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Slade et al. (2009) describe in this issue how the genome of the bacterium Deinococcus radiodurans gets reassembled after being shattered by high-dose radiation. In contrast to the extreme nature of the damage, the steps of repair appear surprisingly ordinary. So, why can't all organisms carry out extreme genome repair?
Assuntos
Reparo do DNA , Deinococcus/genética , Deinococcus/efeitos da radiação , Dano ao DNA , Genoma BacterianoRESUMO
The term "SOS response" was first coined by Radman in 1974, in an intellectual effort to put together the data suggestive of a concerted gene expression program in cells undergoing DNA damage. A large amount of information about this cellular response has been collected over the following decades. In this review, we will focus on a few of the relevant aspects about the SOS response: its mechanism of control and the stressors which activate it, the diversity of regulated genes in different species, its role in mutagenesis and evolution including the development of antimicrobial resistance, and its relationship with mobile genetic elements.
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Spontaneous mutations are important players in evolution. Nevertheless, there is a paucity of information about the mutagenic processes operating in most bacterial species. In this work, we implemented two forward mutational markers for studies in Caulobacter crescentus. We confirmed previous results in which A:T â G:C transitions are the most prevalent type of spontaneous base substitutions in this organism, although there is considerable deviation from this trend in one of the loci analyzed. We also investigated the role of dinB and imuC, encoding error-prone DNA polymerases, in spontaneous mutagenesis in this GC-rich organism. Both dinB and imuC mutant strains show comparable mutation rates to the parental strain. Nevertheless, both strains show differences in the base substitution patterns, and the dinB mutant strain shows a striking reduction in the number of spontaneous -1 deletions and an increase in C:G â T:A transitions in both assays.
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BACKGROUND: Fluoroquinolones such as ciprofloxacin induce the mutagenic SOS response and increase the levels of intracellular reactive oxygen species (ROS). Both the SOS response and ROS increase bacterial mutagenesis, fuelling the emergence of resistant mutants during antibiotic treatment. Recently, there has been growing interest in developing new drugs able to diminish the mutagenic effect of antibiotics by modulating ROS production and the SOS response. OBJECTIVES: To test whether physiological concentrations of N-acetylcysteine, a clinically safe antioxidant drug currently used in human therapy, is able to reduce ROS production, SOS induction and mutagenesis in ciprofloxacin-treated bacteria without affecting antibiotic activity. METHODS: The Escherichia coli strain IBDS1 and its isogenic mutant deprived of SOS mutagenesis (TLS-) were treated with different concentrations of ciprofloxacin, N-acetylcysteine or both drugs in combination. Relevant parameters such as MICs, growth rates, ROS production, SOS induction, filamentation and antibiotic-induced mutation rates were evaluated. RESULTS: Treatment with N-acetylcysteine reduced intracellular ROS levels (by â¼40%), as well as SOS induction (by up to 75%) and bacterial filamentation caused by subinhibitory concentrations of ciprofloxacin, without affecting ciprofloxacin antibacterial activity. Remarkably, N-acetylcysteine completely abolished SOS-mediated mutagenesis. CONCLUSIONS: Collectively, our data strongly support the notion that ROS are a key factor in antibiotic-induced SOS mutagenesis and open the possibility of using N-acetylcysteine in combination with antibiotic therapy to hinder the development of antibiotic resistance.
Assuntos
Acetilcisteína/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Mutagênese/efeitos dos fármacos , Resposta SOS em Genética/efeitos dos fármacos , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Taxa de Mutação , Espécies Reativas de Oxigênio/análiseRESUMO
Resistance to antibiotics is a global health problem. Activation of the SOS response, and the subsequent elevation in mutagenesis, contributes to the appearance of resistance mutations. Among currently used drugs, quinolones are the most potent inducers of the SOS response. In the present study, we show that amikacin inhibits ciprofloxacin-mediated SOS induction and mutagenesis in Pseudomonas aeruginosa.
Assuntos
Amicacina/farmacologia , Ciprofloxacina/farmacologia , Mutação , Pseudomonas aeruginosa/efeitos dos fármacos , Recombinases Rec A/genética , Antibacterianos/farmacologia , Ciprofloxacina/antagonistas & inibidores , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Expressão Gênica , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Mutagênese/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Recombinases Rec A/metabolismo , Resposta SOS em Genética/efeitos dos fármacosRESUMO
The ultraviolet (UV) component of sunlight can damage DNA. Although most solar UV is absorbed by the ozone layer, wavelengths > 300 nm (UVA and UVB bands) can reach the Earth's surface. It is essential to understand the genotoxic effects of UV light, particularly in natural environments. Caulobacter crescentus, a bacterium widely employed as a model for cell cycle studies, was selected for this study. Strains proficient and deficient in DNA repair (uvrA-) were used to concurrently investigate three genotoxic endpoints: cytotoxicity, SOS induction, and gene mutation, using colony-formation, the SOS chromotest, and RifR mutagenesis, respectively. Our findings underscore the distinct impacts of individual UV bands and the full spectrum of sunlight itself in C. crescentus. UVC light was highly genotoxic, especially for the repair-deficient strain. A UVB dose equivalent to 20 min sunlight exposure also affected the cells. UVA exposure caused a significant response only at high doses, likely due to activation of photorepair. Exposure to solar irradiation resulted in reduced levels of SOS induction, possibly due to decreased cell survival. However, mutagenicity is increased, particularly in uvrA- deficient cells.
