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1.
Eur J Pharm Sci ; 146: 105268, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32081832

RESUMO

Sub-inhibitory concentrations (sub-MIC) of antimicrobial agents can lead to genetic changes in bacteria, modulating the expression of genes related to bacterial stress and leading to drug resistance. Herein we describe the impact of sub-MIC of ciprofloxacin and nitrofurantoin on three uropathogenic Escherichia coli strains. Disk-diffusion assays with different antimicrobial agents were tested to detect phenotype alterations, and quantitative real-time PCR (qRT-PCR) was performed to analyze the expression of ompF and recA genes. Significant reduction on the susceptibility to ciprofloxacin and nitrofurantoin was detected on disk diffusion test. The qRT-PCR results revealed a 1.2-4.7 increase in recA expression in all E. coli studied, while the ompF expression varied. Because RecA was pointed as an important component to the development of drug resistance, molecular docking studies were performed with three experimentally known inhibitors of this enzyme. These studies aimed to understand the inhibitory binding mode of such compounds. The results confirmed the ADP/ATP binding site as a potential site of inhibitor recognition and a binding mode based on π-stacking interactions with Tyr103 and hydrogen bonds with Tyr264. These findings can be useful for guiding the search and design of new antimicrobial agents, mainly concerning the treatment of infections with resistant bacterial strains.


Assuntos
Anti-Infecciosos Urinários/farmacologia , Ciprofloxacina/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Escherichia coli/efeitos dos fármacos , Genes Bacterianos , Nitrofurantoína/farmacologia , Recombinases Rec A/efeitos dos fármacos , Escherichia coli Uropatogênica/efeitos dos fármacos , Anti-Infecciosos Urinários/química , Ciprofloxacina/química , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Nitrofurantoína/química , Recombinases Rec A/genética , Escherichia coli Uropatogênica/genética
2.
J Antibiot (Tokyo) ; 72(7): 566-573, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30792518

RESUMO

Antibiotic resistance is considered a global threat to public health. Adaptive resistance mutations and the acquisition of resistance genes by horizontal gene transfer are known to be facilitated by the RecA-dependent SOS response during antibiotic treatment, making RecA inhibitors promising agents for the prevention of antibiotic resistance. However, the impact of RecA inactivation on antibiotic sensitivities remains unclear. Therefore, in this study, we performed high-throughput screening to determine the minimum inhibitory concentrations (MICs) of 217 chemicals, including both antibiotics and toxic chemicals of unknown drug action, in the wild-type MDS42 and the ΔrecA mutant strains of Escherichia coli. The ΔrecA mutant showed increased sensitivity to DNA-damaging agents, DNA replication inhibitors, and chromate stress, as well as to other chemicals, such as S-(2-aminoethyl)-L-cysteine, L-histidine, ruthenium red, D-penicillamine, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), cerulenin, and L-cysteine. Microarray analysis showed further that the ΔrecA mutant had lower expressions of glnK, nac, and glnLG, which encode nitrogen assimilation regulators, as well as amtB, which encodes an ammonium transporter, compared with the wild type. These findings suggest that the ΔrecA mutation affects not only the SOS response but also amino acid metabolism.


Assuntos
Antibacterianos/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Testes de Sensibilidade Microbiana/métodos , Recombinases Rec A/efeitos dos fármacos , Recombinases Rec A/genética , Resposta SOS em Genética/efeitos dos fármacos , Cromatos/toxicidade , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Análise em Microsséries , Mutação , RNA Bacteriano/genética
3.
Microb Pathog ; 128: 342-346, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30682524

RESUMO

2-(2-nitrovinyl) furan is a broad-spectrum antibacterial agent with activity against Gram-positive and Gram-negative bacteria. In this study, the contributions of reactive oxygen species, oxidative DNA damage and glutathione depletion to its activity against Acinetobacter baumannii was investigated. Inactivation of sodB, katG and recA lowered the minimum inhibitory concentration of 2-(2-nitrovinyl) furan. Furthermore, the inactivation increased the superoxide anion radical and hydrogen peroxide contents of 2-(2-nitrovinyl) furan-treated A. baumannii. Antioxidant (thiourea) reversed the elevated levels of superoxide anion radical and hydrogen peroxide. In addition, thiourea lowered the susceptibility of A. baumannii to 2-(2-nitrovinyl) furan. 2-(2-nitrovinyl) furan depleted reduced glutathione (GSH) contents of parental, sodB, katG and recA strains of A. baumannii. NAD+/NADH ratio parental, sodB, katG and recA strains of A. baumannii exposed to 2-(2-nitrovinyl) furan increased significantly. Inactivation of type-I NADH dehydrogenase lowered the reactive oxygen species generation in 2-(2-nitrovinyl) furan-treated A. baumannii. It is evident from this study that 2-(2-nitrovinyl) furan stimulates respiratory chain activity of A. baumannii leading to enhanced ROS generation, which depletes GSH and reacts with Fe2+ to produce hydroxyl radical that damage DNA.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacologia , Dano ao DNA/efeitos dos fármacos , Furanos/farmacologia , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Compostos de Vinila/farmacologia , Antioxidantes/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Catalase/efeitos dos fármacos , Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Radical Hidroxila/metabolismo , Radical Hidroxila/farmacologia , Testes de Sensibilidade Microbiana , NAD/metabolismo , NADH Desidrogenase/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Recombinases Rec A/efeitos dos fármacos , Recombinases Rec A/metabolismo , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Tioureia/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-30519543

