Emergence of resistance from multinucleated bacterial filaments

Julia Bosa, Qiucen Zhangb, Saurabh Vyawaharea, Elizabeth Rogersc, Susan M. Rosenbergc, and Robert H. Austina,1

aDepartment of Physics, Princeton University, Princeton, NJ 08544-0708; bDepartment of Physics, University of Illinois at Urbana–Champaign, Urbana, IL 61801- 3080; and cDepartments of Molecular and Human Genetics, Biochemistry and Molecular Biology, and Molecular Virology and Microbiology, and the Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030

Contributed by Robert H. Austin, November 3, 2014 (sent for review August 26, 2014) can rapidly evolve resistance to via the SOS sized offspring cells (8). However, the mechanisms underlying the response, a state of high-activity DNA repair and mutagenesis. We evolution of filamentous bacteria when the selective pressure is explore here the first steps of this evolution in the bacterium maintained have been poorly described, in part because most studies . Induction of the SOS response by the genotoxic have used stress conditions that were evolutionarily unfavorable and antibiotic ciprofloxacin changes the E. coli rod shape into multichro- instead led to irreversible bacterial growth arrest and/or rapid death. mosome-containing filaments. We show that at subminimal inhib- Bacterial cell-division arrest resulting from inhibition of DNA itory concentrations of ciprofloxacin the bacterial filament divides replication after cipro exposure (2) is caused by the induction of asymmetrically repeatedly at the tip. Chromosome-containing buds a division inhibitor (e.g., SulA in E. coli) (13, 15). sulA gene in- are made that, if resistant, propagate nonfilamenting progeny with duction is part of a set of over 30 induced genes involved in DNA enhanced resistance to ciprofloxacin as the parent filament dies. We repair pathways and error-prone mechanisms, collectively known propose that the multinucleated filament creates an environmental as the SOS response (5, 16, 17), a sequential transcriptional niche where evolution can proceed via generation of improved process that is tightly controlled by two master regulators, LexA mutant chromosomes due to the mutagenic SOS response and pos- and RecA proteins (18). When the replication forks are stalled, sible recombination of the new alleles between chromosomes. Our LexA autoproteolytic cleavage facilitated by RecA protein ini- data provide a better understanding of the processes underlying tiates the SOS stress pathway until the chromosome damage has the origin of resistance at the single-cell level and suggest an anal- been repaired and replication of the chromosome resumes. ogous role to the eukaryotic aneuploidy condition in cancer. However, when the SOS response is fully engaged, the induction of low-fidelity replication polymerases known as error-prone antibiotic resistance | SOS response | filamentation | mutation | evolution polymerases increases the rate of mutation μ during DNA rep- − lication from the normally low value of 10 9 mutations per base −5 acteria can evolve remarkably quickly to be resistant to pair per generation up to a high rate of 10 mutations per base Bantibiotics under certain conditions, as we showed in an pair per generation (19), so that a single bacterial chromosome earlier paper (1) using microfabrication techniques to create such as the 4.6-Mb E. coli chromosome can be expected to have a structured environment of bacteria in a gradient of the genotoxic multiple mutations with each round of replication. Current re- antibiotic ciprofloxacin (cipro), which perturbs chromosome repli- search aims at targeting the error-prone polymerase pathway to cation (2). The need for the structured environment in which steep slow down the evolution of antibiotic resistance and thus im- gradients facilitate the rapid evolution of resistance by multiple prove the long-term viability of some antibiotic drugs (20). mutations confirmed a prediction from recent modeling (3). Our Here we address how filamentation is probably the first step in earlier work on the rapid emergence of cipro resistance in a the evolution of resistance to mutagenic antibiotic exposure, microfabricated complex ecology did not delineate the stages in the internally generating mutant chromosomes via the SOS response emergence of resistance but only presented the ultimate, resistant until a solution is obtained: a mutant chromosome with re- cells (1). However, high-resolution imaging of the cells within the sistance to the stress. We examine how individual bacteria exit device before the full emergence of resistance showed that the cells from the filamentous state caused by subminimal inhibitory went through a transient filamentation phase. Fig. 1 shows images concentrations of the genotoxic antibiotic ciprofloxacin. We from our microecology that Escherichia coli bacteria have changed show that when cipro is maintained at low levels, E. coli filaments their shape into long filamentous structures shortly after exposure to the cipro antibiotic. The filamentation response is not perma- Significance nent, because after 4 h