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Woo et al., Sci. Adv. 2018; 4 : eaat1608 20 June 2018

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C E L L B I O L O G Y

Heterogeneity of spontaneous DNA replication errors in single isogenic Escherichia coli cellsAnthony C. Woo1*†, Louis Faure1‡, Tanja Dapa1§, Ivan Matic1,2¶

Despite extensive knowledge of the molecular mechanisms that control mutagenesis, it is not known how spontaneous mutations are produced in cells with fully operative mutation-prevention systems. By using a mutation assay that allows visualization of DNA replication errors and stress response transcriptional reporters, we examined popula-tions of isogenic Escherichia coli cells growing under optimal conditions without exogenous stress. We found that spontaneous DNA replication errors in proliferating cells arose more frequently in subpopulations experiencing endogenous stresses, such as problems with proteostasis, genome maintenance, and reactive oxidative species production. The presence of these subpopulations of phenotypic mutators is not expected to affect the average mutation frequency or to reduce the mean population fitness in a stable environment. However, these sub-populations can contribute to overall population adaptability in fluctuating environments by serving as a reser-voir of increased genetic variability.

INTRODUCTIONMutations are the primary source of genetic variation, without which there is no evolution. Therefore, understanding how muta-tions arise is fundamental to understanding the evolution of living systems. Scientists broadly classify mutations into two categories: spontaneous mutations and mutations induced by environmental stress. While there is ample evidence about the mechanisms of environmental stress–increased mutation rates, it remains obscure how spontaneous mutations arise. Most of the knowledge about the molecular mechanisms controlling spontaneous mutagenesis comes from studies of mutator alleles, which identify pathways that cells use to prevent mutations (1). These include metabolic control over con-centrations of endogenous and exogenous mutagens, DNA repair systems, deoxynucleotide triphosphate pool balance, the insertion accuracy of DNA polymerases, DNA polymerases’ 3′→ 5′ exonucle-olytic proofreading, and several postreplication systems for repairing mismatches (2, 3). Despite extensive knowledge about these anti-mutator mechanisms, it is still impossible to answer the following question: Which molecular mechanism is responsible for mutant generation when a mutant arises in the population of bacteria whose mutation-prevention systems are operative?

The most reliable information about the actual sources and mech-anisms of spontaneous mutation in cells has come from the studies of antimutator phenotypes. For example, screening for antimutator mutants that reduce spontaneous mutagenesis in carbon- starved, nongrowing, and stationary-phase Escherichia coli populations showed that activation of several stress responses is required for the

mutagenic repair of DNA breaks (4). To date, the only well charac-terized antimutator alleles in growing E. coli cells are mutants of the subunit of the replicative DNA polymerase III (5), suggesting that DNA replication errors are a major source of spontaneous mu-tagenesis in E. coli cells growing under optimal conditions, without exogenous stress. However, these DNA polymerase III antimutator alleles also reduce A:T-to-C:G transition mutations in the mutT mutant genetic background by reducing mispairing of 8-oxo- deoxyguanine with adenine (6). Thus, it is unclear whether sponta-neous DNA replication errors arise in wild-type cells owing to limits of the intrinsic fidelity of DNA polymerase III, which is de-termined by base selection and proofreading activities, or in a sub-population of cells with special phenotypic properties. For example, the cellular amounts of the mutagenic 8-oxo-deoxyguanine, which can increase in some cells, owing to increased reactive oxidative spe-cies production, can be incorporated into DNA by DNA polymerase III (7). Ninio (8) also proposed that mutations could arise at a higher- than-average mutation frequency in subpopulations of cells in which transcription or translation errors attenuate DNA replica-tion fidelity transitorily. Although it was shown that the reduction of translation fidelity by mutations or by antibiotic treatment is muta-genic, to the best of our knowledge, this hypothesis was never tested using wild-type cells growing without exogenous stress (9).

This study aimed to determine the impact of phenotypic vari-ability on spontaneous DNA replication errors in populations of isogenic E. coli cells growing under optimal conditions, without ex-ogenous stress. It is not possible to answer this question by using standard mutation assays, which detect phenotypes resulting from mutations, or by whole genome sequencing (10). Both assays detect mutations, which are heritable, but do not provide information on the physiological state of a cell when mutation occurs. In addition, fitness effects of newly arisen mutations affect both assays. To solve these problems, we used a reporter system that allows visu-alization of the appearance of DNA replication errors in living indi-vidual cells (11–14). This mutation assay is based on the activity of the E. coli mismatch repair system, which corrects 99.99% of all spon-taneous DNA replication errors (15). E. coli mismatch repair in-volves three dedicated proteins: MutS, MutL, and MutH. The MutS protein detects and binds diverse base pair mismatches. The MutL

1INSERM U1001, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine Paris Descartes, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France. 2Centre National de la Recherche Scientifique, 75016 Paris, France.*Present address: Institut Pasteur, Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Department of Microbiology, 25 Rue du Docteur Roux, 75015 Paris, France.†Present address: Institute for Integrative Biology of the Cell, Department of Micro-biology, CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Bâtiment 21, Ave-nue de la Terrasse, 91190 Gif-sur-Yvette cedex, France.‡Present address: Institute of Technology and Innovation, Paris Sciences and Let-ters Research University, F-75005 Paris, France.§Present address: Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal.¶Corresponding author. Email: [email protected]