Assuntos
Caulobacter crescentus , Raios Ultravioleta , Raios Ultravioleta/efeitos adversos , Caulobacter crescentus/genética , Dano ao DNA , Reparo do DNA , MutaçãoRESUMO
UV-induced mutagenesis is, to greater extent, a phenomenon dependent on translesion synthesis (TLS) and regulated by the SOS response in bacteria. Caulobacter crescentus, like many bacterial species, employs the ImuABC (ImuAB DnaE2) pathway in TLS. To have a better understanding of the characteristics of UV-induced mutagenesis in this organism, we performed a whole genome analysis of mutations present in survivors after an acute UVC exposure (300 J/m2). We found an average of 3.2 mutations/genome in irradiated samples, distributed in a mutational spectrum consisting exclusively of base substitutions, including tandem mutations. Although limited in conclusions by the small number of mutations identified, our study points to the feasibility of using whole-genome sequencing to study mutagenesis occurring in experiments involving a single acute exposure to genotoxic agents.
Assuntos
Caulobacter crescentus , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Proteínas de Bactérias/genética , Mutagênese , Dano ao DNA/genética , Reparo do DNA/genéticaRESUMO
Proteus mirabilis is one of the Enterobacteriales species that has been deemed as non-mutable by DNA damaging agents. A genomic analysis of P. mirabilis genomes shows that this species often does not carry pol V-encoding genes in its chromosome, which are responsible for SOS mutagenesis in other bacteria. On the other hand, the highly active umuDC homologs rumAB are carried in the mobile integrative conjugative elements (ICEs) from the SXT/R391 family that are frequently found in this species. Here we show that isolates devoid of SXT/R391 are indeed weakly or non-mutable by UV-light exposure, in contrast to isolates carrying SXT/R391 elements, some of which are mutable under these conditions. SXT/R391-bearing isolates display a variable behavior regarding UV-induced mutagenesis, despite the functionality of rumAB homologs carried by them. We also show that the globally dispersed ICEPmiJpn1 confers UV mutability to otherwise non-mutable isolates and demonstrate that this phenomenon is dependent on rumAB genes. Finally, we investigated whether rumAB genes could play a role in the conjugation of ICEPmiJpn1 and found that these genes improve the conjugation of SXT/R391 by a small margin. Taken together, these results show that the presence of rumAB in SXT/R391 ICEs endows the hosts with damage-inducible mutagenesis ability and promotes a small but significant enhancement in element transfer after exposure to UV light.
Assuntos
Conjugação Genética , Proteus mirabilis , Elementos de DNA Transponíveis , Proteus mirabilis/genética , Raios UltravioletaRESUMO
Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by 'point' mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, sigma(E) acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of sigma(32), which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that sigma(E) promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by sigma(E), and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Resposta SOS em Genética , Fator sigma/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Mutagênese Insercional , Mutação Puntual , Regiões Promotoras Genéticas , Fator sigma/genética , Estresse FisiológicoRESUMO
Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(o(c)) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(o(c)) alleles fully suppress the phenotype of constitutively SOS-"off" lexA(Ind(-)) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(o(c)) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutagênese , Mutação/genética , Resposta SOS em Genética/fisiologia , Western Blotting , Dano ao DNA , Proteínas de Escherichia coli/metabolismo , Óperon Lac/genética , Plasmídeos , Regulação para CimaRESUMO
Integrative conjugative elements (ICEs) are widespread in many bacterial species, often carrying antibiotic resistance determinants. In the present work, we screened a collection of Proteus mirabilis clinical isolates for the presence of type 1 SXT/R391 ICEs. Among the 76 isolates analyzed, 5 of them carry such elements. The complete sequences of these elements were obtained. One of the isolates carried the CMY-2 beta-lactamase gene in a transposon and is nearly identical to the element ICEPmiJpn1 previously described in Japan, and later shown to be present in other parts of the world, indicating global spread of this element. Nevertheless, the Brazilian isolate carrying ICEPmiJpn1 is not clonally related to the other lineages carrying the same element around the world. The other ICEs identified in this work do not carry known antibiotic resistance markers and are diverse in variable gene content and size, suggesting that these elements may be responsible for the acquisition of other advantageous traits by bacteria. Some sequences carried by these elements in Brazilian strains were not previously found in other SXT/R391 variants.