some cells have reverted to a normal phe- notype (Fig. 1B) and have become resistant to high levels of the Understanding how bacteria rapidly evolve under antibiotic se- antibiotic by de novo mutation in genes encoding gyrase A top- lective pressure is crucial to controlling the development of re- oisomerase and efflux pumps (1). We wished to explore the role sistant organisms. We show that initial resistance emerges from filamentation played in the initial stages of antibiotic resistance. successful segregation of mutant chromosomes at the tips of Many microorganisms adopt a filamentous shape caused by filaments followed by budding of resistant progeny. We pro- cell-division arrest yet continue cell-volume growth in response pose that the first stages of emergence of resistance occur via to a variety of stressful environments, including nutrient de- the generation of multiple chromosomes within the filament ficiency (4), extensive DNA damage through the SOS response and are achieved by mutation and possibly recombination be- pathway (5, 6), host innate immune responses (7), desiccation tween the chromosomes. (8), high pressure (9), and antibiotic treatment (10–12). Al- though filamentation has long been considered an overstressed Author contributions: J.B., Q.Z., S.V., E.R., S.M.R., and R.H.A. designed research; J.B., Q.Z., and R.H.A. performed research; J.B., E.R., and S.M.R. contributed new reagents/analytic and/or sick phenotype or solely for completion of DNA repli- tools; J.B., Q.Z., S.M.R., and R.H.A. analyzed data; and J.B., S.M.R., and R.H.A. wrote cation before division (13), it has only recently been described the paper. as a more general survival strategy (7, 8, 14). For example, if the The authors declare no conflict of interest. stress is relieved quickly enough, division of the filamentous cell 1To whom correspondence should be addressed. Email: [email protected]. resumes synchronously and rapidly at regular intervals along the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. entire length of the filament, resulting in multiple viable normal- 1073/pnas.1420702111/-/DCSupplemental.

178–183 | PNAS | January 6, 2015 | vol. 112 | no. 1 www.pnas.org/cgi/doi/10.1073/pnas.1420702111 Downloaded by guest on October 2, 2021 ABfilaments after prolonged growth on low cipro showed less het- erogeneity than that of the buds, since the bulk of parental fil- aments (70%) undergo death as evidenced by their inability to exclude the propidium iodide (PI) dye, reflecting loss of possibly both membrane integrity and efflux mechanisms (Fig. 3B). These findings suggest that filamentation at low cipro is a complex phenomenon that gives rise to short progeny and eliminates the unfit filamentous parent cell. We were most interested by the buds that divide symmetrically without further filamentation and examined whether these buds show a lower level of SOS response. We used a sulA-gfp tran- scriptional reporter in a nongenic chromosomal location in cells Fig. 1. (A) E. coli bacteria form filaments in microhabitats after 3 h of ex- with an intact native sulA gene (22) to quantitatively measure posure to a ciprofloxacin gradient. (B) Cipro-resistant bacteria that have induction of the SOS response within the bacterial filament. We reverted to a normal-length phenotype (arrows) in one of the microhabitats. observed a decrease in the intensity of the gfp expression within buds, indicating that buds had decreased expression of the SOS regulon, as would be expected from the observed increase in undergo asymmetric divisions at the filament tips, giving rise to cipro resistance in the buds and subsequent suppression of the budded resistant offspring cells. The generation of multiple mutant SOS response [Fig. 3 shows that the buds have on average about chromosomes that are successfully segregated toward the fila- 25% less expression of PsulA-gfp than the filaments, whereas the ments tips favors the birth of antibiotic-resistant cells. We discuss intensity of constitutively expressed RFP protein (lac-mrfp1) how this filamentation step is a precursor to the final highly re- shows identical levels both in the buds and in the filaments, in- sistant bacteria in a complex ecology. dicating that protein expression levels are similar in the buds and Results within the filaments]. Because most buds do contain a chromosome, although cipro We focused on the role that the SOS mutational network, trig- directly affects DNA replication by targeting DNA gyrases (23) gered by the presence of stalled replication forks, plays in the and thus perturbs chromosome dynamics (2), we wished to ex- filamentation process and bacterial evolution of drug resistance. amine both the content and the spatial arrangement of the The transient filamentation events were studied on agar plates chromosomes in filamentous cells to understand how low doses containing cipro at varying levels. The primary experiment in- 3 of cipro allow the generation of short viable nucleated offspring. volves spreading a low-density population of less than 10 per mm2 of E. coli bacteria in the presence of 0.125 of the minimal