FIG 2.

Expression of CcsR1–4 is RSP_6037 and Hfq dependent, and elevated levels of the four sRNAs lead to enhanced resistance to oxidative stress. (A) Schematic representation of plasmids used for constitutive overexpression of the CcsR RNAs under the control of the 16S rRNA promoter. The broad-host-range plasmid pRK415 was used as an empty-vector control. (B) The level of CcsR1 expression is decreased in an hfq deletion strain and is negatively influenced by co-overexpression with RSP_6037, as monitored by Northern blotting of total RNA from cultures in the exponential growth phase. Results for wild-type (WT) R. sphaeroides and an hfq deletion strain, each harboring plasmid pRK415, pRCcsR1–4, or pR6037_CcsR1–4, are shown. 5S rRNA served as a loading control. For detection of CcsR1, p-0680a was used. (C and D) Enhanced resistance to oxidative stress caused by paraquat or the organic hydroperoxide tBOOH can be correlated with elevated levels of CcsR RNAs, as demonstrated by zone-of-inhibition assays. Filter disks soaked with either 200 mM paraquat or 700 mM tBOOH were placed on agar plates to suppress bacterial growth. R. sphaeroides wild-type strains carrying plasmid pRK415 or CcsR overexpression plasmids were compared. After 3 days of incubation, the diameters of the zones of inhibition were measured. Results represent the mean and error bars indicate the standard deviation for three independent biological replicates. Asterisks indicate a significant difference in the average zone of inhibition diameter.