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...

The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts [1][2][3] . Here, we report the identification of an enzymatic system, MsrPQ, repairing Met-O containing proteins in the bacterial cell envelope, a compartment particularly exposed to the ROS and RCS generated by the host defense mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a heme-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid (HOCl), a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from Met

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.antibiotic resistance | SOS response | filamentation | mutation | evolution

Methionine ranks among the amino acids most sensitive to oxidation, which converts it to a racemic mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). The methionine sulfoxide reductases MsrA and MsrB reduce free and protein-bound MetSO, MsrA being specific for Met-S-SO and MsrB for Met-R-SO. In the present study, we report that an Escherichia coli metB 1 auxotroph lacking both msrA and msrB is still able to use either of the two MetSO enantiomers. This indicates that additional methionine sulfoxide reductase activities occur in E. coli. BisC, a poorly characterized biotin sulfoxide reductase, was identified as one of these new methionine sulfoxide reductases. BisC was purified and found to exhibit reductase activity with free Met-S-SO but not with free Met-R-SO as a substrate. Moreover, a metB 1 msrA msrB bisC strain of E. coli was unable to use Met-S-SO for growth, but it retained the ability to use Met-R-SO. Mass spectrometric analyses indicated that BisC is unable to reduce protein-bound Met-S-SO. Hence, this study shows that BisC has an essential role in assimilation of oxidized methionines. Moreover, this work provides the first example of an enzyme that reduces free MetSO while having no activity on peptide-bound MetSO residues.

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.