We used standard DAPI staining and live imaging of a specific inhibitory antibiotic concentration (0.125× MIC) of cipro. Time- chromosomal locus, R2 (17), as a reporter of an individual lapse imaging revealed that 99.5% of the bacteria formed long chromosomal copy (SI Appendix, Fig. S2) to enhance chromo- filaments (up to 200 μm) and expanded their length exponen- some counting. R2 loci were visualized by the binding of fluo- tially (Fig. 2A; see Movies S1 and S2 for videos of the growth of rescent TetR-CFP fusions to the tetO operator array located filaments). The remaining 0.5% of the bacteria that did not 21 kb upstream of the lacZ gene (24) (SI Appendix, Fig. S2). elongate or divide in the presence of the antibiotic may be per- Individual chromosomes localize as discrete CFP foci. We found sister cells (21) or dead cells. A lexA3 mutant strain that cannot activate the SOS response and a recB mutant strain, which reduces SOS response induction, general recombination, and AC DNA repair, were not able to filament or grow at these low cipro concentrations, verifying that both filamentation and growth rely on the induction of the LexA-dependent SOS response and possibly on recombination and repair pathways (SI Appendix, Fig. S1A shows the growth of filaments over a 3-h time period and the failure of the lexA3 and recB mutant strains to grow under the same conditions). We observed that the growth of a filament is transiently interrupted by asymmetric division events (Fig. 2B and Movies S1 and S2) that occur repeatably and

predominantly at the tips of the filament (Fig. 2C) and give rise EVOLUTION to short offspring cells that often resumed normal division (Fig. 2B and Movies S1 and S2). This budding process was inherently B distinct from the synchronous, symmetrical division recovery profile of the filament obtained shortly after drug removal (SI Appendix, Fig. S1). Interestingly, some degree of heterogeneity was observed in the bud fate at this low concentration (0.125× MIC) (Fig. 3A): 35% of budded cells divide symmetrically without further fila- mentation, indicating that they are now resistant to 0.125× MIC cipro; 35% cease division and growth; 10% of the buds show abnormal asymmetrical division (minicell formation) with little propagation; and 20% resume filamentation, indicating that they do not show evidence of cipro resistance. Buds that fail to divide Fig. 2. (A) Semilog plot of the contour length of a collection of filaments (n = 20) growing on agar containing 0.125× MIC of cipro vs. time. (B)Example and propagate may either contain a damaged chromosome or of a budding process indicated by the white arrows at either end of a filament. may not contain a chromosome at all [we found that 11% of the Yellow arrows show budded cells resuming normal division. (Scale bar, 5 μm.) buds do not contain a chromosome, by using 4′,6-diamidino- (C) Distribution of budding positions along the filaments (n = 95 cells, average 2-phenylindole (DAPI) staining]. Conversely, the fate of the length 42 ± 16 μm, average time at division 332 ± 71 min).

Bos et al. PNAS | January 6, 2015 | vol. 112 | no. 1 | 179 Downloaded by guest on October 2, 2021 A B of the isolated progeny survived when reexposed to low cipro (0.125× MIC). The frequency of progeny resistant to high levels of cipro (1× MIC) was determined and defined as the number of cipro-resistant colonies that arose after 24 h on solid media containing 1× MIC cipro per viable cell. Progeny showed a 250- − fold increase in resistance frequency [2.8 (±0.3) × 10 6]at1× − MIC cipro compared with that of the parent cells [7.2 (±3) × 10 8] that had not been preexposed to low cipro. This result indi- cates that a subpopulation of budded cells has indeed evolved resistance during the 24-h exposure to low cipro. We assumed C DE that the frequency of persisters was kept low in the progeny population, even after filtration, because progeny either formed filaments or resumed normal division when exposed over again to low cipro. Instead, persisters would have resumed division and growth only once the antibiotic is removed (21). Resistant clones were isolated by selection using subinhibitory (0.5× MIC) or inhibitory (1× MIC) doses of cipro, and a subset of genomic sequences of known resistant genes was analyzed by using targeted genome sequencing (Table 1 and SI Appendix, Table S2). The results showed that the level of resistance was Fig. 3. (A) Distribution of the different fates of the buds: normal symmet- a function of the cipro concentration at selection. Resistance at rical division (red), asymmetrical division (yellow), no division and no growth the bottom of the selection window was associated with over- (black), and filamentation (gray). (B) Death of long filaments after budding, expression of AcrAB efflux pumps, in that mutations in marR imaged after 5 h (Top) and 22 h (Bottom) of low cipro exposure using the were found and none of the other genes sequenced had muta- membrane-impermeable dye propidium iodide. Bright-field (BF), GFP, and tions. Deletion(s) of 1 or 11 nt in the marR gene encoding the μ RFP (PI) images are