Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutantgenerating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (s S ) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small s S -response-''on'' subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents s S induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a ''gambler'' cell subpopulation promote resistance evolution without risking most cells. RESULTS Cipro-Induced MutagenesisWe developed two assays for cipro-induced mutagenesis without cipro selection of the mutants (Figure 1A). In both assays, strains are grown in liquid, each with cipro at its minimum antibiotic concentration (MAC, final colony-forming units [CFU] are 10% of those of no-drug cultures) (Lorian and De Freitas, 1979). These are ''low-dose'' and ''sub-inhibitory'' relative to MICs (CFU %10 À4 of untreated cells). Table S1 shows MACs and MICs for all strains assayed (wild-type MAC, 8.5 ng/mL). Cells are then removed from cipro and plated selectively for colonies resistant to rifampicin (RifR) or ampicillin (AmpR) antibiotics (Figure 1A), and mutation rates are estimated (STAR Methods). RifR arises by specific base-substitutions in the rpoB gene (Figure S1A), and AmpR arises by ampD null mutations in engineered Escherichia coli (Petrosino et al., 2002) (Figures S1B and S1C; STAR Methods). Strikingly, cipro increased RifR and AmpR mutation rates 26-and 18-fold above no-cipro rates (Figure 1B; Table S2 for all mutation rates). The RifR or AmpR mutants are not selected in sub-inhibitory cipro and are at a slight but significant disadvantage (Figure 1C), implying that mutation, not selection of the mutants, is elevated by MAC cipro. Additional controls show negligible cell death in the lowdose cipro (Figures S1D and S2, other controls). ROS-Dependent Mutagenesis Is s S -Dependent Mutagenic Break RepairThe cipro-induced mutagenesis requires ROS and is inhibited by ROS scavenging or preventing agents thiourea (TU) and 2,2 0 -bipyridine (BP) (Figure 1D; Table S2). The following data indicate that the ROS instigate a s S -licensed mutagenic DNA breakrepair (MBR) mechanism triggered by cipro-induced DSBs.MBR is regulated mutagenesis during repair of DSBs, requiring the SOS and s S respon...
Neospora caninum, a recently recognized protozoan parasite of animals, is considered to be a major cause of bovine abortion worldwide. Although its life cycle is not completely known, recent studies suggest that the sexual stage occurs in dogs. The prevalence of sexual reproduction in N. caninum, however, is unknown. We investigated the ability of 3 N. caninum isolates (NC-1, NC-SweB1, and NC-Liverpool) to propagate asexually for approximately 250 parasite generations in a cell line in which they had not been cultured previously. The malthusian parameter of fitness was estimated for each isolate from 10 independent replicates of tachyzoites at the beginning as well as at the end of the experimental period. Derived and ancestral values for mean fitness were compared both within and among NC-1, NC-SweB1, and NC-Liverpool isolates. Results showed a significant increase in mean fitness for the 3 N. caninum isolates at the end of the experimental period. These findings indicate that N. caninum can adapt to new environmental conditions without the help of sexual recombination, supporting the idea that this parasite has, at least potentially, the capacity for maintaining clonal propagation in nature.
32Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, 33 mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the 34 fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations that cause drug resistance by 35 triggering differentiation of a mutant-generating cell subpopulation, using reactive oxygen species 36 (ROS) to signal the sigma-S (σ S ) general-stress response. Cipro-generated DNA breaks activate 37 the SOS DNA-damage response and error-prone DNA polymerases in all cells. However, 38 mutagenesis is restricted to a cell subpopulation in which electron transfer and SOS induce ROS, 39 which activate the σ S response, allowing mutagenesis during DNA-break repair. When sorted, 40 this small σ S -response-"on" subpopulation produces most antibiotic cross-resistant mutants. An 41FDA-approved drug prevents σ S induction specifically inhibiting antibiotic-promoted mutagenesis. 42 Furthermore, SOS-inhibited cell division, causing multi-chromosome cells, is required for 43 mutagenesis. The data support a model in which within-cell chromosome cooperation together 44 with development of a "gambler" cell subpopulation promote resistance evolution without risking 45 most cells. 46 47 48 49 51 break repair, reactive oxygen species (ROS), RpoS (σ S ) stress response, SOS response, 52 starvation stress response, stress-induced mutagenesis, transient differentiation 53 54 100 once antibiotics have gone (Lewis, 2010). Persister formation can occur stochastically, leaving 101 populations ready for a stress that they have not encountered (Balaban et al., 2004), and can also 102 be induced responsively via stress-response regulons including the SOS- (Dorr et al., 2009) and 103 σ S -response (Radzikowski et al., 2016) regulons. It is unknown whether antibiotics induce 104 4 transient differentiation that could promote resistance through mutagenesis, e.g., (Frenoy and 105 Bonhoeffer, 2018). 106Here we show that low, sub-inhibitory doses of cipro induce transient differentiation of a 107 small cell subpopulation with high ROS and σ S -response activity, that generates mutants, 108 including cross-resistant mutants: a "gambler" subpopulation. We show that the ROS promote 109 mutagenesis in gamblers by activating the σ S response, which allows mutagenic repair of cipro-110 triggered DSBs-a novel signaling/differentiating role of ROS in mutagenesis. We elaborate the 111 regulatory chain from cipro to ROS to σ S response to mutant production, and also discover a 112 requirement for SOS-induced inhibition of cell division, causing multiple chromosomes per cell. 113Mathematical analysis supports a model in which multiple chromosomes allow sharing of cellular 114 resources (e.g., recombination, complementation), avoiding deleterious consequences of some 115 mutations during mutagenesis and repair. Thus, multiple chromosomes allow higher mutation 116 rates to be maintained -resulting in faster adaptation. The findings imply a highly regulated, novel 117 transie...
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