Tangled Bank of Experimentally Evolved Burkholderia Biofilms

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Tangled bank of experimentally evolved Burkholderia PNAS PLUS bioﬁlms reﬂects selection during chronic infections

Charles C. Traverse, Leslie M. Mayo-Smith, Steffen R. Poltak, and Vaughn S. Cooper1

Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH 03824

Edited by James M. Tiedje, Michigan State University, East Lansing, MI, and approved November 29, 2012 (received for review April 26, 2012) How diversity evolves and persists in biofilms is essential for Although this species has been identified as the most prevalent and understanding much of microbial life, including the uncertain pathogenic of the Burkholderia cepacia complex species in CF dynamics of chronic infections. We developed a biofilm model patients (14), relatively little is known about how it adapts to enabling long-term selection for daily adherence to and dispersal a biofilm lifestyle or to the CF lung environment (5, 16). from a plastic bead in a test tube. Focusing on a pathogen of the Six B. cenocepacia populations underwent ∼1,050 generations cystic fibrosis lung, Burkholderia cenocepacia, we sequenced of biofilm selection, all of which diversified into similar sets of clones and metagenomes to unravel the mutations and evolution- three distinct, heritable colony morphotypes from a single an- ary forces responsible for adaptation and diversification of a single cestral clone (Fig. 1) (4). At least one of these morphotypes— biofilm community during 1,050 generations of selection. The mu- a small, highly rugose “wrinkly” type (17)—is commonly isolated tational patterns revealed recurrent evolution of biofilm specialists from chronic lung infections of both Burkholderia spp. and from generalist types and multiple adaptive alleles at relatively few Pseudomonas aeruginosa and has been associated with high pa- loci. Fitness assays also demonstrated strong interference compe- tient mortality (7, 17). Each morphotype associates with a dis- tition among contending mutants that preserved genetic diversity. tinct ecological niche and thus can be considered an ecotype; fi fi Metagenomes from ve other independently evolved bio lm lin- collectively, ecotypes segregate nutrients and biofilm space in eages revealed extraordinary mutational parallelism that outlined ways that enhance community productivity (Fig. 1D) (4). The common routes of adaptation, a subset of which was found, sur- persistence of these ecotypes for the duration of the experiment prisingly, in a planktonic population. These mutations in turn were raised several questions about their evolutionary dynamics and surprisingly well represented among mutations that evolved in MICROBIOLOGY fi mutational causes. Did each type evolve once and persist, or did cystic brosis isolates of both Burkholderia and Pseudomonas. multiple mutations produce the same ecotypes? In addition, did These convergent pathways included altered metabolism of cyclic these types generate further diversity and coevolve, much as diguanosine monophosphate, polysaccharide production, tricar- Darwin envisioned for the evolution of his famous “tangled bank” boxylic acid cycle enzymes, global transcription, and iron scaveng- (18) of myriad, interdependent plants and animals? ing. Evolution in chronic infections therefore may be driven by We also sought to test whether mutations favored during pro- mutations in relatively few pathways also favored during labora- longed selection in experimental biofilms occurred in targets similar tory selection, creating hope that experimental evolution may fi illuminate the ecology and selective dynamics of chronic infections to those identi ed during genetic surveys of CF lung isolates, in- and improve treatment strategies. cluding small-colony variants. We applied multiple, complementary genomic approaches to both clones and communities to identify the fi population genomics | microbial ecology | ecotype | clonal interference molecular bases of bio lm adaptation and quantify mutant frequencies over time. These results allowed us to reconstruct a nearly complete evolutionary history of long-term adaptation and diversification acterial evolution during chronic infections, particularly in in one biofilm population with unprecedented resolution. Compar- Bpulmonary infections of persons with cystic fibrosis (CF), fi fi ing these mutations with those found in ve other replicate experi- may involve adaptation to life in bio lms. These aggregations of mental biofilm populations revealed considerable parallelism both in cells on surfaces are more spatially structured and both generate genes known to affect biofilm production and in unexpected path- and experience more diverse environmental conditions than