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

Guelfo JR, Rodríguez-Rojas A, Matic I, Blázquez J - PLoS Genet. (2010)

Bottom Line: 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.Our results indicate that NorM may act as a GO-system backup decreasing AT to CG and GC to TA transversions.Altogether, our results indicate that NorM protects the cell from specific ROS when the GO system cannot cope with the damage.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

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.

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Related in: MedlinePlus

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.
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pgen-1000931-g005: 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.

Mentions: One of the most important effects of an increased intracellular ROS level is protein carbonylation. Thus, we studied the effect of norM expression on protein carbonylation in H2O2-treated and non-treated wild-type and ΔmutT cells. The level of spontaneous protein carbonylation in both the wild-type and mutT non-treated cells growing in the exponential phase was undetectable with the OxyBlot kit. Nevertheless, when submitted to H2O2 pre-treatment, the expression of norM in the multicopy plasmid pCNorM produced a consistent decrease in the amount of carbonylated proteins in both the wild-type NR10831 and the ΔmutT strains (Figure 5A and 5B).


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

Guelfo JR, Rodríguez-Rojas A, Matic I, Blázquez J - PLoS Genet. (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865507&req=5

pgen-1000931-g005: 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.
Mentions: One of the most important effects of an increased intracellular ROS level is protein carbonylation. Thus, we studied the effect of norM expression on protein carbonylation in H2O2-treated and non-treated wild-type and ΔmutT cells. The level of spontaneous protein carbonylation in both the wild-type and mutT non-treated cells growing in the exponential phase was undetectable with the OxyBlot kit. Nevertheless, when submitted to H2O2 pre-treatment, the expression of norM in the multicopy plasmid pCNorM produced a consistent decrease in the amount of carbonylated proteins in both the wild-type NR10831 and the ΔmutT strains (Figure 5A and 5B).

Bottom Line: 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.Our results indicate that NorM may act as a GO-system backup decreasing AT to CG and GC to TA transversions.Altogether, our results indicate that NorM protects the cell from specific ROS when the GO system cannot cope with the damage.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

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.

Show MeSH
Related in: MedlinePlus