Effect of pnp mutations on Hfq and effects of Hfq overexpression in pnp mutants. (A) Overnight cultures of the wild-type strain DJ624 or derivatives harboring an hfq deletion (NRD459), pnp deletion (NRD473) or pnp allele encoding a PNPase with an R93C (NRD453), S437F (NRD457), G489D (NRD493), G436D (NRD494), or R315C substitution (NRD495) were diluted 200-fold in fresh LB medium. Cells were grown at 37°C to an OD600 of ∼1.0, a sample was taken from each culture, and the protein was TCA-precipitated. An equal amount of protein based on the cell density of each culture at the time of sampling was then processed as in Figure 1. (B,C) An overnight culture of the wild-type strain DJ624 (WT; circles), hfq deletion strain NRD459 (Δhfq; squares), or pnp deletion strain NRD473 (Δpnp; triangles) harboring pBAD30 (pBAD; open symbols) or a plasmid expressing hfq from an arabinose inducible promoter (pNRD414; phfq; solid symbols) was diluted 200-fold in fresh LB medium containing ampicillin. Each culture was grown to an OD600 of 0.3–0.4, and arabinose was added. After 5 min of induction, a sample was taken from each culture (0–min samples), and then dipyridyl added. Additional samples were taken as indicated. Sixteen minutes after dipyridyl addition, rifampicin was added to the cultures and samples taken 2, 5, 10, and 20 min after rifampicin treatment. The RNA was extracted and processed as described in Figure 4, using the 5′-biotinylated RyhB, sodB, and ssrA probe. The results in B represent the mean of two experiments, and the standard deviation is indicated by the gray bars. The blots in A and C are representative of two experiments.