ways. Moreover, many of these mutations occurred in genes that well-mixed planktonic populations, leading to increased bio- commonly are mutated among isolates of Burkholderia and diversity (1–4). Biofilm diversity often evolves from founding P. aeruginosa from chronic lung infections, implying that adaptation clones and commonly is observed as variation in colony mor- during chronic infections may be partly driven by selection in biofilms. phology, production of extracellular polymers, motility, and se- creted signals or substrates (3, 5, 6). Because isolates recovered Results from chronic infections of different patients often resemble these This study focused on a single biofilm population [B1, from biofilm-specific variants (7–9), they may reflect adaptation to a larger experiment (4)] in which three distinct colony morphol- similar selective forces in vivo. However, the nature of selection during infections and why multiple types evolve is uncertain; despite several recent surveys of evolution in the lungs of CF patients (10–13), the ecology of chronic infections remains a black Author contributions: C.C.T. and V.S.C. designed research; C.C.T., L.M.M.-S., S.R.P., and box. For this reason, we developed a model enabling long-term V.S.C. performed research; C.C.T. contributed new reagents/analytic tools; C.C.T., L.M.M.-S., selection in biofilms (4) to study how genetic and ecological di- S.R.P., and V.S.C. analyzed data; and C.C.T. and V.S.C. wrote the paper. versity evolves in a structured environment. The authors declare no conflict of interest. Our simple model involves a daily cycle in which cells must This article is a PNAS Direct Submission. form a biofilm on a plastic bead suspended in minimal medium Freely available online through the PNAS open access option. in a test tube (Fig. 1). Colonized beads then serve to inoculate Data deposition: The sequence reported in this paper has been deposited in the National the next tube containing a fresh bead, so bacteria must remain Center for Biotechnology Information Short-Read Archive, http://www.ncbi.nlm.nih.gov/sra adherent during transfer and then disperse to colonize a new (accession no. SRP004277), in accordance with Joint Genome Institute policy. bead. In this environment, we studied adaptation by a soil isolate 1To whom correspondence should be addressed. E-mail: [email protected]. of Burkholderia cenocepacia, an opportunistic pathogen responsible This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. for fatal biofilm-associated lung infections in CF patients (14, 15). 1073/pnas.1207025110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1207025110 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 ogies that correspond with unique ecological functions evolved: studded (S), ruffled (R), and wrinkly (W) (Fig. 1C). The S morphotype is the most abundant and retains the capacity to grow planktonically while forming thin, mostly confluent biofilms; therefore it can be considered a generalist. The R morphotype is a specialist and produces more copious biofilm and fewer planktonic cells. The rarest W type primarily grows as a dense, towering biofilm (Fig. 1). To identify the molecular bases of these ecological differences, we sequenced DNA from mixed communities (metagenomes), the complete genomes of representative clones, and alleles of 60 alternative clones from multiple time points. These analyses (Table 1) revealed that early ecotypes (isolated at generation 315) evolved independently by distinct mutations, whereas late ecotypes (isolated at generation 1,050) derived from a common lineage and subsequently evolved by one to four ecotype- specific mutations. Thus, although the composition of the community—a majority S ecotype and minority R and W ecotypes— remained constant, its genetic structure changed and suggested that R and W types had evolved anew from an S haplotype. Within the S lineage alone, five mutations became detectable within ∼315 generations in a population that never dropped below 5 × 107 cells/ mL (the minimum number of cells adhering to the bead that was transferred). Given this large population size and the fact that the mutation rate did not evolve (4), genetic drift cannot explain the rapid rise of these mutations. Rather, the following features strongly suggest that nearly all mutations were favored by selection: Mutations in coding sequences were mostly nonsynonymous (nonsynonymous/synonymous ratio = 5.5), intergenic mutations were associated with likely promoters, and deletions affected genes that were plausible targets of selection (Table 1). The rapid rise of these lineages combined with the low per-genome muta- − tion rate (<10 2 per genome per generation) also theoretically should hinder hitchhiking of neutral mutations but does not exclude this possibility.

