Northern blot analysis of RyhB, SgrS, and CyaR turnover in pnp mutants. (A,B) Overnight cultures of the wild-type strain DJ624 (circles), the hfq deletion mutant NRD459 (squares), the pnp deletion mutant NRD473 (upward pointing triangles), the pnp point mutant NRD494 (downward pointing triangles), or the Δpnp Δhfq double mutant NRD535 (diamonds) were diluted 200-fold into fresh LB. (C) Overnight cultures of a cyaR deletion strain (NRD531; circles), or isogenic derivatives carrying an hfq deletion (NRD532; squares), a pnp deletion (NRD533; upward pointing triangles), or a pnp point mutation (NRD534; downward pointing triangles), all harboring a plasmid expressing CyaR from a lac promoter (pNRD405), were diluted 200-fold in fresh LB containing ampicillin. All strains were grown at 37°C to an OD600 between 0.3 and 0.4, and dipyridyl (A), α-MG (B), or IPTG (C) was then added to each culture to induce sRNA synthesis. A sample was taken from each culture 15 min after induction. Sixteen minutes after induction, rifampicin was added to each culture, and additional samples were taken at the indicated times. RNA was extracted, fractionated on a polyacrylamide gel, and transferred to a nylon membrane. The Northern blot was developed using the 5′-biotinylated RyhB probe (A), SgrS probe (B), or CyaR probe (C) as described in Materials and Methods. The intensity of the bands in the Northern blots was quantified using the Multi Gauge software. The band intensity for the 0-min sample was set to 100%, and other samples were normalized to the 0-min sample. The results represent the mean of two experiments, and the standard deviation is indicated by the light gray bars.
