RESUMO
8-oxo-7,8-dihydroguanine, commonly referred to as 8-oxoG, is considered one of the most predominant oxidative lesions formed in DNA. Due to its ability to pair with adenines in its syn configuration, this lesion has a strong mutagenic potential in both eukaryotes and prokaryotes. Escherichia coli cells are endowed with the GO system, which protects them from the mutagenic properties of this lesion when formed both in cellular DNA and the nucleotide pool. MutY and MutM (Fpg) DNA glycosylases are crucial components of the GO system. A strong mutator phenotype of the Escherichia coli mutM mutY double mutant underscores the importance of 8-oxoG repair for genomic stability. Here, we report that in Caulobacter crescentus, a widely studied alpha-proteobacterium with a GC-rich genome, the combined lack of MutM and MutY glycosylases produces a more modest mutator phenotype when compared to E. coli. Genetic analysis indicates that other glycosylases and other repair pathways do not act synergistically with the GO system for spontaneous mutation prevention. We also show that there is not a statistically significant difference in the spontaneous levels 8-oxodGuo in E. coli and C. crescentus, suggesting that other yet to be identified differences in repair or replication probably account for the differential importance of the GO system between these two species. Environ. Mol. Mutagen. 61:246-255, 2020. © 2019 Wiley Periodicals, Inc.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , DNA Glicosilases/genética , DNA-Formamidopirimidina Glicosilase/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Mutagênese , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , DNA Glicosilases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Instabilidade Genômica , Guanina/análogos & derivados , Guanina/metabolismoRESUMO
In the present study, we screened a collection of 77 Proteus mirabilis clinical isolates for the presence of mutators, using the frequency of both rifampicin and fosfomycin resistance mutants as markers of spontaneous mutagenesis. We found that none of the strains in our collection are mutators for the rifampicin resistance (RifR) marker. Nevertheless, a significant fraction of the isolates (17%) show high frequencies of fosfomycin resistant mutants (FosR). We show that this increased mutability to FosR correlates with a low level of resistance to Fosfomycin (MICs 8-64µg/ml). These strains also show high frequencies of single step mutants with clinically relevant FosR resistance levels (MIC ≥256µg/ml). Our findings point out to the risk of fosfomycin resistance emergence in P. mirabilis.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Fosfomicina/farmacologia , Mutação , Infecções por Proteus/microbiologia , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/genética , Antibacterianos/uso terapêutico , Fosfomicina/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Taxa de Mutação , Infecções por Proteus/tratamento farmacológicoRESUMO
imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli.
Assuntos
Caulobacter crescentus/metabolismo , Dano ao DNA , Replicação do DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Mutagênese , Resposta SOS em Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genéticaRESUMO
Mutator strains were identified by screening random Tn5 insertion clones of Caulobacter crescentus. We identified clones with robust increases in mutation rates with Tn5 insertions in the mutY, mutS, mutL and uvrD genes, known to act in mutation-preventing pathways in Escherichia coli. Analysis of mutations in the rpoB gene revealed that in both the parental strain and mismatch repair-deficient mutants, A:TâG:C transitions predominate by a large margin over C:GâT:A. We have also investigated the role of the error-prone polymerase encoded by imuC (dnaE2) in spontaneous mutagenesis, and found that a imuC mutant strain shows mutation rates and sequences comparable to the parental strain. Our study characterizes for the first time mutator strains in a member of the alphaproteobacteria group. In spite of the limitations of using a single marker, possible reasons for the observed mutational bias are discussed in the light of the repertoire of DNA repair genes in this bacterium.
Assuntos
Caulobacter crescentus/genética , Reparo de Erro de Pareamento de DNA , Mutagênese , DNA Helicases/genética , Proteínas MutL/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genéticaRESUMO
The SOS response is a universal bacterial regulon involved in the cellular response to DNA damage and other forms of stress. In Caulobacter crescentus, previous work has identified a plethora of genes that are part of the SOS regulon, but the biological roles of several of them remain to be determined. In this study, we report that two genes, hereafter named mmcA and mmcB, are involved in the defense against DNA damage caused by mitomycin C (MMC), but not against lesions induced by other common DNA damaging agents, such as UVC light, methyl methanesulfonate (MMS) and hydrogen peroxide. mmcA is a conserved gene that encodes a member of the glyoxalases/dioxygenases protein family, and acts independently of known DNA repair pathways. On the other hand, epistasis analysis showed that mmcB acts in the same pathway as imuC (dnaE2), and is required specifically for MMC-induced mutagenesis, but not for that induced by UV light, suggesting a role for MmcB in translesion synthesis-dependent repair of MMC damage. We show that the lack of MMC-induced mutability in the mmcB strain is not caused by lack of proper SOS induction of the imuABC operon, involved in translesion synthesis (TLS) in C. crescentus. Based on this data and on structural analysis of a close homolog, we propose that MmcB is an endonuclease which creates substrates for ImuABC-mediated TLS patches.