Phylogeny and Ecological Dynamics of Biofilm Diversification. To determine how well the fully sequenced clones represented the ecotype populations, and to evaluate the accuracy of mutation read depth in the metagenomes as estimates of allele frequency, 10 clones of S, R, and W from the early and late communities were screened for key mutations (Table 1). Read depth in the metagenomes correlated well with mutation frequency among screened clones (linear regression: F = 567.5, df =1, P < 0.0001, r2 = 0.99). Between 10 and 20 mutations were reliably detectable at any given time, and perhaps more if lower-quality reads are considered (Table 1, and Table S1). Importantly, sequencing individual clones linked mutations in the metagenome to certain ecotypes and haplotypes (e.g., M14 and M18 defined new W lineages), and these screening data (Tables S2 and S3) enabled a stepwise, most parsimonious phylogeny of this biofilm population (Fig. 2). By combining this phylogeny with estimates of allele frequency, we developed a model of how the genetic and ecological composition of the community evolved throughout the experiment (Fig. 3). The competitive dynamics of genotypes acting both within and between niches became more apparent

of biofilm selection, an adaptive radiation of biofilm generalists (S) and specialists (R and W) was observed. Subsequently, each ecotype remained detectable throughout the experiment. (C) Each ecotype has distinct growth patterns when grown in monoculture. S occupies the planktonic phase and displays moderate bead attachment, R exhibits less planktonic growth than Fig. 1. Summary of the biofilm experimental evolution and its ecological S and forms thick biofilms, and W forms clumps and produces copious diversification. (A) B. cenocepacia HI2424 was grown in GMM with a 7-mm amounts of biofilm. (D) Ecotypes vary in biofilm production (3); however, polystyrene bead for 24 h. This bead was transferred to a new tube of GMM when S, R, and W are grown together, total biofilm is greater than expected with an oppositely marked bead. Cells that adhered to the first bead needed from their monoculture values and starting frequencies, indicating synergy to disperse and attach to the oppositely marked bead, which then was when all ecotypes are present (Methods). Assays were focused on sequenced transferred to another tube after 24 h of growth. These bead-to-bead clones isolated at 1,050 generations; error bars indicate the 95% confidence transfers were conducted for ∼1,050 generations. (B) After ∼300 generations interval.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1207025110 Traverse et al. Downloaded by guest on September 26, 2021 Table 1. Mutations detected in one population during 6 mo of selection under a daily cycle of biofilm colonization and dispersal and PNAS PLUS their predicted functional significance Ecotype, time Mutation (in generations) Locus Annotation Mutational effect Significance

M1 S, 315 YP_837186 yciR Y355D Cyclic di-GMP M2 YP_837213 Monooxygenase E481D M3 S, 315 S,R,W, 1050 YP_835154 2-oxoglutarate dehydrogenase R204S Metabolism M4 S, 315 S,R,W, 1050 YP_834472 Transcriptional regulator 5′ to Del 38, del 39, L40V Metabolism lactose dehydrogenase M5 S, 315 S,R,W, 1050 yciR and 94 genes downstream Del, 95 genes, including Cyclic di-GMP M1 & M2 M6 S, 315 S,R,W, 1050 YP_834525 manC STOP @ 263 EPS/LPS M7 S,R,W, 1050 YP_835839 5′ of bacterioferritin −10 G → A promoter Iron M8 S, 1050 Loss of 49 diverse genes del, 49 genes M9 S, 1050 YP_837566 Succinate dehydrogenase Synonymous Metabolism M10 R, 1050 YP_834838 DUF88 A209P M11 R, 1050 YP_834185 5′ of mltA −10 G → A promoter Peptidoglycan turnover M12 R, 1050 YP_837420 wspD L35P Cyclic di-GMP M13 W, 1050 YP_837416 wspA A407V Cyclic di-GMP M14 W YP_837421 wspE S726L Cyclic di-GMP M15 R, 315 YP_837186 yciR A106P Cyclic di-GMP M16 W, 315 YP_837416 wspA I196N Cyclic di-GMP M17 W, 315 YP_833988 rpoC A1318V Altered RNAP afﬁnity M18 W YP_835839 5′ of bacterioferritin −35 G → A promoter Iron M19 S,R,W, 1050 YP_838893 McsS Synonymous Osmotic shock M20 All YP_834392 Ferrochelatase (in ancestor) T154A Iron

Ecotypes (S, R, and W) are deﬁned as in Fig. 1, and if the mutation is associated with a particular time point, it was identiﬁed in a fully sequenced isolate. MICROBIOLOGY Most mutations (bold font) were subsequently screened in multiple isolates using Sanger sequencing.