shown. (Scale bar, 2 m.) (C) Microscopic images of fil- MarR efflux pump regulator were identified in 37% of the se- aments and buds expressing sulA-gfp (Top) and constitutively expressed rfp (Bottom) in the same cells after 4 h of growth in low cipro. (Scale bar, 2 μm.) quenced clones. Conversely, at the top of the selection window, (D) Histogram of average sulA-gfp expression within filaments (n = 98 cells). the resistant clones isolated were mainly mutated in the GyrA The red line represents a GFP mean intensity of 528 ± 122 of the population. gyrase subunit, and 87% of the sequenced clones had a single (E) Histogram of average sulA-gfp expression within buds (n = 110 cells). The red line represents a GFP mean intensity of 393 ± 107 of the population. A B that the filaments contain multiple chromosomes (Fig. 4 and SI Appendix, Fig. S2) that are partially or completely segregated, with a linear relation between filament length and chromosome number (Fig. 4A), indicating that as the filament grows it repli- cates multiple chromosomes. The presence of multinucleated filaments occurred only at a low level of cipro (0.125× MIC) (SI Appendix, Fig. S2C). At levels of cipro above 0.125× MIC, the chromosome morphology was severely impaired (SI Appendix, Fig. S2C) and events of budding at the filament tips were rare or nonexistent. At low levels of cipro, we found that some chro- mosomes localize near the tips (three-quarter positions of cell C D length) (Fig. 4B), coinciding with the position of the division sites at the tips (Fig. 1C). Time-lapse experiments showed heteroge- neous dynamics of the chromosomes within the filaments where some chromosomes move tipward as the filaments grow whereas other remain essentially stationary in the midcell regions (Fig. 4 C and D; SI Appendix, Fig. S3; and Movie S3). In addition, two individual chromosomes were eventually observed to alternate between overlapping in a single cluster and then separating into two distinct foci (Fig. 4 C and D and SI Appendix, Fig. S3). We also noticed that the chromosomes within the same filament Fig. 4. (A) Chromosome count as a function of length as measured by DAPI undergo replication or separation of foci in an asynchronous way stain (red crosses) and R2-CFP loci (blue crosses). (B) Distribution of individual C D chromosome (R2-CFP) positions along the filaments after 4 h of growth with (Fig. 4 and ). All together, the chromosome dynamics in 0.125× MIC cipro (n = 50 cells, mean R2 locus counts per cell 4.5 ± 1.9, av- cipro-stressed filaments are complex and do not indicate simple erage filament length 20 ± 4 μm). (C) Time-lapse images of R2-CFP showing replication, but nevertheless the filamentous cell succeeds in complex movement of individual chromosomes along the filament length. coordinating chromosome placement and asymmetric division at (Scale bar, 1 μm.) Each red circle surrounding an R2 locus denotes an individual the tips, leading to the birth of resistant daughter cells. chromosome and results from automated tracking analysis software (Mate- We verified that the offspring evolved resistance in that rials and Methods). White arrowheads indicate events of merging/splitting normal-sized and dividing bacterial buds arose at the tips of the between two chromosomes. The yellow arrowhead indicates a chromosome filaments under continued low cipro treatment, and SOS ex- duplication/separation event. Dashed lines show the cell contours. (D) Distri- bution of R2-CFP locus positions in individual cells shown in C.Traceswere pression levels, as determined by the sulA-gfp reporter, were low. obtained by using a MATLAB-based particle tracking methodology; auto- Wild-type bacteria were cultured in low cipro and filtered using mated (filled circle) or manual tracking (open circles). (Materials and Meth- a sterile 5-μm syringe filter to isolate normal-sized progeny from ods). The black arrow indicates a replication/separation event near the tip. filamentous parent cells (SI Appendix, Fig. S4). About 70 ± 11% Gray arrows show merging and splitting events between two chromosomes.

180 | www.pnas.org/cgi/doi/10.1073/pnas.1420702111 Bos et al. Downloaded by guest on October 2, 2021 Table 1. Targeted-sequencing results showing genes mutated the tips, where they are then budded off by formation of a single in resistant progeny selected at 0.5× MIC and 1× MIC cipro septum. We can guess whether the cessation of the SOS response Cipro at is a global or local event in the cell by estimating how far the SOS selection, No. of mutants No. of mutants division-inhibitor SulA can diffuse during the replication time of Gene ng/mL (base substitution) (base deletion) a chromosome, about 30 min in our case. SulA has a molecular mass of about 18 kDa and measurements of the diffusion co- gyrA 20 0/16 0/16 efficient of similar-size GFP proteins in E. coli have yielded an gyrA 40 7/8 0/8 average value for the diffusion constant of about 7 μm2/s (26), marR 20 0/8 3/8 (marR Δ 1, Δ 11) and hence we would expect that in 30 min the SulA protein marR 40 0/8 0/8 should diffuse about 150 μm, easily the length of a filament. acrR 