Effects of increased and decreased GadY expression on gadX mRNA levels. (A) Map of overlap between GadY small RNA and gadX 3′ UTR. (B) gadX mRNA levels in cells overexpressing GadY. Total RNAs (5 μg each) from wild-type MG1655, MG1655/pRI-GadY, MG1655/pRI, and MG1655 ΔgadX::kan/pRI-GadY(105) grown in LB medium for 4 h were separated, transferred, and probed as described in the legend to Fig. 4B, except that the gadX mRNA was detected with an oligonucleotide (GadX-A1) that was complementary to gadX. (C) GadY RNA levels in gadY −10 promoter mutant strain. Total RNAs (5 μg each) from MG1655 and the gadY −10 mutant strain (GSO109) grown in LB medium for 6 h were separated, transferred, and probed as described in the legend to Fig. 1B. (D) gadX mRNA levels in cells with decreased GadY expression. Total RNAs (5 μg each) from MG1655 and the gadY −10 mutant grown in LB medium for 6 h were separated, transferred, and probed as described in the legend to Fig. 4B. The samples were run alongside Millenium RNA size markers, which were probed separately. The gadX mRNA migrated at the expected size of approximately 1 kb. The Northern blot in panel D was overexposed compared to that in panel B to allow detection of the gadX transcript from MG1655. The nonspecific hybridization to the 16S rRNA detected with the long exposure of panel D served as a loading control.