and suggested a more fluid community structure. In addition, R type. Because this newer haplotype displaced earlier S hap- new mutants coexisted with their ancestors for hundreds of lotypes, the frequencies of M1 and M2 declined but remained generations, in contrast to the sequential replacement that often detectable (Fig. 3A). A single base-pair deletion (M6) in manC, occurs in unstructured, homogeneous environments. Each eco- a gene associated with exopolysaccharide (EPS) and LPS pro- type also remained genetically diverse, often by different muta- duction, occurred next in this dominant lineage and then gave rise tions in genes of similar putative function. to yet another new R and a W variant. These niche invasions in- The S type was the first to achieve high frequency and, as volved three new R lineages and one new W lineage (Fig. 3C;the suggested by the genome of an early S clone, evolved by se- mutations responsible for these invasions are unknown) that drove quential mutations that eventually became globally successful. the original R resident (M15) extinct but did not noticeably affect The dominant S lineage originated with an SNP in yciR (M1), the original W lineage. The early W lineage also had acquired two which encodes a protein with three domains: a PAS-sensor, new mutations (M17 and M18). The R and W biofilm specialist a GGDEF diguanylate cyclase, and an EAL phosphodiesterase ecotypes therefore evolved recurrently from a single, dominant (19); this mutation therefore likely affected metabolism of cyclic S lineage. diguanosine monophosphate (c-di-GMP). Shortly thereafter, The final defining mutation of the dominant lineage (M7) was a second SNP (M2) occurred 27 genes downstream from this the most successful mutation in the experiment and altered the initial mutation on the same haplotype, and this lineage in- promoter of the iron-storage gene bacterioferritin. Remarkably, creased to ∼95% of the population (Fig. 3). However, before this M18 that evolved on a W haplotype altered the same promoter lineage could fix, a different yciR SNP occurred and produced (Table 1); the dynamics during this period suggest that M7 may the first R ecotype (M15). Similarly, another SNP in a homolog have evolved in response to M18. M18 was detected between 315 of wspA (M16) occurred in association with the first detected W and 525 generations and was associated with a large gain in W ecotype. The wsp operon has been characterized extensively in frequency (increase in red fraction in Figs. 3 and 4) to ∼30% of Pseudomonas species (20, 21) for its production of a wrinkly the entire population. Subsequently, the rise of M7 in the main S spreader phenotype related to c-di-GMP metabolism. These lineage associated with the decline of W ecotypes to their orig- mutations imply that varied c-di-GMP metabolism was the cause inal ∼10% (decline in red fraction at 600 generations, Figs. 3 and of ecological differentiation and set the stage for prolonged co- 4). Coincidentally, all R types lacking a bacterioferritin mutation existence of S, R, and W (Fig. 3B). went extinct, and only new R mutants evolving from the M7 Mutations on the S lineage continued to accumulate but also haplotype (e.g., M10) preserved this ecotype. However, the fre- began to produce new R and W lineages (Fig. 3B). The next S quency of the W lineage with mutation M18 apparently did not mutation was an SNP in the tricarboxylic acid (TCA) cycle in- change when two new W lineages invaded from the M7 haplo- termediate enzyme, 2-oxoglutarate dehydrogenase (OGDH, M3), type later in the experiment (Fig. 3), presumably because each of which displaced its direct ancestor and also produced a new R these lineages had a similar bacterioferritin mutation. Once variant (Fig. 3B). Then a peculiar set of mutations (M4 and M5) again, the new W types evolved by single mutations in wsp followed in an uncertain order: M4 consisted of a two-codon de- homologs, wspA (M13) and wspE (M14), similar to the original letion in a transcriptional regulator, and M5 was a large deletion W lineage. These dynamics highlight the global importance of that removed the mutated yciR locus and the new M1 and M2 acquiring a mutation in the bacterioferritin promoter sequence alleles. This new M3/M4/M5 haplotype also spawned a successful and the capacity for small-colony variants to re-evolve.

Traverse et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 contending adaptive lineages. Each mutant displayed fitness advantages that would displace the ancestor before 70 generations in the absence of clonal interference; in actuality, none of these alleles fixed, and their frequencies increased approximately 10-fold more slowly in the evolving community (Table 2). However, the slight variation in fitness among mutants versus the ancestor did M12 M9 not explain how selection favored the mutants’ rapid succession. In M14 M8 contrast, far greater fitness differences among haplotypes were M11 evident from competitions with the most derived (M3–M7) hap- M13 lotype, which was considerably fitter versus an early-evolved mutant than versus the original ancestor. The fitness ranks of M10 evolved haplotypes were consistent with the mutational order and demonstrated a large advantage of the yciR deletion (M5).

