FIG 4.

Identification of small noncoding RNAs that alter T3SS gene expression. (A) P. aeruginosa strain PAK carrying a P_exsD-lacZ transcriptional reporter was transformed with a vector control and the sRNA expression vectors indicated in Table S1 (note that only 237 of the 512 putative sRNAs were tested). Strains were cultured under inducing (+ EGTA) conditions for T3SS gene expression in medium supplemented with 0.2% arabinose to induce sRNA expression and assayed for P_exsD-lacZ reporter activity. The reporter values represent the activity of each sRNA relative to the vector control. (B) sRNAs that inhibited or stimulated P_exsD-lacZ reporter activity greater than 2-fold (indicated by red lines in panel A) were selected for further analyses. Expression vectors for sRNA 18, 179, 182, 183, 214, 351, and 483 were introduced into strains PA14 and PA103 carrying the P_exsD-lacZ reporter and assayed as described in panel A for reporter activity. sRNA 0161, previously shown to inhibit T3SS gene expression (32), and the Hfq expression vector were included as controls. The reported values represent the activity of each sRNA or pHfq relative to each parental strain carrying the vector control. The reported values with the standard error represent the average of at least three experiments. *, P < 0.01.