Mutations in the mutY gene of Escherichia coli enhance the frequency of targeted G:C → T:A transversions induced by a single 8-oxoguanine residue in single-stranded DNA

Oxidative damage to guanine in DNA results in the formation of 8-oxoguanine, which has been shown to induce G → T transversions targeted to this site. The mutagenicity of this lesion was studied in several mutator strains of Escherichia coli, using single-stranded DNA containing a single 8-oxoguanine residue. The frequencies of targeted G → T transversions increased markedly in mutY strains, while this mutagenic event was not affected in mutM or mutS strains. Introdution of a mutM mutation into a mutY strain caused a somewhat higher frequency of G → T transversions than that in the mutY strain and the effect of a mutS mutation was marginal. We conclude that the mutY gene plays a crucial role in preventing targeted G → T mutations derived from misreplication of the 8-oxoguanine-containing template DNA.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh⁸Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh⁸Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh⁸Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh⁸Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.     

UV-sensitivity and repair of UV-damages in Salmonella of wild type.

Summary The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E. coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4–5 times more sensitive than wild type E. coli and their inactivation curve is similar to that for E. coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E. coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.     

DNA repair and sequence context affect 1O2-induced mutagenesis in bacteria

Electronic excited molecular oxygen (singlet oxygen, ¹O₂) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by ¹O₂ in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a ¹O₂-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G→T and G→C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with ¹O₂-induced DNA damage are linked to the context of the damaged sequence.     

Helicobacter pylori genes involved in avoidance of mutations induced by 8-oxoguanine

Chromosomal rearrangements and base substitutions contribute to the large intraspecies genetic diversity of Helicobacter pylori. Here we explored the base excision repair pathway for the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), a ubiquitous form of oxidized guanine. In most organisms, 8-oxoG is removed by a specific DNA glycosylase (Fpg in bacteria or OGG1 in eukaryotes). In the case where replication of the lesion yields an A/8-oxoG base pair, a second DNA glycosylase (MutY) can excise the adenine and thus avoid the fixation of the mutation in the next round of replication. In a genetic screen for H. pylori genes complementing the hypermutator phenotype of an Escherichia coli fpg mutY strain, open reading frame HP0142, a putative MutY coding gene, was isolated. Besides its capacity to complement E. coli mutY strains, HP0142 expression resulted in a strong adenine DNA glycosylase activity in E. coli mutY extracts. Consistently, the purified protein also exhibited such an activity. Inactivation of HP0142 in H. pylori resulted in an increase in spontaneous mutation frequencies. An Mg-dependent AP (abasic site) endonuclease activity, potentially allowing the processing of the abasic site resulting from H. pylori MutY activity, was detected in H. pylori cell extracts. Disruption of HP1526, a putative xth homolog, confirmed that this gene is responsible for the AP endonuclease activity. The lack of evidence for an Fpg/OGG1 functional homolog is also discussed.     

Mosaic DNA Imports with Interspersions of Recipient Sequence after Natural Transformation of Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of half of the human population, causing gastritis, ulcers, and cancer. H. pylori is naturally competent for transformation by exogenous DNA, and recombination during mixed infections of one stomach with multiple H. pylori strains generates extensive allelic diversity. We developed an in vitro transformation protocol to study genomic imports after natural transformation of H. pylori. The mean length of imported fragments was dependent on the combination of donor and recipient strain and varied between 1294 bp and 3853 bp. In about 10% of recombinant clones, the imported fragments of donor DNA were interrupted by short interspersed sequences of the recipient (ISR) with a mean length of 82 bp. 18 candidate genes were inactivated in order to identify genes involved in the control of import length and generation of ISR. Inactivation of the antimutator glycosylase MutY increased the length of imports, but did not have a significant effect on ISR frequency. Overexpression of mutY strongly increased the frequency of ISR, indicating that MutY, while not indispensable for ISR formation, is part of at least one ISR-generating pathway. The formation of ISR in H. pylori increases allelic diversity, and contributes to the uniquely low linkage disequilibrium characteristic of this pathogen.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

Insertion Sequence-Driven Evolution of <Emphasis Type="Italic">Escherichia coli</Emphasis> in Chemostats