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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protein interacts with the MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination DNA site. Finally, coordinated helicase/exonuclease activities remove the error from the newly synthesized strand. The mutation assay used in this study is based on the property of the MutL protein to polym-erize on the sites of unrepaired mismatches (13). Tagging the MutL protein with a fluorescent protein allows monitoring DNA replica-tion errors, which appear as fluorescent foci, in single living cells. MutL fluorescent foci allow detection of DNA replication errors independently of their future effect on phenotype: beneficial, neu-tral, deleterious, or even lethal. This mutation assay is also unaffected by inheritance of mutations by the offspring of mutant cells because MutL fluorescent focus disappears when the next DNA replication cycle separates the two DNA strands and mutations are fixed. We coupled the MutL-based mutation assay with different reporters for the growth phase and stress response induction. Results of our study indicate that most spontaneous DNA replication errors in proliferat-ing cells arise in subpopulations of cells suffering from endogenous stresses.

RESULTSQuantification of DNA replication errors in proliferating cellsWe used previously constructed yellow fluorescent protein (YFP)–MutL reporter fusion, but to be able to combine MutL-based mutation reporter with green or yellow fluorescence–based gene expression reporters, we also constructed mCherry fusion to the wild-type mutL gene (table S1). We confirmed that the mCherry-labeled MutL pro-tein retains its normal function, by measuring the frequency of rifampicin-resistant mutants in the strain whose chromosomal mutL gene was deleted and that carried only the labeled MutL derivative (table S2). We also verified that there was no statistically significant difference in the MutL foci frequency between YFP-MutL and mCherry-MutL reporter strains (fig. S1A and table S3). To detect all DNA replication errors, we introduced MutL reporter fusions into a mutH strain. In this strain, which is MutS- and MutL-proficient, mismatches can be detected but cannot be repaired (13). We first examined MutL foci frequency in populations of mutH cells during growth in rich Luria Broth (LB) medium using fluorescence mi-croscopy (Fig. 1A). As expected, there were more DNA replication errors in exponentially growing cells than in cells approaching the stationary phase (Fig. 1, B and C).

We decided to follow DNA replication fidelity in cells during the exponential growth phase, that is, cell culture with the optical den-sity at 600 nm (OD600) at 0.2. At this growth phase, cells are meta-bolically active and at maximum growth rate. First, we confirmed that MutL foci are tagging DNA replication errors by introducing the antimutator allele dnaE911 (5), which enhances the fidelity of the DNA polymerase III (table S2). The dnaE911 allele significantly decreased the frequency of the MutL foci (Fig. 1D and table S3). Second, we confirmed that the detected MutL foci were not artifac-tual aggregates of the MutL protein fusions independent of mis-matches, by inactivating the mutS gene that codes for the mismatch binding protein (13). We did not observe any MutL focus in the mutS mutH mutant cells (Fig. 1D and table S3). Third, because the formation of MutL foci requires the MutS protein, some mismatches may not be tagged by MutL in the cells with an insufficient amount of the MutS protein (16, 17). To test this possibility, we overpro-duced the MutS protein and observed that the MutL foci frequency

increased slightly but significantly (Fig. 1D and table S3). This find-ing indicated that the amount of MutS protein in some cells was insufficient for efficient mismatch repair detection, thus resulting in increased mutagenesis because of undetected and therefore unre-paired DNA replication errors. The cellular amount of the MutL protein is not limiting in this study because the mutL gene is driven by an inducible promoter (13).

Next, because cell size varies according to growth phase—that is, growing cells are bigger than stationary-phase cells—we examined the correlation between cell size and occurrence of MutL foci. We found that cells with ≥1 MutL foci were bigger than cells with no focus (fig. S1, B and C). For this reason, all data presented hereinafter were normalized to cell size. To further examine the link between growth phase and DNA replication fidelity, we coupled the MutL-based assay with fluorescent transcriptional reporters of the rrnB ribosomal RNA operon. We used fusion of the P1rrnB or P2rrnB operon promoters to the genes coding for green and yellow fluorescent proteins, respectively. P1 promoter activity increases with growth rate, while the P2 promoter is active during transitions, that is, at the point of entry and outgrowth from the stationary phase (18, 19). We found that cells having ≥1 MutL focus had significantly higher P1-driven reporter fluorescence than cells without foci (Table 1). There was no significant difference in P2-driven reporter fluores-cence between cells having ≥1 and no MutL foci (Table 1 and fig. S2), whereas there was a positive correlation between the expression levels of the P1-driven reporter and MutL foci (Fig. 2A). Finally, there were fourfold more cells with MutL foci among the top 10% of the population showing strong P1-driven reporter fluorescence when compared to the 10% of the cells that showed weak reporter fluorescence (Fig. 2C and table S3). Together, these data indicated that DNA replication errors occurred more frequently in actively growing cells.