RESUMO

The SOS response is a conserved response to DNA damage that is found in Gram-negative and Gram-positive bacteria. When DNA damage is sustained and severe, activation of error-prone DNA polymerases can induce a higher mutation rate than is normally observed, which is called the SOS mutator phenotype or hypermutation. We previously showed that zinc blocked the hypermutation response induced by quinolone antibiotics and mitomycin C in Escherichia coli and Klebsiella pneumoniae. In this study, we demonstrate that zinc blocks the SOS-induced development of chloramphenicol resistance in Enterobacter cloacae. Zinc also blocked the transfer of an extended spectrum beta-lactamase (ESBL) gene from Enterobacter to a susceptible E. coli strain. A zinc ionophore, zinc pyrithione, was ~100-fold more potent than zinc salts in inhibition of ciprofloxacin-induced hypermutation in E. cloacae. Other divalent metals, such as iron and manganese, failed to inhibit these responses. Electrophoretic mobility shift assays (EMSAs) revealed that zinc, but not iron or manganese, blocked the ability of the E. coli RecA protein to bind to single-stranded DNA, an important early step in the recognition of DNA damage in enteric bacteria. This suggests a mechanism for zinc's inhibitory effects on bacterial SOS responses, including hypermutation.


Assuntos
Farmacorresistência Bacteriana/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Transferência Genética Horizontal , Resposta SOS em Genética/efeitos dos fármacos , Zinco/farmacologia , Cloranfenicol/farmacologia , Ciprofloxacina/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples , Proteínas de Ligação a DNA/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/genética , Escherichia coli/genética , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Mutação/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Piridinas/farmacologia , Recombinases Rec A/efeitos dos fármacos , beta-Lactamases/genética
5.
Mol Microbiol ; 40(4): 909-16, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11401698

RESUMO

Cell division normally follows the completion of each round of chromosome replication in Escherichia coli. Transcription of the essential cell division genes clustered at the mra region is shown here to depend on continuing chromosomal DNA replication. After chromosome replication was blocked by either nalidixic acid treatment or thymine starvation, the transcription of these cell division genes was repressed significantly. This suggests a way in which cell division is controlled by chromosome replication.


Assuntos
Cromossomos Bacterianos , Proteínas do Citoesqueleto , Replicação do DNA , Proteínas de Escherichia coli , Escherichia coli/citologia , Escherichia coli/genética , Transcrição Gênica , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Citoesqueleto/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Família Multigênica , Mutação , Ácido Nalidíxico/farmacologia , Regiões Promotoras Genéticas , Recombinases Rec A/efeitos dos fármacos , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Ribossômicas/efeitos dos fármacos , Proteínas Ribossômicas/genética , Timidina/metabolismo
6.
Mol Microbiol ; 35(6): 1443-53, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10760145

RESUMO

In addition to being an essential component of trans-lesion synthesis, the UmuD'C complex is an antagonist of RecA-mediated homologous recombination. When constitutively expressed at an elevated concentration, the UmuD'C complex sensitizes recA+ bacteria to DNA damage, whereas it has no effect on bacteria expressing a RecA [UmuR] protein that overcomes recombination inhibition. Using as a genetic screen enhanced cell killing on mitomycin plates, we isolated novel umuD' and umuC mutations that restored mitomycin sensitivity to recA D112G [UmuR] bacteria overproducing the UmuD'C complex. The mutations were named [Rin++] because a characterization in a recA+ as well in a recA D112G background showed that they enhanced UmuD'C-promoted recombination inhibition in two assays, conjugational recombination and recombinational repair of palindrome-containing DNA. The [Rin++] mutations affect five amino acids, G25D, S28T, P29L, E35K, and T95R, in UmuD' and seven, F10L, Y270C, K277E, F287L, F287S, K342Q and F351I, in UmuC. These amino acids might play a key role in the UmuD'C anti-recombination activity. None of the [Rin++] mutations enhanced UmuD'C-promoted mutagenic bypass of UV lesions, in contrast, several lead to a defect in this process. In this study, we discuss a few molecular mechanisms that could account for the recombination and mutagenesis phenotypes of a mutant UmuD'C [Rin++] complex.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Recombinação Genética , Aminoácidos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Mitomicina/farmacologia , Mutação , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/efeitos da radiação , Recombinases Rec A/efeitos dos fármacos , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Raios Ultravioleta
7.
Biophys J ; 69(6): 2728-38, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8599679

RESUMO

The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.