Insertion sequence (IS) elements are present in almost all bacterial genomes and are mobile enough to provide genomic tools to differentiate closely related isolates. They can be used to estimate genetic diversity and identify fitness-enhancing mutations during evolution experiments. Here, we determined the genomic distribution of eight IS elements in 120 genomes sampled from Escherichia coli populations that evolved in glucose- and phosphate-limited chemostats by comparison to the ancestral pattern. No significant differential transposition of the various IS types was detected across the environments. The phylogenies revealed substantial diversity amongst clones sampled from each chemostat, consistent with the phenotypic diversity within populations. Two IS-related changes were common to independent chemostats, suggesting parallel evolution. One of them corresponded to insertions of IS1 elements within rpoS encoding the master regulator of stress conditions. The other parallel event was an IS5-dependent deletion including mutY involved in DNA repair, thereby providing the molecular mechanism of generation of mutator clones in these evolving populations. These deletions occurred in different co-existing genotypes within single populations and were of various sizes. Moreover, differential locations of IS elements combined with their transpositional activity provided evolved clones with different phenotypic landscapes. Therefore, IS elements strongly influenced the evolutionary processes in continuous E. coli cultures by providing a way to modify both the global regulatory network and the mutation rates of evolving cells.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB

Background and Aims: Fluoroquinolone‐containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone‐resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin‐resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin‐resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β-lactamase fusion proteins (BlaM⁺) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.     

Characterization of the DNA Adenine 5′-GATC-3′ Methylase <Emphasis Type="Italic">Hpy</Emphasis>IIIM from <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

The effect of inactivation of the 5-GATC-3 methylase HpyIIIM in Helicobacter pylori (H. pylori) on mismatch repair, adherence, and in vitro fitness was examined. Chromosomal DNA from 90 H. pylori strains was isolated, and restriction enzyme digestion indicated all strains examined possess HpyIIIM. Wild-type H. pylori and a strain with an inactive HpyIIIM were found to have rifampicin mutation frequencies of 2.93 × 10^–7 and 1.05 × 10^–7 (p > 0.05), respectively, indicating that HpyIIIM does not appear to be important in mismatch repair. Adherence of H. pylori in an in vitro model cell system was also unaffected by inactivation of HpyIIIM. Inactivation of HpyIIIM did not result in a decrease in fitness, as determined by liquid in vitro competition experiments.     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.     

Escherichia coli Nth and human hNTH1 DNA glycosylases are involved in removal of 8-oxoguanine from 8-oxoguanine/guanine mispairs in DNA

The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C→C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.     

Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells, with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.     

Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli

Summary A fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508, dnaA167, (dnaA5, dnaA46, dnaA211), dnaA205, dnaA204, dnaA203. The mutations dnaA601, dnaA602, dnaA603, dnaA604 and dnaA606 were found to map very close to each other and close to dnaA205 in the middle third of the dnaA gene. In analysing the dominance relationship all 13 dnaA mutations were found to be recessive to the wild type. Characteristic phenotypes of the dnaA(Ts) mutants, like reversibility of the temperature inactivation of the dnaA protein, cold sensivity of haploid or of merodiploid strains and suppressibility by rpoB mutations, are found to correlate with clusters of mutations within the gene.     

The CrdRS (HP1365-HP1364) Two-Component System Is Not Involved in pH-Responsive Gene Regulation in the <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and G27

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease. However, urease-independent mechanisms are likely to contribute to acid adaptation. pH-responsive gene regulation in this organism is mediated by a two-component system (HP0166-HP0165) designated ArsRS and the metal-dependent regulators NikR and Fur. Recently, it was reported that another two-component system termed CrdRS (HP1365-HP1364) is required for pH-responsive regulation of the major acid-resistance systems in the H. pylori strain J99. By the analysis of crdRS null mutants of the H. pylori strains 26695 and G27, we show that low pH induction of both the urease and the amidase genes occurs in the absence of crdRS in these strains, suggesting substantial strain-specific differences in the regulation of a major virulence determinant.     

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H₂O₂, and the rpoB mutant frequencies were calculated. The frequency of the H₂O₂-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.