DNA replication errors in subpopulations of cells suffering from endogenous stressesThere is growing awareness that there is important cell-to-cell phe-notypic heterogeneity in isogenic bacterial populations exposed to the same environmental conditions (20, 21). To reveal subpopula-tions of cells suffering from endogenous stresses, we used transcrip-tional reporters for the induction of three stress response regulons. First, we used a recA gene promoter reporter, which is induced by genotoxic stresses as part of the LexA-regulated SOS response (fig. S3) (22). Second, we used the ibpA gene promoter as a reporter for the induction of RpoH-regulated heat shock response (fig. S3) (23). Third, we followed the induction of the katG gene promoter, which is induced as part of the OxyR regulon by hydrogen peroxide (fig. S3) (7). We observed cell-to-cell heterogeneity of the expression of all three reporters (Fig. 2).

Next, by using the MutL-based mutation assay, we examined whether DNA replication errors emerge more frequently in sub-populations of cells experiencing endogenous stresses. We found that cells having ≥1 MutL focus showed significantly higher mean fluorescence for all three transcriptional reporters than cells with-out MutL foci in the mutH background (Table 1). The number of cells with ≥1 MutL focus in the top 10% of the population show-ing strong reporter fluorescence was three-, four-, and twofold higher for the SOS, heat shock, and oxidative stress reporters, re-spectively, than in the 10% of the cells with weak reporter fluores-cence (Fig. 2, F, I, and L, and table S3). Therefore, DNA replication

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errors occur at a higher frequency in subpopulations of endogenous-ly stressed cells.

We observed the strongest increase in DNA replication errors in cells that exhibited induced SOS and heat shock responses. Pennington and Rosenberg (24) showed that SOS response can be induced in subpopulations of E. coli cells growing in stable environ-mental conditions. It is also well known that several LexA-controlled genes, particularly those coding for translesion synthesis DNA polymerases, increase mutagenesis (22). However, much less is known about DNA replication errors in cells exhibiting heat shock response induction. Heat shock response can be induced by tem-perature shift or by the accumulation of denatured proteins due to translation errors (25). In our experiments, we incubated cells at

constant, optimal growth temperature. Thus, we tested whether an increase in spontaneous translation error rates might be associated with an increase in DNA replication errors.

The impact of the treatments with translation error–inducing antibiotics on DNA replication errorsWe first tested whether increased translation errors increase MutL foci frequency, by treating cells with aminoglycoside antibiotics streptomycin and paromomycin (fig. S4). We have chosen amino-glycoside antibiotics because they are known to increase translation errors (26). In addition, Ren et al. (27) also show that streptomycin increases DNA replication errors. We found that treatment of E. coli cells with sub-minimum inhibitory concentrations of both anti-biotics significantly increased the frequency of the MutL foci (fig. S4). We also found that the introduction of the dnaE911 antimutator allele abolished streptomycin-induced increase in MutL foci fre-quency (fig. S5). The fact that there were no MutL foci in mutS- deficient cells showed that streptomycin did not induce aggregation of the MutL protein fusions. Finally, the overproduction of MutS protein did not have any impact on the MutL foci frequency in streptomycin-treated cells. This result indicated that the increase in DNA replication errors was not due to the decreased amount of the MutS protein.

The incidence of the spontaneous translation and DNA replication errorsTherefore, it is clear that decrease in translation fidelity by exoge-nous stresses results in decrease of DNA replication fidelity. How-ever, we wanted to test whether there are more DNA rep lication errors in cells having high spontaneous translation errors. Spontaneous

Fig. 1. Visualization and quantification of DNA replication errors at a single-cell level. (A) MutL foci in the mutH strain examined under a microscope during the exponential phase. Each fluorescent focus represents one unrepaired DNA replication error. (B) Growth kinetics of E. coli MG1655 cells. OD600 values of 0.2 (mid-exponential), 0.8 (late exponential), and 1.4 (early stationary) were selected for further analysis. (C) Quantification of DNA replication errors of mutH cells in different growth phases. Cells in the mid-exponential phase had statistically significantly more DNA replication errors than in two other growth phases (see table S3). (D) Quantification of DNA replication errors in different strains during the mid-exponential phase. All strains were statistically significant different from the mutH strain (see table S3).

Table 1. Mean fluorescence of different transcriptional reporters among cells with ≥1 and no MutL focus. We observed a statistically significant difference in the fluorescence between cells with ≥1 and no MutL focus among all reporters except for P2rrnB-YFP.