Time ** Evidently, selection in this bioﬁlm system is strongly context

(Not to scale) dependent, favoring mutants that are superior in both the current * M7 M18 biotic environment comprising multiple lineages and in the fluc- * M6 tuating abiotic conditions. * Nonetheless, the extraordinarily high fitness advantages of M4/M5 M17 evolved mutants in pairwise competition far exceed their realized benefits in the community, as judged either from the population Ancestor M3 dynamics of the entire metagenome or among alleles within Studded M15 Ruffled a niche (Table 2). The fact that many contending adaptive lin- M16 eages are found at appreciable frequencies throughout the ex- M1/M2 Wrinkly * Unknown R periment indeed may have diminished the net advantage of any ** Unknown W given haplotype. In addition, more complex frequency- or density-dependent interactions among favored genotypes, such as fi fi Fig. 2. Phylogeny of adaptation and diversi cation in a model bio lm the cross-feeding interactions described previously (4), may have population. Haplotypes were assembled by screening 30 clones from 525 slowed the rise of adaptive alleles and preserved genetic diversity generations and 30 clones from 1,050 generations at variable loci in the fi metagenome sequences of the community, and their most parsimonious within the bio lm community. phylogeny was constructed. The ancestor (black line) is rapidly displaced by new mutant ecotypes depicted in different colors; lineages that do not Genetics and Physiology of Biofilm Adaptation. The functional sig- continue were outcompeted by superior lineages within their niche. “Un- nificance of the mutations associated with adaptation to pro- known” lineages are ecotypes of uncertain genetic composition, i.e., the longed biofilm selection highlights the challenges faced by mutation defining the R or W morphology is unknown. Burkholderia in this system and the changes that were favored. Four major pathways of adaptation emerged, described below, but some mutations do not fall into these categories, suggesting By retrospective sampling of the population throughout its that selective forces in this biofilm system are manifold and may evolution, we found that the relative frequency of each morpho- preserve many alternative alleles. type, and hence the balance of their respective ecological strate- Altered regulation of c-di-GMP. Three yciR mutations (M1, M5, and gies, indeed fluctuated over time (Fig. 4). Shifting morphotype M15) were critical to the early evolution of the biofilm community frequencies were tightly paired with changing allele frequencies, (Fig. 3). M1 (Y355D) occurred in a highly conserved residue of most often when new mutants of the S lineage invaded and dis- the GGDEF domain and was associated with S morphology. placed existing S, R, or W inhabitants (Fig. 3). This pattern of M15 (A106P) was found in the PAS sensor domain of yciR and in ecological and genetic invasion from the numerically dominant S isolation defined R morphology; thus, alternative yciR alleles niche into the more specialized R and W niches suggests that fi high-biofilm specialists evolve from generalists, but not vice versa. de ned morphological and ecological differences in this system. Therefore the highly productive, synergistic late community (Fig. The third yciR mutation (M5) deleted this locus and 94 other genes and was likely mediated by an IS116-family insertion se- 1D) (4) mostly comprises ecotypes that are immediate genetic ′ relatives with only one or two mutations defining them. quence at the 3 end of this lesion. This mutation was also associated with S morphology, but its ecological dominance over the Clonal Interference Within and Among Niches. Each ecotype remained two yciR SNPs remains to be explored. Curiously, disrupting this genetically diverse throughout the experiment (Fig. 3), indicating gene in another Burkholderia species had been shown previously that beneficial mutations were relatively common and likely to decrease both biofilm production and quorum sensing (24). competed with one another (22). This dynamic of clonal inter- In general, wrinkly colony morphologies nearly always are ference can effectively reduce the relative benefit of any single associated with increased c-di-GMP concentrations and in mutation or haplotype and may allow multiple lineages to per- Pseudomonas may be caused by mutations that lead to auto- sist. Theory suggests that such competition also can produce a induction of an associated diguanylate cyclase enzyme, WspR simultaneous sweep of multiple linked mutations (e.g., mutations (21). Four nonsynonymous mutations were detected in this op- M3–M6, Fig. 3A), because more than one beneficial mutation eron, each in different clones, including two in wspA (M13 and may be required to prevail in competition (22, 23). M16) and one each in wspD (M12) and wspE (M14). All wsp We began to quantify the selective forces acting on each mutations were associated with either an R or W morphotype: mutational step leading to the dominant haplotype by compe- wspA and wspE alleles were found in W clones, and wspD was tition between these mutants and two reference clones: with the found in the successful R lineage (Fig. 3B). The wspE mutation founding ancestor and with a highly adapted intermediate S occurred in an active site for phosphorylation and hence is likely clone (Table 2). Competition versus the original ancestor pro- to alter signaling of this pathway. Presumably, each of these four vides a common reference to judge fitness in the absence of mutations adaptively alters the levels of c-di-GMP in this system, other beneficial variation, whereas competition versus a well- a subject of ongoing study, and also defines particular ecological adapted “contemporary” mutant illustrates selective forces among roles and leads to niche subdivision.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1207025110 Traverse et al. Downloaded by guest on September 26, 2021 PNAS PLUS A 1 .75 .50

Frequency .25.2 0 M1M2 M3 M4 M5M6 M7

M15 B M12 M11 R M10 * Ecotype Niche Breadth * * M1 M2

M3 M4 S M5 M8 M9 ** M6 M7

M14 Mutation frequency M13 W Ancestor M18 M16 M17

0 315 525 735 10500 Time (generations) MICROBIOLOGY C

Studded Ruffled Wrinkly

Fig. 3. Population genomics and ecological structure of the bioﬁlm community over time. Allele frequencies were determined at four time points (vertical dashed lines), and dynamics were interpolated. (A) Frequency of majority mutations belonging to the dominant haplotype throughout the community. (B) Mutational dynamics within and among niches. Each color transition represents a new haplotype (labeled as in Table 1), and color breadth shows haplotype frequency in the community, to scale. The earliest mutants arose on the ancestral genotype, and subsequent mutations evolved within the ecotypes that subdivided the community. Lines crossing ecotype boundaries (light blue lines) represent invasion of the dominant S haplotype into the R or W niche associated with novel mutations. Horizontal light blue lines highlight the ecological boundaries that evolved within this community. Additional low-frequency mutations were detected in the metagenomes and are reported in Table S1, and other mutants likely evolved before the ﬁrst samples. *, R isolate with unknown niche-specifying mutation; **, W isolate with unknown niche-specifying mutation. (C) Dynamics of niche invasions by mutants of S over time. Each blue arrow represents the invasion of an S type into an R or W niche.