20 0/8 0/8 However, the budding process is local and not evenly distributed acrR 40 0/8 0/8 along the length of the filament, indicating that either SulA does rbsA 20 0/8 0/8 not diffuse freely in the cytoplasm or other players such as the rbsA 40 0/8 0/8 Min division control proteins are determining the local decision sulA 20 0/4 0/4 to bud. Identifying the molecular actors that govern the decision The number of mutants over the number of sequenced clones (e.g., 4, 8, of local budding will be of valuable interest to answer the or 16) is indicated. question of why asymmetric division is important to generating resistant progeny. This view differs from current paradigms for cell-cycle mutation in gyrA (GyrA S83A), which is located in the quinolone checkpoint control in bacteria and other organisms. Cell fila- resistance-determining region, a hot spot region for mutations mentation during SOS is thought to reflect the need to complete (25). Mutations in gyrA or marR are likely to be sufficient to DNA replication before cell division to protect chromosomes after promote intermediate-level resistance of the tested clones (Fig. DNA damage (13). Similarly, filamentation allows repair of DNA 5). The rest of the cipro-resistant clones presumably carry a re- damage by recombination between chromosomes by keeping sistance-conferring mutation(s) at a place not sequenced. more than one chromosome per cell until repair is completed The MIC of resistant progeny was 2.5× increased (10 clones (27). Similar DNA-protection roles underlie eukaryotic cell-cycle analyzed) but remained five times lower than that of the high- checkpoint control (28). We suggest that in addition to DNA level resistant strain QZ030, which evolved four mutations both preservation, cell filamentation may accelerate evolution by in gyrA and the efflux pumps when cultured in a complex envi- adding recombination, which is possible only with more than one ronment (1). Although the budded progeny show enhanced re- chromosome per cell, to the increased mutagenesis documented sistance to cipro, they do not show the full resistance observed in during SOS. Recombination between chromosomes can potentially the cells that grow in the microfabricated devices, indicating separate beneficial mutations from linked deleterious mutations or that the budded progeny are part-way along the path to a fully unrepaired DNA damage. The dramatic acceleration of evolution enhanced resistance (Fig. 5 compares the resistance of filtered by recombination accompanying mutagenesis, relative to muta- bud progeny to cipro with that of the strain of bacteria QZ030 genesis without recombination, is supported by modeling (29, 30). retrieved from our previous experiment, where evolution to re- Coupling of mutagenesis with recombination is observed sistance to cipro emerged in a complex gradient in a meta- throughout life, including being predicted in cancer genomes population, as opposed to strains extracted from this experiment, (31, 32), and observed in the immune system (33), yeast (34, 35) where evolution occurs in a fixed concentration of cipro). In all, and bacteria (17, 36–38). Because increased mutagenesis cre- the data reveal that a low level of adaptive evolution to low cipro ates both deleterious and beneficial mutations, recombination can occur in a few hours within a single filament. that unlinks these can be beneficial. Discussion Our data show that there are five fundamental features that characterize the growth of the bacterial filaments during exposure to subminimal inhibitory concentrations of cipro (Fig. 6): (i)Ex- posure to 0.125× MIC cipro initiates the SOS response (Fig. 3C and Fig. S1); (ii) the filament contour length L(t) increases exponen- tially with time (Fig. 2A); (iii) the filaments contain multiple copies

of the bacterial chromosome (Fig. 4); (iv) the filamenting process is EVOLUTION transiently interrupted by a budding process that generates resistant progeny at the tips (Fig. 1 and Movies S1 and S2); and (v)most filaments die, but not some of the budded progeny (Fig. 3B). Such an evolutionary path to antibiotic resistance encom- passes two remarkable events that occur during the filamentation process: the presumably local decision to transiently exit from SOS and bud a resistant daughter cell from the end of the fila- ment, and the generation of multiple chromosomes that evolve resistance via mutagenesis and possibly recombination within a single filament. The budding events are presumably triggered by the local cessation of the SOS response in the vicinity of an evolved re- sistant chromosome with the mutations found in Table 1, be- cause the SOS response is responsible for the filamentation phenotype caused by a lack of septation. It could be either that Fig. 5. Resistance of the wild-type strain, a QZ030 hyperresistant isolate (1), better-adapted chromosomes are generated near the tips or that and three different progeny clones to cipro as measured by growth curves. better-adapted chromosomes generated internally migrate toward The cipro concentration at which growth was monitored is indicated.