Reporter

Mean fluorescence per cell ± SEM (AU) Wilcoxon

signed-rank test (P)Cells with no

MutL focusCells with ≥1 MutL focus

P1rrnB-GFP 79 ± 1.0 88 ± 1.3 <0.05

P2rrnB-YFP 180 ± 1.9 188 ± 2.2 >0.05

PkatG-GFP 3.2 ± 0.03 3.4 ± 0.03 <0.05

PrecA-YFP 7.3 ± 0.10 8.2 ± 0.20 <0.05

PibpA-GFP 4.9 ± 0.05 5.2 ± 0.05 <0.05

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Fig. 2. DNA replication errors in various subpopulations. We coupled the MutL-based assay with the following fluorescent transcriptional promoter reporters: P1rrnB (A to C), PkatG (D to F), PrecA (G to I), and PibpA (J to L). We plotted the mean fluorescence detected per cell against the number of foci per cell (A, D, G, and J). Heteroge-neity of fluorescence detected in isogenic cells (B, E, H, and K). We further examined the cells having the strongest (top 10% of the population) and the weakest (the bottom 10% of the population) fluorescence for the distribution of the number of foci per cell (C, F, I, and L). We observed a statistically significant difference between cells with ≥1 and no MutL foci among all reporters (see Table 1 and table S3). AU, arbitrary units.

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translation error rates are at least an order of magnitude higher than transcription and DNA replication error rates (8). Drummond and Wilke estimated that more than 20% of individual proteins in E. coli contain at least one misincorporated amino acid in an average pro-tein length of around 300 amino acid residues (28). These high translation error rates allow monitoring translation fidelity at the single-cell level (29). For this study, we constructed a translation er-ror reporter based on the introduction of a stop codon in the gfp gene. Stop codons cause termination of translation and ribosome detachment from the messenger RNA. However, functional green fluorescent protein (GFP) can be produced not only by translation errors but also by transcription errors. To construct the reporter, which is not affected by transcription errors, we introduced two mu-tations in the gfp gene generating the UAG stop codon from the ly-sine AAA codon. Production of functional GFP due to translation

errors therefore occurs at the transfer RNA level. Double mutation renders very unlikely that transcription errors produce functional mRNA. We then introduced the mutated GFP reporter in the chro-mosome with a Ptet promoter (Fig. 3). As a control for the activity of the Ptet promoter, the promoter was coupled with a functional mCherry gene in a nearby locus of the chromosome. We confirmed the validity of the translation error reporter by treating cells with sub- inhibitory concentrations of streptomycin, an antibiotic known to increase mistranslation, as well as by introducing an rpsD mutation, which is known to increase translation errors (30). We also ob-served cell-to-cell heterogeneity of the spontaneous translation er-rors (Fig. 3 and fig. S6).

Next, we coupled the translation error reporter with the MutL-based assay in the mutH strain, and we found that the top 5% of the cells showing strong translation error reporter fluorescence had

Fig. 3. Translation error reporter and DNA replication errors in cells with different levels of translation errors. (A) Construct of the chromosomal translation error reporter. We introduced two point mutations in the sfgfp gene, which is under control of the Ptet promoter, creating the UAG stop codon from the lysine AAA codon. As a control, a functional mCherry gene, which is also under control of the Ptet promoter, was added. (B to D) Validation of the translation error reporter. (B) Correlation be-tween the fluorescence signals from mCherry and sfGFP. We found a significant but weak correlation, and we examined 334 cells. (C) The fluorescence signals increased in cells with an rpsD mutation, which is known to increase translation errors, and when cells were treated with sub-minimal inhibitory concentrations of streptomycin, an antibiotic known to increase mistranslation. (D) Representative flow cytometry histogram showing GFP intensity in cells after heat shock treatment from exponentially growing culture (OD600 = 0.2) to later growth phase (OD600 = 0.6). (E) The MutL-based assay was coupled with a translation error reporter in the mutH strain and the wild-type (WT) strain. The 5% of the cells with the strongest translation error reporter fluorescence (high translation error) had statistically significant more cells with ≥1 focus than the 5% of the cells with the weakest fluorescence (low translation error) in both strains (see table S3).

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significantly more MutL foci than the 5% of the cells with the weakest translation error reporter fluorescence (Fig. 3D and table S3). We did not observe this when we compared the top 10% of the cells show-ing strong fluorescence versus the 10% with the weakest fluorescence (fig. S6C and table S3). Therefore, the association between sponta-neous translation errors and DNA replication errors is observed only in small subpopulations, which corroborated a hypothesis based on theoretical calculations (8).

Finally, we verified whether an observed increase in the frequen-cy of MutL foci between the top 5% of the cells showing the stron-gest and the top 5% of the cells showing the weakest translation error reporter fluorescence observed in the mutH background could also be observed in the mismatch repair–proficient background. We found that an increase in the frequency of MutL foci was even stronger, that is, 4.6-fold versus 2.1-fold (Fig. 3E). Overproduction of the MutS protein slightly (but not significantly) reduced this in-crease (fig. S7 and table S3), just like in the streptomycin-treated cells (fig. S5). This suggests that an increase in translation errors af-fects some proteins participating in the mismatch repair–mediated elimination of DNA replication errors other than MutS and MutL proteins, such as UvrD helicase and different exonucleases.