Shifts in central metabolism. Three beneficial mutations, M2, M3, in LPS biosynthesis. Late S, R, and W clones produced less biofilm and M4 (Tables 1 and 2), likely altered central metabolism. M2 is when a functional manC was added on a plasmid (Fig. S1). We a mutation in a gene annotated as FAD-binding monooxygenase. hypothesize that this mutation effectively directs mannose toward This type of enzyme may be involved in electron transport, and exopolysaccharide and away from energy production and may in- altering the levels of FAD(H) within the cell may provide crease adherence and coherence as a response to disrupting LPS a growth advantage in the selective environment. M3 is a non- biosynthesis, as has been reported elsewhere (26, 27). synonymous mutation in OGDH (M3), and M4 is a two-codon Enhanced iron competition. The two different mutations in the bac- deletion from a LysR-like transcriptional regulator that is 5′ to terioferritin promoter sequence (M18 and M7) occurred in the −35 a gene encoding lactate dehydrogenase (M4). These mutations and −10 σ factor-binding domains, respectively. Quantitative RT- could enhance growth in galactose minimal medium, but they PCR of genotypes with the M7 mutation exhibited increased tran- also may reflect selection for biofilm production. scription at this locus (Fig. S2), indicating that these mutants gained Altered mannose metabolism related to EPS and LPS production and capacity for iron storage. Because the experimental environment adherence. The globally successful mutation M6 created a pre- was not supplemented with iron, this resource likely became limit- mature stop codon in a multidomain gene (manC) that is in- ing. Another iron-associated mutation evolved in the late commu- volved in mannose metabolism and is located within a gene nity in a gene encoding ferredoxin (Table S1), providing further cluster related to LPS biosynthesis. The gene is bifunctional, with evidence of selection for altered iron metabolism. Thus, competi- an N-terminal mannose-1-phosphate guanylyl transferase do- tion for iron occurred both within and between niches in the biofilm main and a C-terminal mannose-6-phosphate isomerase (PMI) community, which favored mutantswitheitherM7orM18,dis- (25). A single-nucleotide deletion occurred in a poly-A tract (a placed mutants lacking these alleles, and facilitated invasion from relatively rare sequence in this genome, given its 67% GC con- the S niche to the R and W niches. tent) that preserved the N-terminal domain but truncated the C-terminal PMI domain and likely generated polar effects on the Evolutionary Parallelism Among Six Independently Evolved Biofilm rest of the operon. The effects of this disruption are potentially Populations. Although we have not assembled a detailed evolu- widespread, because downstream genes are expected to participate tionary model of all six biofilm populations (as in Fig. 3), mutations

Traverse et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 M3/M4/M5/M6 these alleles. Alternatively, different alleles in the same gene may be favored by planktonic or bioﬁlm growth. However, none Ancestor of the wsp mutations associated with R and W types were 1.0 M7 Studded detected, and three unique mutations in the quorum-sensing Ruffled regulator cepR (28) evolved in this planktonic population. In Wrinkly general, the high genetic parallelism in adaptive targets suggested that adaptation in this model proceeded along relatively few pathways, despite progressive ecological complexity and the ease of selecting adaptive mutants (which evolve in a matter of M13 days) from the ancestral clone in this system. 0.5 M1/M2 and M10/ M14 Convergent Evolution with CF Lung Isolates. The same four classes

Frequency M18 M11/ of mutations found in our replicate populations were found in M12 studies of isolates of Burkholderia dolosa and Pseudomonas aeruginosa that evolved during CF infections (Table 4) (10–12). We make this comparison cautiously, because more mutations were 0.0 reported in these clinical studies than in this system and because 0 200 400 600 800 1000 database matches may be uncertain. However, the degree of Generations overlap for these functions is remarkable and is statistically im- probable to have occurred by chance alone, given the number of Fig. 4. Ecotype frequencies in biofilm population B1 over time as evaluated observed mutations in each study and the number of genes as- from plate counts. As certain mutations became detectable (arrows and sociated with each function (Table S6 and SI Methods). Moreover, mutation labels), changes in morphotype frequencies were observed. none of the mutations that uniquely evolved in the planktonic population were shared by these studies. Smith et al. (12) reported associated with adaptation were identified in the early and late six independent wspF mutations in their Pseudomonas aeruginosa samples of all populations. Exceptional parallelism among study, which highlights altered c-di-GMP regulation in certain adaptive targets occurred: all six populations experienced muta- infectious lineages. Another study of P. aeruginosa reported two tions in the four major functional categories outlined previously more mutations in this operon, wspE and wspC (Table 4) (11), (Table 3 and Table S4). At least 26 mutations involved in c-di- although these mutations could be explained by chance. Eighteen GMP metabolism occurred, including seven independent muta- distinct mutations in the wsp operon were selected among our tions in the yciR gene, 18 independent mutations in the wsp op- replicate populations. In addition, five OGDH mutations were eron, and one in an unclassified diguanylate cyclase. Another detected in 112 clinical B. dolosa isolates, suggesting the impor- commonly mutated area across the populations was a gene cluster tance of altered TCA cycle enzyme activity. Likewise, OGDH involved in LPS biosynthesis, including mutation M6. Twenty mutations evolved in all six biofilm populations and were associ- independent mutations occurred within this gene cluster among ated with increased fitness in population B1. all six populations, thus suggesting that altered LPS may be One of the more remarkable examples of convergent evolu- adaptive in biofilms. Mutations in OGDH also were successful in tion involved a gene cluster responsible for synthesizing O-an- each population, highlighting this step in the TCA cycle as limiting tigen LPS, in which manC (mutation M6) is found. Lieberman for the ancestor in this environment. Other classes of mutations et al. (10) identified 19 mutations in this locus across 112 clinical occurred in replicate populations as well, including in RNA isolates and one additional mutation in another manC homolog. polymerase subunits rpoC and rpoD (n = 5) and in a galactose Among the six experimental biofilm populations studied here, 20 metabolism operon (n =11). independent mutations in the homologous and well-conserved A population evolved under planktonic conditions in the same gene cluster were found. Mutations in manC evolved in both B. medium, selected because it was the only one of six to evolve an dolosa (n = 2) and B. cenocepacia (n = 4). The remainder of the additional colony type, was sequenced also. This population also mutations occurred in similar but not identical sugar transferase revealed some parallelism with the biofilm lines, including three genes in both organisms. Similarly, LPS-associated mutations mutations in the LPS gene cluster, two mutations in rpoC, and have been detected in clinical P. aeruginosa isolates in the wbpA, one mutation in yciR (Table S5). These results imply that ad- wbpW (a manC homolog), and wbpZ genes (11, 29). Given this aptation to the high-galactose environment lacking the bead (but exceptional mutational parallelism found among relatively few preserving all other conditions, including constant 37 °C and the studies, it is likely that some of the same forces that drive biofilm potential for biofilm growth on the test tube walls that may have adaptation also contribute to adaptation of these pathogens been shed and preserved during transfer) may explain some of within the CF lung.

