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...

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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.

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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...

The increasing rates of global extinction due to human activities necessitate studies of the ability of organisms to adapt to the new environmental conditions resulting from human disturbances. We investigated the evolutionary adaptation of a microalga to sudden environmental change resulting from exposure to novel toxic chemical residues. A laboratory strain of Dictyosphaerium chlorelloides (Naum.) Kom. and Perm. (Chlorophyceae) was exposed to increasing concentrations of the modern contaminant 2,4,6‐trinitrotoluene (TNT). When algal cultures were exposed to 30 mg·L−1 TNT, massive lysis of microalgal cells was observed. The key to understanding the evolution of microalgae in such a contaminated environment is to characterize the TNT‐resistant variants that appear after the massive lysis of the TNT‐sensitive cells. A fluctuation analysis demonstrated unequivocally that TNT did not facilitate the appearance of TNT‐resistant cells; rather it was found that TNT‐resistant cells appeared spontaneously by rare mutations under nonselective conditions, before exposure to TNT. The estimated mutation rate was 1.4 × 10−5 mutants per cell division. Isolated resistant mutants exhibited a diminished fitness in the absence of TNT. Moreover, the gross photosynthetic rate of TNT‐resistant mutants was significantly lower than that of wild‐type cells. Competition experiments between resistant mutants and wild‐type cells showed that in small populations, the resistant mutants were driven to extinction. The balance between mutation rate and the rate of selective elimination determines the occurrence of about 36 TNT‐resistant mutants per million cells in each generation. These scarce resistant mutants are the guarantee of potential for adaptation.

Riboviruses (RNA viruses without DNA replication intermediates) are the most abundant pathogens infecting animals and plants. Only a few riboviral infections can be controlled with antiviral drugs, mainly because of the rapid appearance of resistance mutations. Little reliable information is available concerning i) kinds and relative frequencies of mutations (the mutational spectrum), ii) mode of genome replication and mutation accumulation, and iii) rates of spontaneous mutation. To illuminate these issues, we developed a model in vivo system based on phage Qß infecting its natural host, Escherichia coli . The Qß RT gene encoding the Read-Through protein was used as a mutation reporter. To reduce uncertainties in mutation frequencies due to selection, the experimental Qß populations were established after a single cycle of infection and selection against RT − mutants during phage growth was ameliorated by plasmid-based RT complementation in trans . The dynamics of Qß genome replication were confirmed to reflect the linear process of iterative copying (the stamping-machine mode). A total of 32 RT mutants were detected among 7,517 Qß isolates. Sequencing analysis of 45 RT mutations revealed a spectrum dominated by 39 transitions, plus 4 transversions and 2 indels. A clear template•primer mismatch bias was observed: A•C>C•A>U•G>G•U> transversion mismatches. The average mutation rate per base replication was ≈9.1×10 −6 for base substitutions and ≈2.3×10 −7 for indels. The estimated mutation rate per genome replication, μ g , was ≈0.04 (or, per phage generation, ≈0.08), although secondary RT mutations arose during the growth of some RT mutants at a rate about 7-fold higher, signaling the possible impact of transitory bouts of hypermutation. These results are contrasted with those previously reported for other riboviruses to depict the current state of the art in riboviral mutagenesis.

Summary• Adaptation of Spirogyra insignis (Chlorophyceae) to growth and survival in an extreme natural environment (sulphureous waters from La Hedionda Spa, S. Spain) was analysed by using an experimental model.• Photosynthetis and growth of the alga were inhibited when it was cultured in La Hedionda Spa waters (LHW), but after further incubation for several weeks, the culture survived due to the growth of a variant that was resistant to LHW.• A Luria-Delbrück fluctuation analysis was carried out to distinguish between resistant filaments arising from rare spontaneous mutations and resistant filaments arising from other mechanisms of adaptation. It was demonstrated that the resistant filaments arose randomly by rare spontaneous mutations before the addition of LHW (preselective mutations). The rate of spontaneous mutation from sensitivity to resistance was 2.7 × 10 − 7 mutants per cell division.• Since LHW resistant mutants have a diminished growth rate, they are maintained in nonsulphureous natural waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, recurrence of rare spontaneous preselective mutations ensures the survival of the alga in sulphureous waters. Abbreviations, maximum fluorescence of light-adapted cells; F t , steady state fluorescence of light-adapted cells; LHW, La Hedionda Spa water; N 0 , initial number of cells in a culture; N t , final number of cells in a culture after a time period, P 0 , proportion of cultures showing no mutant cells in a fluctuation analysis; Φ PSII , effective quantum yield from PSII; µ, mutation rate.