Effect of recA inactivation on mutagenesis of Escherichia coli exposed to sublethal concentrations of antimicrobials

Thi, Thuy Do; López, Elena; Rodríguez‐Rojas, Alexandro; Rodríguez-Beltrán, Jerónimo; Couce, Alejandro; Guelfo, Javier R.; Castañeda-García, Alfredo; Blázquez, Jesús

doi:10.1093/jac/dkq496

Cited by 149 publications

(142 citation statements)

References 47 publications

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“…Likewise, inactivation of the recA gene of E. coli is known to increase bacterial susceptibility to several antibiotics (22,50). We obtained similar results in tests of the susceptibility of the A. baumannii recA mutant to different groups of antimicrobials (Table 3).…”

Section: Discussionsupporting

confidence: 74%

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

et al. 2011

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RecA is the major enzyme involved in homologous recombination and plays a central role in SOS mutagenesis. In Acinetobacter spp., including Acinetobacter baumannii, a multidrug-resistant bacterium responsible for nosocomial infections worldwide, DNA repair responses differ in many ways from those of other bacterial species. In this work, the function of A. baumannii RecA was examined by constructing a recA mutant. Alteration of this single gene had a pleiotropic effect, showing the involvement of RecA in DNA damage repair and consequently in cellular protection against stresses induced by DNA damaging agents, several classes of antibiotics, and oxidative agents. In addition, the absence of RecA decreased survival in response to both heat shock and desiccation. Virulence assays in vitro (with macrophages) and in vivo (using a mouse model) similarly implicated RecA in the pathogenicity of A. baumannii. Thus, the data strongly suggest a protective role for RecA in the bacterium and indicate that inactivation of the protein can contribute to a combined therapeutic approach to controlling A. baumannii infections.

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Section: Discussionsupporting

confidence: 74%

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

et al. 2011

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“…on May 10, 2018 by guest http://aac.asm.org/ nation (41), and the induction of mutagenic polymerases that participate in the SOS response (32,37,44). In the present work, colony staining with diaminobenzidine indicated that colonies growing on LB plates supplemented with CIP accumulated considerably more hydrogen peroxide than those developed in the absence of antibiotic (data not shown).…”

supporting

confidence: 48%

Effect of Ciprofloxacin Concentration on the Frequency and Nature of Resistant Mutants Selected from Pseudomonas aeruginosa mutS and mutT Hypermutators

Morero

Monti

Argaraña

2011

Antimicrob Agents Chemother

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The rapid emergence of drug resistance upon treatment of Pseudomonas aeruginosa infections with fluoroquinolones is a serious concern. In this study, we report the effect of hypermutability on the mutant selection window for ciprofloxacin (CIP) by comparing the hypermutator MPAO1 mutS and mutT strains with the wild-type strain. The mutant selection window was shifted to higher CIP concentrations for both hypermutators, presenting the mutS strain with a broader selection window in comparison to the wild-type strain. The mutation prevention concentrations (MPC) determined for mutT and mutS strains were increased 2-and 4-fold over the wild-type level, respectively. In addition, we analyzed the molecular bases for resistance in the bacterial subpopulations selected at different points in the window. At the top of the window, the resistant clones isolated were mainly mutated in GyrA and ParC topoisomerase subunits, while at the bottom of the window, resistance was associated with the overexpression of MexCD-OprJ and MexAB-OprM efflux pumps. Accordingly, a greater proportion of multidrug-resistant clones were found among the subpopulations isolated at the lower CIP concentrations. Furthermore, we found that the exposure to CIP subinhibitory concentrations favors the accumulation of cells overexpressing MexCD-OprJ (due to mutations in the transcriptional repressor NfxB) and MexAB-OprM efflux pumps. We discuss these results in the context of the possible participation of this antibiotic in a mutagenic process.

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“…All of these compounds strongly induce RFP expression of the reporter. Sulfanilamide inhibits the synthesis of purines and pyrimidines and, due to imbalance of the substrates for DNA synthesis, activates the SOS response, as visualized by application of another LexA-regulated reporter, recA::gfp (39,40). The moderate induction of RFP expression by rifampin is of interest.…”

Section: Discussionmentioning

confidence: 99%

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

Osterman

Komarova

Shiryaev

et al. 2016

Antimicrob Agents Chemother

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e In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.T he spread of antibiotic resistance genes among pathogenic bacteria is leading to a gradual decrease in the efficiency of known antibiotics. Substantial efforts have been invested in platforms for new antibiotic development (1). High-throughput screening (HTS) is a major method for the discovery of new chemical scaffolds for drug discovery. However, in the search for new antibiotics, HTS demonstrated low efficiency (for a discussion, see references 2 and 3). Acceleration of the antibiotic development pipeline demands increased efficiency in the identification of mechanisms of action with both HTS of chemical libraries and screening of natural compounds. Ideally, the mechanism of action should be determined while screening for antibacterial activity. One of the major challenges in high-throughput screening is the development of a cost-effective procedure that could maximize information output while concomitantly minimizing the number of pipetting steps and the reagent costs.The most efficient way to reveal the mechanism of action is the application of reporter strains (for a recent review, see reference 4). However, the majority of the reporter strains developed thus far aim to identify a narrow group of chemically related compounds, such as tetracyclines (5), macrolides (6, 7), or ␤-lactams (8). Broader-spectrum reporters based on stress response promoters are also available (9-11). A combination of several reporter strains could help to classify more mechanisms of action, but that would require multiple experiments for a single substance being tested.The majority of antibiotics currently in clinical use target the cell wall, DNA, or protein biosynthesis. For the latter two mechanisms ...

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Effect of recA inactivation on mutagenesis of Escherichia coli exposed to sublethal concentrations of antimicrobials

Abstract: The fact that inactivation of recA reduces mutagenicity and/or increases the activity of a large number of antimicrobials supports the hypothesis that RecA inhibition might have favourable effects on antibiotic therapy.

Cited by 149 publications

References 47 publications

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

Effect of Ciprofloxacin Concentration on the Frequency and Nature of Resistant Mutants Selected from Pseudomonas aeruginosa mutS and mutT Hypermutators

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

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