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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 some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations are a common route to clinically relevant beta-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial beta-lactam resistance mutations using the well-developed genetics of E. coli by integrating enterobacterial ampRC genes into the E. coli chromosome. Like the cells of other enterobacteria, E. coli cells acquire beta-lactam resistance by ampD mutation. Here we show that starvation and stress responses provoke ampD beta-lactam resistance mutagenesis. When starved on lactose medium, Lac(-) strains used in mutagenesis studies accumulate ampD beta-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for lac reversion in these cells. Surprisingly, the stress-induced ampD mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike lac mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the ampD system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.
Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Mutação/genética , Resistência beta-Lactâmica/genética , Proteínas de Bactérias/genética , Carbono/deficiência , Carbono/fisiologia , Análise Mutacional de DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Mutação da Fase de Leitura , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , N-Acetil-Muramil-L-Alanina Amidase/genéticaRESUMO
Bacterial resistance is a severe threat to global public health. Exposure to sub-lethal concentrations has been considered a major driver of mutagenesis leading to antibiotic resistance in clinical settings. Ciprofloxacin is broadly used to treat infections caused by Pseudomonas aeruginosa, whereas increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin has been reported for the reference strain, PAO1, in vitro. In this study we report increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin for another reference strain, PA14-UCBPP, and lower mutagenesis for clinical isolates when compared to the reference strain. This unexpected result may be associated with missense mutations in imuB and recX, involved in adaptive responses, and the presence of Pyocin S2, which were found in all clinical isolates but not in the reference strain genome. The genetic differences between clinical isolates of P. aeruginosa and the reference PA14-UCBPP, often used to study P. aeruginosa phenotypes in vitro, may be involved in reduced mutagenesis under sub-lethal concentrations of CIP, a scenario that should be further explored for the understanding of bacterial fitness in hospital environments. Moreover, we highlight the presence of a complete umuDC operon in a P. aeruginosa clinical isolate. Even though the presence of umuDC did not contribute to a significant increase in mutagenesis, it highlights the dynamic exchange of genetic material between bacterial species in the hospital environment.
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Oxidative stress generated by hydrogen peroxide is faced by bacteria when encountering hostile environments. In order to define the physiological and regulatory networks controlling the oxidative stress response in the free-living bacterium Caulobacter crescentus, a whole transcriptome analysis of wild type and ΔoxyR strains in the presence of hydrogen peroxide for two different exposure times was carried out. The C. crescentus response to H2O2 includes a decrease of the assimilative sulfate reduction and a shift in the amino acid synthesis pathways into favoring the synthesis of histidine. Moreover, the expression of genes encoding enzymes for the depolymerization of polyhydroxybutyrate was increased, and the RpoH-dependent genes were severely repressed. Based on the expression pattern and sequence analysis, we postulate that OxyR is probably directly required for the induction of three genes (katG, ahpCF). The putative binding of OxyR to the ahpC regulatory region could be responsible for the use of one of two alternative promoters in response to oxidative stress. Nevertheless, OxyR is required for the expression of 103 genes in response to H2O2. Fur and part of its regulon were differentially expressed in response to hydrogen peroxide independently of OxyR. The non-coding RNA OsrA was upregulated in both strains, and an in silico analysis indicated that it may have a regulatory role. This work characterizes the physiological response to H2O2 in C. crescentus, the regulatory networks and differentially regulated genes in oxidative stress and the participation of OxyR in this process. It is proposed that besides OxyR, a second layer of regulation may be achieved by a small regulatory RNA and other transcriptional regulators.
Assuntos
Caulobacter crescentus/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Peróxido de Hidrogênio/efeitos adversos , Fatores de Transcrição/genética , Proteínas de Bactérias/genética , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Estresse Oxidativo , Análise de Sequência de RNA/métodos , Estresse FisiológicoRESUMO
The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Regulon/genética , Resposta SOS em Genética/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Caulobacter crescentus/metabolismo , Dano ao DNA , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Técnicas de Amplificação de Ácido Nucleico , Regiões Operadoras Genéticas/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Sítio de Iniciação de Transcrição , Raios UltravioletaRESUMO
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.
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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
DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.
Assuntos
Caulobacter crescentus/genética , Dano ao DNA , Mutagênese , Óperon , Resposta SOS em Genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Caulobacter crescentus/metabolismo , Caulobacter crescentus/efeitos da radiação , DNA Polimerase III/classificação , DNA Polimerase III/genética , DNA Polimerase III/fisiologia , Genoma Bacteriano , Filogenia , Raios UltravioletaRESUMO
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.