A MATE-family efflux pump rescues the Escherichia coli 8-oxoguanine-repair-deficient mutator phenotype and protects against H(2)O(2) killing

Abstract

Hypermutation may accelerate bacterial evolution in the short-term. In the long-term, however, hypermutators (cells with an increased rate of mutation) can be expected to be at a disadvantage due to the accumulation of deleterious mutations. Therefore, in theory, hypermutators are doomed to extinction unless they compensate the elevated mutational burden (deleterious load). Different mechanisms capable of restoring a low mutation rate to hypermutators have been proposed. By choosing an 8-oxoguanine-repair-deficient (GO-deficient) Escherichia coli strain as a hypermutator model, we investigated the existence of genes able to rescue the hypermutable phenotype by multicopy suppression. Using an in vivo-generated mini-MudII4042 genomic library and a mutator screen, we obtained chromosomal fragments that decrease the rate of mutation in a mutT-deficient strain. Analysis of a selected clone showed that the expression of NorM is responsible for the decreased mutation rate in 8-oxoguanine-repair-deficient (mutT, mutY, and mutM mutY) strains. NorM is a member of the multidrug and toxin extrusion (MATE) family of efflux pumps whose role in E. coli cell physiology remains unknown. Our results indicate that NorM may act as a GO-system backup decreasing AT to CG and GC to TA transversions. In addition, the ability of NorM to reduce the level of intracellular reactive oxygen species (ROS) in a GO-deficient strain and protect the cell from oxidative stress, including protein carbonylation, suggests that it can extrude specific molecules-byproducts of bacterial metabolism-that oxidize the guanine present in both DNA and nucleotide pools. Altogether, our results indicate that NorM protects the cell from specific ROS when the GO system cannot cope with the damage.

Figure 1. The papillation assay.

The strain GLF1 ΔmutT::Kan Mu cts produces red colonies (Ara⁻) on arabinose-tetrazolium chloride agar plates. Ara⁺ revertants are spontaneously produced by mutation and appear as white microcolonies growing out of the surface of the main red colonies. The Ara⁻→Ara⁺ reversion rate can be visualized by the number of white papillae appearing per colony on tetrazolium-arabinose plates incubated for 7 days. A: the mutator strain GLF1 ΔmutT::Kan Mu cts forms colonies with a high number of Ara⁺ papillae (white); B: the strain GLF1 ΔmutT::Kan Mu cts harboring the mini-Mu plasmid with the chromosomal fragment containing norM forms colonies with a low number of papillae.

Figure 2. ORFs (arrows) present in the isolated chromosomal fragment.

Figure 3. Viability after H₂O₂ treatment.

The data represent survival percentages after 30 min of 50 mM H₂O₂ treatment. Data for strains NR10831, NR10831ΔnorM, NR10831ΔmutT, and NR10831ΔsodB harboring either the empty vector pCA24N (black) or the plasmid expressing norM, pCNorM, (gray) are shown. Survival is represented as the percentage of cfu after H₂O₂ treatment relative to before treatment. The error bars indicate one standard error of the mean of four independent replicates.

Figure 4. ROS levels in E. coli wild-type and norM, mutT and sodB derivatives.

Representative histograms plotting the spontaneous fluorescence of 15,000 non-treated (A–D) and H₂O₂-treated (E–H) cells, revealed by DHR, as measured by flow cytometry. Cells containing either the empty vector or the norM-containing plasmid are represented as red or black lines, respectively. A and E: wild-type strain (non treated and H₂O₂-treated, respectively); B and F: norM-deficient strain; C and G: mutT-deficient strain; D and H: sodB-deficient strain.

Figure 5. Protein carbonylation.

Carbonylation is observed in the wild-type and mutT-derivative strains containing either the empty vector or the plasmid expressing norM, following treatment with 10mM H₂O₂ for 15 min. A: Bar graph quantifying the protein carbonylation (femtomoles of DNP) in cells containing the empty vector pCA24N (black bars) or the norM-plasmid pCNorM (gray bars) in the wild-type (left bars) and mutT strains (right bars). The data are the mean values from four separate experiments and error bars represent one standard error. B: Representative blot showing the accumulation of protein carbonyl groups in H₂O₂ challenged cells.