The active site and S1 domain of PNPase are both important for sRNA stabilization and target regulation. (A) Analysis of RyhB. A strain harboring a PBAD::sodB′-′lacZ fusion (WT, NRD537) or derived strains: Δpnp (NRD1114), PNPase ΔKHΔS1 (NRD1116), PNPase ΔS1 (NRD1151), or PNPase S437-9A (NRD1117) were grown to exponential phase. RyhB expression was then induced from the chromosome by the addition of dipyridyl. Rifampicin was added after 15 min. (B) Analysis of CyaR. Transcription of the sRNA was induced from a lac-based promoter on a plasmid using a ΔcyaR strain carrying PBAD::ompX′-′lacZ (WT, NRD377), or derived mutants (Δpnp, NRD677; ΔKH ΔS1, NRD1044; ΔS1, NRD1147; S437-9A, NRD1045). (C) Analysis of GcvB. The sRNA was expressed from its native chromosomal locus in the strains from A. Northern blots were performed in duplicate and quantified with standard error of mean (SEM) indicated for RyhB (A), CyaR (B), or GcvB (C). Strains from A and B harboring an empty vector or a plasmid expressing the negatively regulating sRNAs, RyhB (D), or CyaR (E), respectively, were used in a β-galactosidase assay. (F) β-Galactosidase assays were performed on a wild-type or ΔgcvB strain harboring a PBAD::dppA′-′lacZ fusion or derived strains (WT, NRD1382; Δpnp, NRD1383; ΔKH ΔS1, NRD1384; S437-9A, NRD1385; ΔgcvB, NRD1388; Δpnp ΔgcvB, NRD1389; ΔKH ΔS1 ΔgcvB, NRD1390; S437-9A ΔgcvB, NRD1391). The percentage of β-galactosidase activity relative to the strain lacking expression of the given sRNA is indicated above each bar.