Table 2. Fitness estimates of evolutionary steps within the dominant S haplotype Evolved S haplotype Fitness vs. ancestor Fitness vs. M3-M7 Fitness from whole metagenome Fitness from niche metagenome

M1–M2 0.45 (0.065) −0.96 (0.19) 0.053 0.066 M1–M3 0.40 (0.061) −0.84 (0.22) 0.0091 0.029 M3–M5 0.51 (0.034) 0.15 (0.14) 0.0082 0.030 M3–M6 0.57 (0.067) −0.32 (0.17) 0.015 0.035 M3–M7 0.52 (0.068) – 0.014 0.015

Haplotypes are defined by mutations listed in Table 1. Empirical fitness (columns 1 and 2) was calculated by direct competitions between clones (column 1, 5–9 replicates, column 2, 11 or 12 replicates) and is reported as selective rate constants with 95% confidence intervals in parentheses. Haplotypes vary modestly in competition versus the ancestor (F = 4.38, P = 0.0058) but vary widely in their fitness versus the most derived genotype (F = 46.0 P < 0.0001), demonstrating very strong selection among co-occurring mutants. Much lower realized fitness differences were inferred from metagenomes (columns 3 and 4) as changes in mutation frequency over time, either throughout the community (column 3) or within niches associated with ecotypes (column 4).

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1207025110 Traverse et al. Downloaded by guest on September 26, 2021 Table 3. Mutations shared among the six evolved bioﬁlm populations, categorized by putative function PNAS PLUS Function Locus Annotation No. of mutations

Transcription YP_833988 rpoC 3 YP_837516 rpoD 2 Galactose metabolism NC 5′ of galactose metabolism operon 7 YP_834263 iciR family (regulator of galactose metabolism) 3 YP_834264 2-keto-3-deoxy-galactonokinase 1 (galactose metabolism) LPS gene cluster YP_834517 dTDP-glucose 4,6-dehydratase (3′ of manC)1 YP_834518 Glucose-1-phosphate thymidylyltransferase 3 YP_834524 Glycosyltransferase 1 YP_834525 manC 4 YP_834526 GDP-mannose 4,6-dehydratase 10* YP_834528 fkbM, methyltransferase (5′ of manC)2 YP_834530 Glycosyl transferase, family 2 1 YP_834532 Glycosyltransferase 1 YP_834533 capB, polysaccharide synthesis 1 OGDH YP_835153 OGDH E1 7 YP_835154 OGDH E2 2 c-di-GMP 95-gene deletion yciR deletion 1 YP_837186 yciR 7 YP_839310 GGDEF, PAS/PAC Sensor 1 YP_837415 wspR 1 YP_837416 wspA 6 YP_837420 wspD 2 YP_837421 wspE 9

Parallel evolution in each functional category occurred much more often than expected by chance (Table S6). The full list of mutated MICROBIOLOGY positions is found in Tables S1 and S4. NC, noncoding. *One mutation is the likely polar effect of a deletion and premature stop in the gene found 5′ in the operon.