Bos et al. PNAS | January 6, 2015 | vol. 112 | no. 1 | 181 Downloaded by guest on October 2, 2021 Materials and Methods Imaging. Long time-lapse movies (images recorded every 2 min for 10 h) of low-density bacteria [JEK1036 (gfp+)] growing on LB agar pads containing 5 ng/mL cipro and isopropyl β-D-1-thiogalactopyranoside (IPTG) were made using a Nikon 90i microscope equipped with a Nikon Plan Apo 60× objective and a Rolera-XR cooled CCD camera and processed with Micro-Manager (NIH, www.micro-manager.org) and ImageJ (NIH) software. All other images were collected on a Nikon Eclipse 90 microscope equipped with a Nikon Plan Apo 100× objective and an Andor Neo camera and processed with Nikon NIS-Elements software. In all experiments, cells were transferred from liquid culture to 1.4% agarose-padded slides containing LB medium (or 0.5× LB for strain AB1157), with low ciprofloxacin and IPTG when required. A coverslip Fig. 6. Basic model for the initial stages of resistance in filamentous cells. was placed on the pad and then sealed with valap (1 vol vaseline/1 vol lanolin/ Light gray cells are initial sensitive cells, dark gray cells have double-strand 1 vol paraffin). Filament death was visualized by fluorescence microscopy breaks and stalled replication forks, green cells have evolved moderate an- using propidium iodide, a membrane-impermeable nucleic acid stain com- tibiotic resistance, and red cells are undergoing death. Chromosomes are monly used as a cell-death marker. Nucleoids in the filaments were visualized shown within the cells. by adding DAPI (live-cell DNA staining) directly to the agarose pads. R2 loci were visualized in E. coli AB1157 cells placed on 1.4% agarose-padded slides containing 0.5× diluted LB medium (to reduce fluorescence background). Further, the chromosomes display a distinct dynamics pattern Images were recorded every 2 min right after the cells were placed on the in the same filament. Events of merging/splitting between two agarose pad in the no-cipro conditions, or every 5 min after the cells were chromosomes were frequently observed during the experiment allowed 2 h of growth on the agarose pad in the low-cipro conditions. See SI Appendix, Materials and Methods for details of experimental protocols. (Fig. 4C; SI Appendix, Fig. S3; and Movie S2), possibly reflecting the intimate interplay between DNA replication and genetic Bacterial Strains and Growth. E. coli strains were cultured at 37 °C in liquid LB information exchange during cipro exposure. In addition, repli- medium and on an LB agar (1.7%) plate (solid), unless noted. In all experi- cation of sister loci did not occur synchronously, as shown in ments, cipro antibiotic was added at a subminimal inhibitory concentration Fig. 4C, where one chromosome out of six was captured while of 5 ng/mL (0.125× MIC) referred to as “low cipro,” unless indicated (see replicating, and suggests potential differential DNA damage SI Appendix, Materials and Methods for details of strain growth). Strains load between the chromosomes (39). Although we found these and primers used in this study are listed in SI Appendix, Tables S1 and observations captivating, it remains unclear to us why there is S2, respectively. such heterogeneity in the chromosome dynamics. One can Minimum Inhibitory Concentration Determination. The MIC defined as the speculate that the nonstationary chromosomes are the chromo- lowest concentration of ciprofloxacin that prevents any growth of the somes with increased fitness (probably containing mutations bacteria is known to be 40 ng/mL. For each strain, a volume of 3 mL of conferring cipro resistance) that would not be eliminated but overnight culture was used to inoculate, in triplicate, 150 μLofLBmedium chosen for replication. Instead, stationary and/or nonreplicating containing increasing concentrations of ciprofloxacin (0, 5, 10, 15, 20, 25, chromosomes that are likely to contain neutral or deleterious 30, and 40 ng/mL) using 96-well flat-bottom plates. Growth was moni- mutations would eventually be eliminated in the dying filament tored by reading the absorbance at 630 nm every 20 min for 10 h of in- or would recombine to separate beneficial mutations from linked cubation at 37 °C with shaking in a ChroMate 4300 Microplate Reader (Awareness Technology). MICs for ciprofloxacin-resistant offspring clones deleterious damage and hence be chosen for survival (Fig. 3B). (10 clones analyzed) were determined using the same conditions described Therefore, the multiple chromosomes that are generated in the above; in addition, wells contained LB plus ciprofloxacin at 50, 100, 200, filament could enhance the chances of producing resistant and 500 ng/mL. chromosomes as well as allow repair. The ultimate decision to release the progeny would be done by pinching off the successful Determination of Cipro-Resistant Mutant Frequency. The frequency of the mutant chromosome that is resistant to the stress by local loss of cipro-resistant (ciproR) mutant was determined