Experimental setup and validation of the microarray data. (A) Schematic illustration of the experimental layout used in this microarray study. cDNAs from the WT (MG1655), ppGpp0 (CF1693), ΔdksA (AAG95), and ppGpp0 ΔdksA (AAG98) strains, labeled with either Cy3 or Cy5, were hybridized to microarray slides in the different combinations shown. The origins of the arrows show the Cy3-labeled samples, whereas the destinations show the Cy5-labeled samples. The number on the arrow shows the number of replicates for each combination. (B) Statistical MA plots of the normalized raw data obtained from each WT/mutant comparison. M represents the fold change in gene expression between WT and mutant (log2 values), and A represents the average spot intensity. (C) RT-PCR analysis of transcription of 10 selected genes from the WT (MG1655), ppGpp0 (CF1693), ΔdksA (AAG95), and ppGpp0 ΔdksA (AAG98) strains. The amounts of total RNA used as the template were 10 ng for tnaA; 20 ng for hns, ompT, and uspA; 40 ng for fadL; 200 ng for flu, flhD, fliC, and fimA; and 400 ng for fliA. hns was included as a control to confirm that equivalent amounts of template were used. Samples from bacterial cultures grown in LB medium to an OD600 of 1.5 at 37°C were used for all the studies shown.