Assuntos
DNA Viral/ultraestrutura , Estrutura Secundária de Proteína , Recombinases Rec A/química , Recombinases Rec A/ultraestrutura , Thermus/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Bacteriófago phi X 174 , Escherichia coli/metabolismo , Análise de Fourier , Substâncias Macromoleculares , Microscopia Eletrônica , Modelos Estruturais , Polimorfismo Genético , Recombinases Rec A/efeitos dos fármacos
8.
Proc Natl Acad Sci U S A ; 92(8): 3478-82, 1995 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-7724585

RESUMO

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Recombinases Rec A/metabolismo , Recombinação Genética/fisiologia , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/metabolismo , Compostos de Alumínio/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Fluoretos/metabolismo , Fluoretos/farmacologia , Modelos Genéticos , Ácidos Nucleicos Heteroduplexes/metabolismo , Ligação Proteica/efeitos dos fármacos , Recombinases Rec A/efeitos dos fármacos , Cloreto de Sódio/farmacologia
9.
FEBS Lett ; 333(1-2): 89-95, 1993 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-8224177

RESUMO

RecB and RecA proteins play key roles in the process of DNA recombination in Escherichia coli and both possess DNA unwinding activities which can displace short regions of duplex DNA in an ATP-dependent manner in vitro. We have examined the effect of the most abundant DNA adduct caused by the chemotherapeutic agent cis-diamminedichloroplatinum(II) on those activities. For this purpose, we have constructed a partially duplex synthetic oligonucleotide containing the intrastrand d(GpG) crosslink positioned at a specific site. We report here that both the DNA strand separating and DNA-dependent ATPase activities of the RecB protein are inhibited by the d(GpG) cis-DDP adduct. In contrast, neither the unwinding nor the ATPase activities of RecA protein appear to be perturbed by this lesion.


Assuntos
Cisplatino/farmacologia , DNA Helicases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Exodesoxirribonucleases/metabolismo , Recombinases Rec A/metabolismo , Sequência de Bases , Cisplatino/metabolismo , DNA/metabolismo , DNA Helicases/efeitos dos fármacos , Fosfatos de Dinucleosídeos/metabolismo , Escherichia coli/efeitos dos fármacos , Exodesoxirribonuclease V , Exodesoxirribonucleases/efeitos dos fármacos , Cinética , Dados de Sequência Molecular , Recombinases Rec A/efeitos dos fármacos
10.
Tsitologiia ; 31(12): 1478-84, 1989 Dec.
Artigo em Russo | MEDLINE | ID: mdl-2700258

RESUMO

A study was made of the adaptive response to methylmethane sulfonate (MMS) in E. coli. (18 strains of B, WP2, and H/r30 groups, including three strains of bacteria with pKM101 plasmid). The adaptation of wild type cells and uvrA- and uvrB- mutants to non-lethal concentrations of MMS (10-30 mkg/ml during 90-120 min) leads to a significant increase in their resistance to lethal MMS concentrations (10-30 mM for 10-120 min): the dose modifying factor (DMF) being 1.5-1.8. In single recA or lexA mutants (or double recA uvr- and lexA uvr- mutants) the efficiency of adaptive response to MMS was significantly lower: the DMF being 1.1-1.2. In Bs-1 gamma R strain with intragenic suppressor of lexA gene the adaptive response efficiency was the same as in B/r (recA+lexA+) strain. There is no adaptive response to MMS in polA- strains. The adaptive response to MMS in E. coli is different from that to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methylnitrosourea (MNM), because in these two cases it is absolutely lexA-recA dependent. It is supposed that a partial recA-lexA dependence of the adaptive response to MMS in E. coli may be due to a specific MMS-induced lethal damage that induces an adaptive repair non-related to the system of recA-lexA-independent adaptive responses to MNNG and MNM. The presence of a plasmid of drug resistance pKM101 exerts no influence on the value, efficiency and recA-lexA-dependence of the adaptive response of E. coli to MMS.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Proteínas de Bactérias/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Recombinases Rec A/efeitos dos fármacos , Proteínas Repressoras/efeitos dos fármacos , Serina Endopeptidases , Fatores de Transcrição/efeitos dos fármacos , Adaptação Fisiológica/genética , Proteínas de Bactérias/genética , Relação Dose-Resposta a Droga , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Genes Bacterianos/efeitos dos fármacos , Genes Bacterianos/genética , Mutação , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Recombinases Rec A/genética , Proteínas Repressoras/genética , Fatores de Tempo
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