DISCUSSIONHere, we explored the origins of spontaneous mutations in rapid-ly growing E. coli cells using the MutL-based assay that allows de-tection of DNA replication errors occurring in single cells, while simultaneously assaying induction of stress responses and transla-tion fidelity. Results of our study indicate that most spontaneous DNA replication errors in proliferating cells arise in subpopulations of cells suffering from endogenous stresses, that is, problems with proteostasis, genome maintenance, and reactive oxidative species production.

Endogenous stresses may be induced by toxic metabolic by- products, spontaneous chemical alteration of cellular components, and stochastic fluctuations in the abundance of the low-copy pro-teins, all of which can also affect genome integrity and fidelity of DNA replication (31–33). For instance, Pennington and Rosenberg (24) observed that around 0.9% of the exponentially growing E. coli cells have spontaneously induced SOS response, which is mainly induced by double-strand DNA breaks. Cellular protection and repair systems fix most damages, but damages in some cells may escape repair and cause mutations. Jee et al. (34) observed that over-production of catalase, which eliminates H2O2, decreases eightfold spontaneous mutation rates in populations of growing E. coli cells. This suggests that there is either high reactive oxidative species pro-duction or suboptimal capacity to deal with this stress in some cells. The latter possibility is supported by the observation that fluctua-tions in O6-alkylguanine transferase protein abundance can com-promise repair of spontaneous mutagenic DNA alkylation damages in E. coli and create a subpopulation of cells with increased muta-tion rates (35). Here, we also observed that MutS protein amount might be limiting for the spontaneous DNA replication error detec-tion in growing cells.

The quantity of proteins can vary not only because of the ran-dom distribution of the low-abundance protein during cell division but also because of high translation errors that may produce mutated and truncated proteins, which may have loss of function, partial function, and dominant negative properties. The resulting subopti-

mal quantity and/or quality of proteins implicated in DNA repli-cation, DNA repair, and other cellular functions may affect sponta-neous mutation rates. We found that there are significantly more DNA replication errors in subpopulations of cells having high translation errors in both mismatch repair–proficient and –deficient strains.

Spontaneous mutation rates are kept at a minimum level because the probability that one newly generated mutation is deleterious or lethal is much higher than the probability that it is beneficial (36). The presence of subpopulations of phenotypic mutators, similar to those we described in this study, is not expected to affect the average mutation frequency and therefore to reduce the population mean fitness in a stable environment. Therefore, there should be no selec-tive pressure for the evolution of molecular mechanisms, which will further decrease subpopulations of phenotypic mutators. Neverthe-less, these subpopulations can contribute to the population adapt-ability in fluctuating environments because they represent a reservoir of increased genetic variability (37). In particular, their presence could be important when a combination of several mutations is required for adaptation, such as to cross fitness valleys (38).

MATERIALS AND METHODSBacterial strains and growth conditionsBacterial strains used in this study are described in table S1. All strains were derivatives of the E. coli K12 MG1655 strain. When needed, mu-tants were constructed by P1 transduction of alleles from the INSERM U1001 laboratory strain collection or the Keio collection (39).

Overnight liquid cultures were grown in LB medium (Difco) at 37°C with 150 rpm shaking. When needed, medium was supplemented with antibiotics (Sigma-Aldrich) at the following concentrations: chloramphenicol (30 g/ml), ampicillin (100 g/ml), kanamycin (50 g/ ml), norfloxacin (0.04 g/ml), paromomycin (5 g/ml), rifam-picin (100 g/ml), and streptomycin (1 or 5 g/ml).

Construction of the MutL-based DNA replication error reporterThe construction of mCherry fusions to wild-type mutL expressed from the Plac promoter on the pET-32a–type plasmid is described below. The plasmid encoding mCherry-MutL was made by using primers 5′-GCAGATCTATTAAAGAGGAGAAAGGTACCATGGT-GAGC-3′ and 5′-ATGAATTCGCCAGATCCTGATCCAGATCCT-GAGCCGCTCTTGTACAGCTCGTCCATGCCG-3′ to amplify the fragment encoding mCherry from the template p37pRD131 (labo-ratory collection). These primer pairs contain a Bgl II and an Eco RI restriction site at the N terminus and the C terminus, respectively. The polymerase chain reaction (PCR)–amplified fragments were digested by Bgl II and Eco RI and cloned in the pYFP-MutLCmR (13), creating the plasmid pmCherry-MutLCmR. Then, using the gene replacement technique, the plasmid pmCherry-MutLCmR was used as a template to integrate the mCherry-mutL-cat under a lac-tose promoter into the chromosome. Primers that include sequences homologous to the chromosomal lactose region were used to amplify the mCherry-mutL-cat. The primer 5′-TGTGTGGAATTGTGAGC-GGATAACAATTTCACACAGGAAACAGCTATGGTGAG-CAAGGGCGA-3′ contains a 45-bp sequence homologous to the lactose promoter (from −1 to −45), and the primer 5′-CACCAGACCAACTG-GTAATGGTAGCGACCGGCGCTCAGCTGTTAGCAGCCG-GATCTCAGTG-3′ contains a 40-bp sequence homologous to the

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lacZ C terminus (from 3026 to 3065). The amplified PCR fragments were introduced to the MG1655 mutL::frt strain by transformation con-taining the plasmid pKD46. Clones with a chloramphenicol-resistant and nonfunctional lacZ gene were selected and named AW052.

