Polynucleotide phosphorylase protects Escherichia coli against oxidative stress

Abstract

Escherichia coli polynucleotide phosphorylase (PNPase) primarily functions in RNA degradation. It is an exoribonuclease and integral component of the multienzyme RNA degradosome complex [Carpousis et al. (1994) Cell 76, 889]. PNPase was previously shown to specifically bind a synthetic RNA containing the oxidative lesion 8-hydroxyguanine (8-oxoG) [Hayakawa et al. (2001) Biochemistry 40, 9977], suggesting a possible role in removing oxidatively damaged RNA. Here we show that PNPase binds to RNA molecules of natural sequence that were oxidatively damaged by treatment with hydrogen peroxide (H(2)O(2)) postsynthetically. PNPase bound oxidized RNA with higher affinity than untreated RNA of the same sequence, raising the possibility that it may act against a wide variety of lesions. The importance of such a protective role is illustrated by the observation that, under conditions known to cause oxidative damage to cytoplasmic components, PNPase-deficient cells are less viable than wild-type cells. Further, when challenged with H(2)O(2), PNPase-deficient cells accumulate 8-oxoG in cellular RNA to a greater extent than wild-type cells, suggesting that this RNase functions in minimizing oxidized RNA in vivo. Introducing the pnp gene encoding PNPase rescues defects in growth and RNA quality of the pnp mutant cells. Our results also suggest that protection against oxidative stress is an intrinsic function of PNPase because association with the RNA degradosome or with RNA helicase B (RhlB) is not required.

Figure 1

PNPase binds oxidized RNA with high affinity. Crude cell extracts were incubated with RNA beads. After washing, bead-bound proteins were eluted, separated by SDS–PAGE, and visualized with SYPRO Ruby. The S100 extracts from wild type (WT) and a mutant lacking PNPase (77.1 kDa) and RNase G (55.4 kDa) were used. The concentrations of H₂O₂ used for the treatment of RNA are shown on top of the lanes. Migration of protein size markers is shown on the left. Purified PNPase protein was run in lane 6.

Figure 2

Cell viability and RNA oxidation upon treatment with paraquat. (A) Exponentially grown cultures of wild-type and pnp mutant strains were normalized to OD_550nm = 0.002 and then serially diluted 1:5. The diluted cultures were inoculated on YT agar plates containing 0, 0.4, and 0.6 mM paraquat. (B) The level of 8-oxoG was determined in total RNA isolated from exponentially grown wild-type cultures after treatment with paraquat at the indicated concentration and time. (C) The level of 8-oxo-dG was determined in total DNA isolated from exponentially grown wild-type cultures after treatment with 1 mM paraquat at the indicated time.

Figure 3

PNPase-deficient cells are hypersensitive to H₂O₂ and PMS. (A) Sensitivity of wild-type (WT) and pnp mutant cells to H₂O₂. Cultures were grown, diluted, and inoculated as described in Figure 2A. (B) Sensitivity of wild-type and pnp mutant cells to phenazine methosulfate (PMS). (C) Sensitivity of wild-type and pnp mutant cells to H₂O₂ determined by cell density. Cultures were grown to exponential phase in liquid YT medium, treated with indicated concentrations of H₂O₂ for 90 min in a 96-well plate (n = 4), and incubated at 37 °C with shaking at 150 rpm. Cell density was measured in a plate reader at OD_550nm and is expressed as the percent of an untreated control. (D) Sensitivity to H₂O₂ determined by colony forming units (cfu). Exponentially growing cultures were challenged with the indicated concentrations of H₂O₂ for 90 min. The cultures were then diluted, plated on YT agar plates, and incubated overnight at 37 °C. The data are expressed as the percent of an untreated control.

Figure 4

Activity of PNPase under various treatment conditions. E. coli cultures were grown in YT medium. At OD_550nm = 0.5, the cultures of the wild-type strain were treated with various chemicals at concentrations shown in the legend. Cells were collected at the indicated time points. PNPase polymerization activity was measured by ADP incorporation into RNA as described in Materials and Methods using 18 μg of protein from cell extracts in a 50 μL reaction. The averages of two measurements and standard errors are shown.

Figure 5

H₂O₂-induced RNA damage is increased by PNPase deficiency, but H₂O₂ hydrolysis is not. Exponentially grown wild-type and pnp mutant cultures were treated with or without 1 mM H₂O₂. Total RNA was isolated by TRI Reagent in (A) and by phenol–chloroform extraction in (B) at the indicated time points. The level of 8-oxoG in RNA was determined by HPLC. Means of two measurements and standard errors are shown. (C) Wild-type and pnp mutant cultures were grown to OD_550nm = 0.5 in YT medium, and H₂O₂ was added to a final concentration of 0.5 mM. Samples were taken at the indicated time points and assayed for H₂O₂ concentration using the Amplex Red kit.

Figure 6

Introduction of pnp gene rescue response to H₂O₂ insult. (A) pKAK7 harboring the pnp gene (23) and the control plasmid pBR322 were introduced into the wild-type and pnp mutant cells, respectively. Midlog cultures containing 50 μg/mL ampicillin were serially diluted and inoculated on YT agar plates with or without 0.5 mM H₂O₂ to determine H₂O₂ sensitivity. (B) Total RNA was isolated 15 min after treatment with or without 0.5 mM H₂O₂, and the level of 8-oxoG in RNA was determined by HPLC. The mean of duplicate experiments and standard errors are shown. (C) Cultures at OD_550nm = 0.5 (0 min) were treated for 1 h with or without 1 mM H₂O₂. PNPase activity was measured by ADP incorporation into RNA using 6 μg of protein in each 50 μL reaction. The averages of two measurements and standard errors are shown.

Figure 7

Responses of strains defective in PNPase-associated proteins to H₂O₂ treatment. (A) Sensitivity of wild-type, rneΔ(844–1045), and their respective pnp derivative cells to H₂O₂. Exponentially grown cultures were serially diluted 1:3 and inoculated to YT plates with or without 0.5 mM H₂O₂. (B) Sensitivity of wild-type, rhlB, and their respective pnp derivative cells to H₂O₂. Exponentially grown cultures were serially diluted by 1:5 and inoculated to YT plates with or without 0.5 mM H₂O₂.