Genetic analysis of the requirements for SOS induction by nalidixic acid in Escherichia coli

Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Gene (Impact Factor: 2.14). 09/2005; 356(1):69-76. DOI: 10.1016/j.gene.2005.04.029
Source: PubMed


Nalidixic acid, the prototype antibacterial quinolone, induces the SOS response by a mechanism that requires the RecBCD nuclease/helicase. A key step inferred for this induction pathway is the conversion of a drug-induced gyrase cleavage complex into a DNA break that can be processed by RecBC. We tried to clarify the nature of this step by searching for additional gene products that are specifically necessary for SOS induction following nalidixic acid treatment. A transposon library of approximately 19,000 insertion mutants yielded 18 mutants that were substantially reduced for SOS induction following nalidixic acid but not UV treatment, and which were also hypersensitive to nalidixic acid. All 18 mutants turned out to have insertions in recB or recC. As expected, recA insertion mutants were uncovered as being uninducible by either nalidixic acid or UV treatment. Insertions in 11 other genes were found to cause partial defects in SOS induction by one or both pathways, providing possible leads in understanding the detailed mechanisms of SOS induction. Overall, these results suggest that nalidixic acid-induced DNA breaks are generated either by RecBC itself, by redundant activities, and/or by an essential protein that could not be uncovered with transposon mutagenesis.

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Available from: Kenneth N Kreuzer, Feb 19, 2015
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    • "Rapid lethality is increased by the lexA Ind-allele, and recombination-deficient E. coli strains are hypersensitive to quinolones [27]. The RecBCD nuclease/helicase also seems to be required for SOS induction by quinolones, as demonstrated with nalidixic acid [28]. Interestingly, DSBs may also be repaired by a non-homologous end joining (NHEJ) mechanism that comprises break recognition, end processing, and ligation activities. "
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    ABSTRACT: Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide. Exposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 microg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 microg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 microg/ml but scarce after 10 microg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose. This procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.
    BMC Microbiology 05/2009; 9(1):69. DOI:10.1186/1471-2180-9-69 · 2.73 Impact Factor
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    • "The homologous recombination function of RecA was found to be required for repair of topo I cleavage complex. Induction of the SOS response by the recombination deficient RecA718 protein was found to be insufficient for repair of topo I mediated DNA cleavage (65) Quinolones are known to induce the SOS response of E. coli via the RecBCD pathway (66). Double-strand DNA breaks and chromosomal fragmentation occur after trapping of the cleavage complex between the gyrase A subunits and both strands of DNA (67,68). "
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    ABSTRACT: Bacterial topoisomerase I is a potential target for discovery of new antibacterial compounds. Mutant topoisomerases identified by SOS induction screening demonstrated that accumulation of the DNA cleavage complex formed by type IA topoisomerases is bactericidal. Characterization of these mutants of Yersinia pestis and Escherichia coli topoisomerase I showed that DNA religation can be inhibited while maintaining DNA cleavage activity by decreasing the binding affinity of Mg(II) ions. This can be accomplished either by mutation of the TOPRIM motif involved directly in Mg(II) binding or by altering the charge distribution of the active site region. Besides being used to elucidate the key elements for the control of the cleavage-religation equilibrium, the SOS-inducing mutants of Y. pestis and E. coli topoisomerase I have also been utilized as models to study the cellular response following the accumulation of bacterial topoisomerase I cleavage complex. Bacterial topoisomerase I is required for preventing hypernegative supercoiling of DNA during transcription. It plays an important role in transcription of stress genes during bacterial stress response. Topoisomerase I targeting poisons may be particularly effective when the bacterial pathogen is responding to host defense, or in the presence of other antibiotics that induce the bacterial stress response.
    Nucleic Acids Research 12/2008; 37(3):731-7. DOI:10.1093/nar/gkn936 · 9.11 Impact Factor
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    • "The results mentioned above clearly underscore that application of certain drugs acting as DNA damaging agents promote a variety of consequences linked to SOS induction that affect pathogenicity. The molecular action of fluoroquinolones, although not understood in full detail, probably resides in the generation of double-stranded breaks and activation of RecA through disruption of their target topoisomerases (Newmark et al., 2005; Polhaus and Kreuzer, 2005; Malik et al., 2006). The link to SOS induction by 4-quinolones has been known for more than a decade, and the consequences described above should perhaps come as no surprise. "
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    ABSTRACT: The SOS response that responds to DNA damage induces many genes that are under LexA repression. A detailed examination of LexA regulons using genome-wide techniques has recently been undertaken in both Escherichia coli and Bacillus subtilis. These extensive and elegant studies have now charted the extent of the LexA regulons, uncovered many new genes, and exposed a limited overlap in the LexA regulon between the two bacteria. As more bacterial genomes are analysed, more curiosities in LexA regulons arise. Several notable examples include the discovery of a LexA-like protein, HdiR, in Lactococcus lactis, organisms with two lexA genes, and small DNA damage-inducible cassettes under LexA control. In the cyanobacterium Synechocystis, genetic and microarray studies demonstrated that a LexA paralogue exerts control over an entirely different set of carbon-controlled genes and is crucial to cells facing carbon starvation. An examination of SOS induction evoked by common therapeutic drugs has shed new light on unsuspected consequences of drug exposure. Certain antibiotics, most notably fluoroquinolones such as ciprofloxacin, can induce an SOS response and can modulate the spread of virulence factors and drug resistance. SOS induction by beta-lactams in E. coli triggers a novel form of antibiotic defence that involves cell wall stress and signal transduction by the DpiAB two-component system. In this review, we provide an overview of these new directions in SOS and LexA research with emphasis on a few themes: identification of genes under LexA control, the identification of new endogenous triggers, and antibiotic-induced SOS response and its consequences.
    Molecular Microbiology 01/2007; 62(5):1228-38. DOI:10.1111/j.1365-2958.2006.05444.x · 4.42 Impact Factor
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