Construction of the translation error reporterThe construction of the translation error reporter is described be-low. Primers 5′-CGCCATCATACCCAACGTAACAAAACTCAG-GCACGTTCCCGATATCAAATTACGCCCCGC-3 ′ and 5 ′ -GAAAGAATAATGCAAAACAGAAAATGGATTTT-GACCTCGCGATGCCTCTAGATTTAAATG-3′ that contain a se-quence homologous to yzgL were used to amplify the fragment encoding the cat-sfgfp from strain RB166. The PCR fragments were integrated into the chromosome of strain MG1655, resulting in strain TD049. Primers 5′-CGCCATCATACCCAACGTAACAA AAC-TCAGGCACGTTCCCGATATCAAATTACGCCCCGC-3′ and 5 ′ -GAAAGAATAATGCAAAACAGAAAATGG A T T T T -GACCTCGCCATATGAATATCCTCCTTAG-3′ that contain a sequence homologous to yzgL were used to amplify the fragment encoding the cat-sfgfp-kan from strain RB166, and the PCR frag-ments were integrated into the chromosome of strain MG1655, resulting in strain TD050. Next, the primer 5′-ATGGCCGACTCT-GGTGACTACCCTGACCTAGGGTGTTCAGTTAAGAC-CCACTTTCACATT-3′, which contains a mutated stop codon switched from AAA to UAG at the codon coding for the 53rd amino acid in the flanking homologous regions of the sfgfp gene, and the primer 5 ′ -AATCATGCTGCTTCATGTGATCCGGGTAACGA-GAAAAACACTAAGCACTTGTCTCCTG-3′ were used to amplify the tetracycline-resistant gene, tet, and introduced into TD049 by lambda Red recombination (40). By this, the downstream part of the sfgfp gene was replaced by the tet gene. The primer 5′-CTGACC-CTGAAATTCATCTGCACTACCGGTTAGCTGCCGG-3′, which also carried a mutated stop codon switched from AAA to UAG in the flanking homologous regions of the sfgfp gene, and the primer 5 ′ -GAAAGAATAATGCAAAACAGAAAATGG A T T T T -GACCTCGC-3′ were then used to amplify the downstream part of the sfgfp gene and kan from TD050 and introduce it into the cat-sfgfp-tet construct. By this, the tet gene was replaced with the downstream part of the sfgfp gene and kan, resulting in the kanamycin-resistant but tetracycline-sensitive strain TD299. Finally, primers 5′-CGC-CATCATACCCAACGTAACAAAACTCAGGCACGTTCCCGATATC-GACGTCTAAGAAAC-3′ and 5′-GAAAGAATA ATGCAAAACA-GAAAATGGATTTTGACCTCGCCATATGAATATCC TC CTTAG-3′ were used to transfer the stop codon- sfgfp-kan construct from TD299 into MG1655, resulting in strain TD339.

Determination of rifampicin-resistant mutant frequenciesLB medium was inoculated with fewer than 100 cells from over-night culture so that there were no preexisting rifampicin mutants in the starting inoculum. Cells were grown in LB medium overnight with shaking at 37°C. Cells with appropriate dilutions were plated on LB agar medium to determine the total number of colony-forming units and on a selective medium, LB agar medium supplemented with rifampicin (100 g/ml), to detect rifampicin-resistant mutants. Colonies were counted after 24 hours of incubation at 37°C.

Microscopy and image analysisTester strains were grown in LB medium with 0.1 mM isopropyl--d- thiogalactopyranoside overnight at 37°C with shaking. Over-

night culture was diluted 1:400 (v/v) and incubated at 37°C with shaking. Cells from exponentially growing culture (OD600 = 0.2) were washed with phosphate-buffered saline (PBS) and inoculated onto a solid matrix of minimal medium agarose in microscope cav-ity slides, as described previously (14). Cells were examined with a 100× objective on an Axiovert 200 inverted microscope (Carl Zeiss) equipped with a Photometrics CoolSNAP camera (Princeton In-struments). Images were recorded and analyzed by MetaMorph software and ImageJ software, respectively.

Flow cytometry and cell sortingBacterial cultures were grown in LB medium overnight with shaking at 37°C. Overnight cultures were diluted 1:400 (v/v) and incubated at 37°C with shaking. Different treatments were applied to cells from exponentially growing culture (OD600 = 0.2): streptomycin (1 g/ml), norfloxacin (0.04 g/ml), and 5 mM hydrogen peroxide. Cells were washed with PBS and analyzed with a cytometer (Beckman Coulter) when the cultures reached an OD600 of 0.6.

To test the impact of heat shock response on the reporters, cul-tures were grown overnight with shaking at 30°C. Overnight cul-tures were diluted 1:400 (v/v) and incubated at 30°C with shaking until the cultures reached an OD600 of 0.2. Then, cultures were grown at 42°C with shaking, and cells were analyzed with a cytometer when cultures reached an OD600 of 0.6. FlowJo (LLC) was used to ana-lyze data.