and defined as the number of × the SOS response. Further in-depth mechanistic studies of the ciproR colonies that arose after 24 h of growth on solid media containing 1 MIC cipro, per viable cell. Three independent cultures of wild-type strains interplay between processes of stress-induced mutagenesis, re- were subcultured at 1:100 dilution from an overnight starter culture and combination, chromosome partitioning, and local budding are grown to midexponential phase. A volume of 0.1 mL of cell culture was needed to deepen our understanding of how antibiotic resistance plated on LB agar containing low cipro antibiotic and incubated at 37 °C for stems from a single multinucleated bacterium. The chromosome 24 h. The bacterial lawn was resuspended in 5 mL LB using a sterile scraper. dynamics in an elongating filament might possibly resemble The resulting cell culture contained a mixed population of filaments and a form of Turing machine (40), where the “computation” of fit- short bud offspring. When needed, the cell culture was recovered by fil- μ ness is like the writing of mutations on a chromosome and erasure tration through a 5- m sterile syringe filter to harvest the progeny and remove the filamentous cells. All cultures (mixed-population or filtered of marks would correspond to the elimination of chromosomes progeny) were serially diluted, and 0.1-mL samples of appropriate dilutions that were less fit and to recombination between chromosomes were plated onto nonselective plates to determine total viable cell counts, as reverting deleterious new mutations, and the halting of the well as on plates containing cipro 1× MIC (40 ng/mL) to score high-level Turing machine upon solution of the problems would be repre- ciproR mutants. CiproR frequencies were calculated by dividing the number sented by the pinching off of the mutant, more fit chromosome. of high-level cipro-resistant mutants by the total number of viable bacteria. However, this is highly speculative at this point in our work. A total of three experiments was conducted independently. Means of mu- ”±” Perhaps a phenomenon similar to bacterial multichromosome tant frequency are given in the text and the values represent SD. resistance occurs in aneuploid cells. For instance, cancer cells – Sequencing. Cipro-resistant colonies were selected on LB plates containing typically contain 60 90 chromosomes (41). Aneuploidy is a either cipro 0.5× MIC (20 ng/mL) or 1× MIC (40 ng/mL), after 24 h incubation ubiquitous feature of cancer that may contribute to a more rapid at 37 °C. See SI Appendix, Materials and Methods for details of the ex- accumulation of mutations, increasing survival and proliferation perimental protocol. (42–45). Therefore, the strategy of generating multiple mutant chromosomes within a single cell may represent a widespread ACKNOWLEDGMENTS. We thank Thomas Silhavy for helpful comments on and conserved mechanism for the rapid evolution of genome the manuscript and Douglas Austin for helping in writing the custom MATLAB code. This project was supported by National Cancer Institute change in response to unfavorable environments (i.e., chemo- Grant U54CA143803 (to R.H.A.) and National Institutes of Health Grant R01- therapy drugs and antibiotics). GM53158 (to S.M.R.).

182 | www.pnas.org/cgi/doi/10.1073/pnas.1420702111 Bos et al. Downloaded by guest on October 2, 2021 1. Zhang Q, et al. (2011) Acceleration of emergence of bacterial antibiotic resistance in 24. Wang X, Lesterlin C, Reyes-Lamothe R, Ball G, Sherratt DJ (2011) Replication and connected microenvironments. Science 333(6050):1764–1767. segregation of an Escherichia coli chromosome with two replication origins. Proc Natl 2. Diver JM (1989) Quinolone uptake by bacteria and bacterial killing. Rev Infect Dis Acad Sci USA 108(26):E243–E250. 11(Suppl 5):S941–S946. 25. Yoshida H, Bogaki M, Nakamura M, Nakamura S (1990) Quinolone resistance- 3. Hermsen R, Hwa T (2010) Sources and sinks: A stochastic model of evolution in het- determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob erogeneous environments. Phys Rev Lett 105(24):248104. Agents Chemother 34(6):1271–1272. 4. Pine L, Boone CJ (1967) Comparative cell wall analyses of morphological forms within 26. Parry BR, et al. (2014) The bacterial cytoplasm has glass-like properties and is fluidized the genus Actinomyces. J Bacteriol 94(4):875–883. by metabolic activity. Cell 156(1-2):183–194. 5. Radman M (1975) SOS repair hypothesis: Phenomenology of an inducible DNA repair 27. Recchia GD, Aroyo M, Wolf D, Blakely G, Sherratt DJ (1999) FtsK-dependent and – which is accompanied by mutagenesis. Basic Life Sci 5A:355–367. -independent pathways of Xer site-specific recombination. EMBO J 18(20):5724 5734. 6. Bos J, Yakhnina AA, Gitai Z (2012) BapE DNA endonuclease induces an apoptotic-like 28. Paulovich AG, Toczyski DP, Hartwell LH (1997) When checkpoints fail. Cell 88(3): – response to DNA damage in Caulobacter. Proc Natl Acad Sci USA 109(44): 315 321. 18096–18101. 