Overexpression of Rae1 mitigates the induction of S1025 and bmrX expression in response to chloramphenicol. (A) Northern blot analysis of total RNA upon addition of subinhibitory concentrations of Cm to exponentially growing cultures at 0.5 µg/mL and 2.5 µg/mL. Chloramphenicol was added for the times indicated. (B) Northern blot analysis of total RNA isolated from a Δrae1 strain containing either an empty vector (pDG) or a plasmid expressing the WT protein Rae1 (pRae1) at mid-log phase in rich medium supplemented, or not, with 0.5 µg/mL chloramphenicol. Quantifications of the impact of Rae1 overexpression on mRNA levels from two independent experiments are indicated. (A,B) Northern blots were probed in a sequential order: oligo CC2344 (bmrC), oligo CC1589 (yrzI), and then with a probe complementary to 16S rRNA (CC058) as a loading control. For yrzI, the correspondence between bands and transcripts is given to the right of the blot: the P1-T4 and the P3-T4 correspond to primary transcripts from the P1 and P3 promoters to the main operon terminator (T4). R-T4 refers to the 521-nt species extending from 21 nt upstream of the yrzI coding sequence to the T4 terminator.