Samples for cell sorting were incubated overnight at 37°C with shaking. Overnight cultures were diluted 1:400 (v/v) and grown at 37°C with shaking until cultures reached an OD600 of 0.2. Cultures were then washed and resuspended in PBS before sorting using a cell sorter (BD FACSAria). Cells were analyzed for GFP expression before cell sorting to determine the populations to be sorted. Gates were drawn to separate the 5% of the cells expressing lower levels of GFP and the 5% of the cells expressing higher levels of GFP. Cells (7 × 105) were collected for each population. Cells were concentrated and analyzed with a microscope after cell sorting.

Statistical analysesAll statistical analyses were performed using the software MATLAB.

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/6/eaat1608/DC1fig. S1. MutL reporters, cell area, and number of foci per cell.fig. S2. DNA replication errors and transcriptional promoter reporter P2rrnB.fig. S3. Induction of fluorescent transcriptional promoter reporters.fig. S4. Determination of minimum inhibitory concentration (MIC) of aminoglycosides for the E. coli wild-type strain MG1655 and the impact of sub-MIC of aminoglycoside on DNA replication errors.fig. S5. DNA replication errors of different strains with sub-MIC of streptomycin.fig. S6. DNA replication errors and translation error reporter.fig. S7. DNA replication errors and translation errors in wild-type cells with excess MutS proteins.table S1. Bacterial strains and plasmids.table S2. The frequency of appearance of rifampicin mutants in mismatch repair–proficient and –deficient strain.table S3. Chi-square test results of MutL foci frequency in this study.

REFERENCES AND NOTES 1. J. H. Miller, Mutators in Escherichia coli. Mutat. Res. 409, 99–106 (1998). 2. T. A. Kunkel, DNA replication fidelity. J. Biol. Chem. 279, 16895–16898 (2004). 3. J. W. Schroeder, P. Yeesin, L. A. Simmons, J. D. Wang, Sources of spontaneous

mutagenesis in bacteria. Crit. Rev. Biochem. Mol. Biol. 53, 20–48 (2018).

on October 12, 2019


Dow

nloaded from

http://advances.sciencemag.org/cgi/content/full/4/6/eaat1608/DC1

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8 of 8

4. A. A. M. Al Mamun, M.-J. Lombardo, C. Shee, A. M. Lisewski, C. Gonzalez, D. Lin, R. B. Nehring, C. Saint-Ruf, J. L. Gibson, R. L. Frisch, O. Lichtarge, P. J. Hastings, S. M. Rosenberg, Identity and function of a large gene network underlying mutagenic repair of DNA breaks. Science 338, 1344–1348 (2012).

5. A. R. Oller, R. M. Schaaper, Spontaneous mutation in Escherichia coli containing the dnaE911 DNA polymerase antimutator allele. Genetics 138, 263–270 (1994).

6. R. M. Schaaper, Suppressors of Escherichia coli mutT: Anitimutators for DNA replication errors. Mutat. Res. 350, 17–23 (1996).

7. J. A. Imlay, Oxidative stress. EcoSal Plus 3, (2009). 8. J. Ninio, Transient mutators: A semiquantitative analysis of the influence of translation

and transcription errors on mutation rates. Genetics 129, 957–962 (1991). 9. L. Boe, Translational errors as the cause of mutations in Escherichia coli. Mol. Gen. Genet.

231, 469–471 (1992). 10. K. T. Nishant, N. D. Singh, E. Alani, Genomic mutation rates: What high-throughput

methods can tell us. Bioessays 31, 912–920 (2009). 11. L. Robert, J. Ollion, J. Robert, X. Song, I. Matic, M. Elez, Mutation dynamics and fitness

effects followed in single cells. Science 359, 1283–1286 (2018). 12. M. Elez, L. Robert, I. Matic, Method for detecting and studying genome-wide mutations in

single living cells in real time. Methods Mol. Biol. 1736, 29–39 (2018). 13. M. Elez, M. Radman, I. Matic, Stoichiometry of MutS and MutL at unrepaired mismatches

in vivo suggests a mechanism of repair. Nucleic Acids Res. 40, 3929–3938 (2012). 14. M. Elez, A. W. Murray, L.-J. Bi, X.-E. Zhang, I. Matic, M. Radman, Seeing mutations in living

cells. Curr. Biol. 20, 1432–1437 (2010). 15. M. G. Marinus, DNA mismatch repair. EcoSal Plus 5, (2012). 16. G. Feng, H.-C. T. Tsui, M. E. Winkler, Depletion of the cellular amounts of the MutS and

MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells. J. Bacteriol. 178, 2388–2396 (1996).

17. A. Gutierrez, L. Laureti, S. Crussard, H. Abida, A. Rodríguez-Rojas, J. Blázquez, Z. Baharoglu, D. Mazel, F. Darfeuille, J. Vogel, I. Matic, -Lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity. Nat. Commun. 4, 1610 (2013).

