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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

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.
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pgen-1000931-g001: 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.

Mentions: As stated in the Materials and Methods section, the mini-Mu system produces a plasmid overexpression library of chromosomal fragments by homologous recombination between two adjacent mini-Mu transposons. We screened for GO− strains with a reduced mutation rate using the colony papillation screen described in figure 1. The screen of the mini-Mu library (about 1,500 clones) yielded several colonies with a clearly reduced number of white Ara+ papillae on agar plates containing arabinose and tetrazolium chloride. One of them, showing a papillation pattern similar to the wild-type strain was chosen for further study (Figure 1). Plasmid DNA was purified, retransformed into the original ΔmutT strain and retested with the Ara−→Ara+ reversion assay. The ends of the fragment contained in the mini-Mu plasmid were sequenced and the chromosomal region between them inferred. This region included 16 genes, 8 with assigned functions (gloA, rnt, lhr, sodB, purR, cfa, ribC and norM) (Figure 2). In principle, we considered sodB, which encodes the Fe-dependent superoxide dismutase, and norM, which encodes an orthologue of the MATE family [21]–[23], as major candidates responsible for the decreased mutation rate in the mutT-deficient background. Overexpression of sodB may reduce the level of reactive oxygen species (ROS) in the cell, leading to a reduced level of 8-oxo-dG, which consequently compensates for the absence of MutT activity. On the other hand, NorM is the prototype of the MATE family of cation-coupled transporters, which characteristically possess 12 putative transmembrane domains and have been reported in all three kingdoms of life [21]. Expression of NorM conferred resistance to several agents, such as norfloxacin, aminoglycosides and ethidium bromide, via a mechanism requiring the proton motive force [24]. Interestingly, MATE proteins have been described as exporters of toxic organic cations and guanidine [25], rendering NorM an excellent candidate for the export of oxidative precursor molecules.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000931-g001: 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.
Mentions: As stated in the Materials and Methods section, the mini-Mu system produces a plasmid overexpression library of chromosomal fragments by homologous recombination between two adjacent mini-Mu transposons. We screened for GO− strains with a reduced mutation rate using the colony papillation screen described in figure 1. The screen of the mini-Mu library (about 1,500 clones) yielded several colonies with a clearly reduced number of white Ara+ papillae on agar plates containing arabinose and tetrazolium chloride. One of them, showing a papillation pattern similar to the wild-type strain was chosen for further study (Figure 1). Plasmid DNA was purified, retransformed into the original ΔmutT strain and retested with the Ara−→Ara+ reversion assay. The ends of the fragment contained in the mini-Mu plasmid were sequenced and the chromosomal region between them inferred. This region included 16 genes, 8 with assigned functions (gloA, rnt, lhr, sodB, purR, cfa, ribC and norM) (Figure 2). In principle, we considered sodB, which encodes the Fe-dependent superoxide dismutase, and norM, which encodes an orthologue of the MATE family [21]–[23], as major candidates responsible for the decreased mutation rate in the mutT-deficient background. Overexpression of sodB may reduce the level of reactive oxygen species (ROS) in the cell, leading to a reduced level of 8-oxo-dG, which consequently compensates for the absence of MutT activity. On the other hand, NorM is the prototype of the MATE family of cation-coupled transporters, which characteristically possess 12 putative transmembrane domains and have been reported in all three kingdoms of life [21]. Expression of NorM conferred resistance to several agents, such as norfloxacin, aminoglycosides and ethidium bromide, via a mechanism requiring the proton motive force [24]. Interestingly, MATE proteins have been described as exporters of toxic organic cations and guanidine [25], rendering NorM an excellent candidate for the export of oxidative precursor molecules.

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