29. Hadany L, Beker T (2003) On the evolutionary advantage of fitness-associated re- – 7. Justice SS, Hunstad DA, Seed PC, Hultgren SJ (2006) Filamentation by Escherichia coli combination. Genetics 165(4):2167 2179. subverts innate defenses during urinary tract infection. Proc Natl Acad Sci USA 30. Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91(22):10747–10751. 103(52):19884–19889. 31. Nik-Zainal S, et al.; Breast Cancer Working Group of the International Cancer Genome 8. Stackhouse RR, Faith NG, Kaspar CW, Czuprynski CJ, Wong AC (2012) Survival and Consortium (2012) Mutational processes molding the genomes of 21 breast cancers. of Salmonella enterica serovar Enteritidis filaments induced by reduced Cell 149(5):979–993. water activity. Appl Environ Microbiol 78(7):2213–2220. 32. Roberts SA, et al. (2012) Clustered mutations in yeast and in human cancers can arise 9. Zobell CE, Cobet AB (1964) Filament formation by Escherichia coli at increased hy- from damaged long single-strand DNA regions. Mol Cell 46(4):424–435. drostatic pressures. J Bacteriol 87(3):710–719. 33. Di Noia JM, Neuberger MS (2007) Molecular mechanisms of antibody somatic hy- 10. Rolinson GN (1980) Effect of beta-lactam antibiotics on bacterial cell growth rate. permutation. Annu Rev Biochem 76:1–22. J Gen Microbiol 120(2):317–323. 34. Magni GE, Von Borstel RC (1962) Different rates of spontaneous mutation during 11. Ryan DM, Monsey D (1981) Bacterial filamentation and in vivo efficacy: A comparison mitosis and meiosis in yeast. Genetics 47(8):1097–1108. of several cephalosporins. J Antimicrob Chemother 7(1):57–63. 35. Strathern JN, Shafer BK, McGill CB (1995) DNA synthesis errors associated with dou- 12. Miller C, et al. (2004) SOS response induction by beta-lactams and bacterial defense ble-strand-break repair. Genetics 140(3):965–972. against antibiotic lethality. Science 305(5690):1629–1631. 36. Sung HM, Yasbin RE (2002) Adaptive, or stationary-phase, mutagenesis, a component 13. Huisman O, D’Ari R (1981) An inducible DNA replication-cell division coupling of bacterial differentiation in Bacillus subtilis. J Bacteriol 184(20):5641–5653. – mechanism in E. coli. Nature 290(5809):797 799. 37. Harris RS, Longerich S, Rosenberg SM (1994) Recombination in adaptive mutation. 14. Justice SS, Hunstad DA, Cegelski L, Hultgren SJ (2008) Morphological plasticity as Science 264(5156):258–260. – a bacterial survival strategy. Nat Rev Microbiol 6(2):162 168. 38. Rosenberg SM, Longerich S, Gee P, Harris RS (1994) Adaptive mutation by deletions in 15. Bi E, Lutkenhaus J (1993) Cell division inhibitors SulA and MinCD prevent formation of small mononucleotide repeats. Science 265(5170):405–407. – the FtsZ ring. J Bacteriol 175(4):1118 1125. 39. Donley N, Thayer MJ (2013) DNA replication timing, genome stability and cancer: Late 16. Michel B (2005) After 30 years of study, the bacterial SOS response still surprises us. and/or delayed DNA replication timing is associated with increased genomic in- PLoS Biol 3(7):e255. stability. Semin Cancer Biol 23(2):80–89. 17. Al Mamun AA, et al. (2012) Identity and function of a large gene network underlying 40. Turing AM (1995) Lecture to the London Mathematical Society on 20 February 1947. – mutagenic repair of DNA breaks. Science 338(6112):1344 1348. MD Comput 12(5):390–397. 18. Butala M, Zgur-Bertok D, Busby SJ (2009) The bacterial LexA transcriptional repressor. 41. Rajagopalan H, Nowak MA, Vogelstein B, Lengauer C (2003) The significance of un- Cell Mol Life Sci 66(1):82–93. stable chromosomes in colorectal cancer. Nat Rev Cancer 3(9):695–701. 19. Cirz RT, et al. (2005) Inhibition of mutation and combating the evolution of antibiotic 42. Gordon DJ, Resio B, Pellman D (2012) Causes and consequences of aneuploidy in resistance. PLoS Biol 3(6):e176. cancer. Nat Rev Genet 13(3):189–203. 20. Lee AM, Ross CT, Zeng BB, Singleton SF (2005) A molecular target for suppression of 43. Selmecki AM, Dulmage K, Cowen LE, Anderson JB, Berman J (2009) Acquisition of the evolution of antibiotic resistance: Inhibition of the Escherichia coli RecA protein aneuploidy provides increased fitness during the evolution of antifungal drug re- by N(6)-(1-naphthyl)-ADP. J Med Chem 48(17):5408–5411. sistance. PLoS Genet 5(10):e1000705. 21. Lewis K (2010) Persister cells. Annu Rev Microbiol 64:357–372. 44. Duesberg P, Stindl R, Hehlmann R (2000) Explaining the high mutation rates of cancer 22. Pennington JM, Rosenberg SM (2007) Spontaneous DNA breakage in single living cells to drug and multidrug resistance by chromosome reassortments that are cata- Escherichia coli cells. Nat Genet 39(6):797–802. lyzed by aneuploidy. Proc Natl Acad Sci USA 97(26):14295–14300. 23. Hooper DC (1999) Mechanisms of fluoroquinolone resistance. Drug Resist Updat 2(1): 45. Merlo LM, Wang LS, Pepper JW, Rabinovitch PS, Maley CC (2010) Polyploidy, aneu- 38–55. ploidy and the evolution of cancer. Adv Exp Med Biol 676:1–13. EVOLUTION

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