18. H. D. Murray, R. L. Gourse, Unique roles of the rrn P2 rRNA promoters in Escherichia coli. Mol. Microbiol. 52, 1375–1387 (2004).

19. M. S. Bartlett, R. L. Gourse, Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli. J. Bacteriol. 176, 5560–5564 (1994).

20. M. E. Lidstrom, M. C. Konopka, The role of physiological heterogeneity in microbial population behavior. Nat. Chem. Biol. 6, 705–712 (2010).

21. K. M. Davis, R. R. Isberg, Defining heterogeneity within bacterial populations via single cell approaches. Bioessays 38, 782–790 (2016).

22. L. A. Simmons, J. J. Foti, S. E. Cohen, G. C. Walker, The SOS regulatory network. EcoSal Plus 3, (2008).

23. J. L. Rodrigues, L. R. Rodrigues, Potential applications of the Escherichia coli heat shock response in synthetic biology. Trends Biotechnol. 36, 186–198 (2018).

24. J. M. Pennington, S. M. Rosenberg, Spontaneous DNA breakage in single living Escherichia coli cells. Nat. Genet. 39, 797–802 (2007).

25. E. Gur, D. Biran, E. Z. Ron, Regulated proteolysis in Gram-negative bacteria—How and when? Nat. Rev. Microbiol. 9, 839–848 (2011).

26. L. Brakier-Gingras, P. Phoenix, The control of accuracy during protein synthesis in Escherichia coli and perturbations of this control by streptomycin, neomycin, or ribosomal mutations. Can. J. Biochem. Cell Biol. 62, 231–244 (1984).

27. L. Ren, M. S. Rahman, M. Z. Humayun, Escherichia coli cells exposed to streptomycin display a mutator phenotype. J. Bacteriol. 181, 1043–1044 (1999).

28. D. A. Drummond, C. O. Wilke, The evolutionary consequences of erroneous protein synthesis. Nat. Rev. Genet. 10, 715–724 (2009).

29. M. Meyerovich, G. Mamou, S. Ben-Yehuda, Visualizing high error levels during gene expression in living bacterial cells. Proc. Natl. Acad. Sci. U.S.A. 107, 11543–11548 (2010).

30. D. I. Andersson, K. Bohman, L. A. Isaksson, C. G. Kurland, Translation rates and misreading characteristics of rpsD mutants in Escherichia coli. Mol. Gen. Genet. 187, 467–472 (1982).

31. P. C. Burcham, Internal hazards: Baseline DNA damage by endogenous products of normal metabolism. Mutat. Res. 443, 11–36 (1999).

32. C. L. Linster, E. Van Schaftingen, A. D. Hanson, Metabolite damage and its repair or pre-emption. Nat. Chem. Biol. 9, 72–80 (2013).

33. P. J. Choi, L. Cai, K. Frieda, X. S. Xie, A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442–446 (2008).

34. J. Jee, A. Rasouly, I. Shamovsky, Y. Akivis, S. R. Steinman, B. Mishra, E. Nudler, Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing. Nature 534, 693–696 (2016).

35. S. Uphoff, N. D. Lord, B. Okumus, L. Potvin-Trottier, D. J. Sherratt, J. Paulsson, Stochastic activation of a DNA damage response causes cell-to-cell mutation rate variation. Science 351, 1094–1097 (2016).

36. E. Denamur, I. Matic, Evolution of mutation rates in bacteria. Mol. Microbiol. 60, 820–827 (2006).

37. H. K. Alexander, S. I. Mayer, S. Bonhoeffer, Population heterogeneity in mutation rate increases the frequency of higher-order mutants and reduces long-term mutational load. Mol. Biol. Evol. 34, 419–436 (2017).

38. J. W. Drake, Too many mutants with multiple mutations. Crit. Rev. Biochem. Mol. Biol. 42, 247–258 (2007).

39. T. Baba, T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. L. Wanner, H. Mori, Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

40. K. A. Datsenko, B. L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97, 6640–6645 (2000).

AcknowledgmentsFunding: This work was supported by FRM Grant DBF20160635736. A.C.W. was supported by a postdoctoral fellowship from Labex “Who am I?” Idex ANR-11-IDEX-0005-01/ANR-11-LABX-0071, Idex-Sorbonne Paris Cité. Author contributions: A.C.W. and I.M. designed the study. A.C.W. and L.F. performed experiments and analysis. A.C.W., L.F., and T.D. constructed strains. A.C.W. and I.M. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

Submitted 30 January 2018Accepted 14 May 2018Published 20 June 201810.1126/sciadv.aat1608

Citation: A. C. Woo, L. Faure, T. Dapa, I. Matic, Heterogeneity of spontaneous DNA replication errors in single isogenic Escherichia coli cells. Sci. Adv. 4, eaat1608 (2018).

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cellsEscherichia coliHeterogeneity of spontaneous DNA replication errors in single isogenic Anthony C. Woo, Louis Faure, Tanja Dapa and Ivan Matic

DOI: 10.1126/sciadv.aat1608 (6), eaat1608.4Sci Adv

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