusion of positions −600 to −1, the P1 reporter is a fusion of positions −600 to −375, and the P2 reporter is a fusion of positions −262 to −1 to luciferase (numbers are relative to the start of ORF-ATG). Each data point is an average of six independent determinations. The variability is shown with error bars in both graphs. RLU, relative luminescence units (bioluminescence counts per minute, normalized to the OD₆₀₀). The right panel shows the growth curve of wild-type cells in the experimental conditions used in the present study. The ODs are shown for time points of 20 min, starting at 90 min after the inoculation of each culture. (D) Steady-state RpoS levels in MG1655 and ΔiraD strains throughout growth. Samples were taken at ODs indicated, and TCA-precipitated as described in the methods section.