Discussion ability of bacteria to both attach and disperse has not been Adaptive diversification and niche partitioning have been ob- performed. Our previous observation that three distinct ecotypes served previously in bacterial populations that were evolved ex- evolved and persisted in each of six replicate evolved biofilm perimentally in structured environments (30–33), so the evolved populations (4) led us to anticipate that each type represented diversity in this biofilm model is unsurprising. However, we lack a distinct lineage that had evolved continuously by endemic understanding of the population genetic dynamics of biofilm alleles. We also found that ecotype frequencies varied over time, communities over the longer term and particularly how newly and biofilm communities became more productive (4), suggesting established lineages might coevolve and shape their environ- that the ecotypes had coevolved. However, a detailed population ments. Further, to our knowledge, experimental selection for the genomic analysis of one biofilm community, made possible by

Table 4. Convergence among mutations found in isolates from chronic pulmonary infections of CF patients, as reported for B. dolosa (10) and P. aeruginosa (11, 12) and mutations that evolved in this experimental system (Table 3) B. dolosa Annotation Mutations P. aeruginosa Annotation Mutations

Transcription BDAG_04154 rpoD 4 PA14_07520 rpoD 1 BDAG_02789 rpoB 1 PA14_08760 rpoB 2 BDAG_02788 rpoC 2 PA14_08780 rpoC 1 PA4462 rpoN 5 PA4270 rpoB 1 LPS gene cluster BDAG_02317 Glycosyltransferase 10 PA14_71910 wbpZ 2(ns) BDAG_02321 Glycosyltransferase 6 PA14_71970 wbpW (manC)1(ns) (wbpX homolog) BDAG_02323 manC 2 PA3159 wbpA 1(ns) BDAG_02820 manC homolog 1 OGDH BDAG_00384 OGDH E3 1 BDAG_01761 OGDH E1 2 BDAG_01898 OGDH E3 2 Cyclic di-GMP BDAG_04135 diguanylate cyclase 1 (ns) PA14_16450 wspC 1(ns) PA14_16470 wspE 1(ns) PA14_31330 EAL 1 (ns) PA14_56790 GGDEF/EAL 1 (ns) PA3703 wspF 6

Mutations in the same row are not necessarily orthologs. Convergent evolution between systems occurred more often than expected by chance for all functional categories except those labeled ns (not signiﬁcant) (Table S6 and SI Methods).

Traverse et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 repeated sequencing of clones and the evolving community as produce robust biofilms, and many form small-colony variants (5, a whole, revealed more complex dynamics. Particularly notable 7, 9, 38), and, notably, the colony morphologies described in the was an example of competition over limiting iron between eco- literature (38–41) as related to infection outcomes are strikingly types that eventually remodeled the community, but contrary to similar to those that evolved in this system. Thus, it is remarkable expectations new specialist ecotypes actually had evolved re- that the causes of phenotypic variation during this experimental currently from the source population of S (Fig. 3). These high- biofilm selection and during chronic infections both involved biofilm, small-colony variants evolved by a variety of single mutations that affected the TCA cycle, iron metabolism, and mutations in relative few pathways (often in wsp). More broadly, RNA polymerase. For example, mutations in OGDH occurred ecotype niche breadth correlated with evolutionary potential, frequently in a study of B. dolosa (10) and in this system (Table because generalist S genotypes gave rise to new specialist R and 4), and this function was up-regulated in two independent studies W types, but the converse did not occur. of clinical P. aeruginosa (13, 42). This form of altered central The order of adaptive mutations in each lineage also revealed metabolism may be favored by evolution in biofilms. Iron ac- the prevailing selective forces over time. At the start of the ex- quisition also is known to be a major selective pressure in the CF periment, cells that adhered better to the plastic bead were fa- lung (10–12), and it appears to be central to the dynamics of this vored; consequently, the first three successful ecotypes acquired system, suggesting that perhaps the biofilm environment rather mutations in genes (M1, M15, and M16) that are likely re- than an iron-limited host may favor these changes. Convergent sponsible for controlling levels of c-di-GMP, which is known to mutations in subunits of RNA polymerase (RNAP) in clinical underlie transition to a sessile lifestyle (20). These alleles also B. dolosa and P. aeruginosa (10–12) and in our study are more altered colony morphology and excluded the ancestor type, difficult to explain, given their global effects on transcription. suggesting that variation in c-di-GMP production is sufficient for Mutations in major σ factors and polymerase subunits have been adaptive differentiation and the subdivision of biofilm labor, widely reported during long-term selection in chemostats (43) perhaps by varying the tendency to adhere or disperse. and occurred in our planktonic control population as well; these Next, selection for enhanced metabolic efficiency likely domi- changes may optimize global gene regulation for better growth in nated, because mutations occurred that affected central metab- minimal medium (44, 45) that requires more anabolism. RNAP olism and polysaccharide biosynthesis. Each of these mutations mutations in isolates from infections have been associated with appeared to expand the potential of the S ecotype (Fig. 4) and selection for antibiotic resistance but alternatively may be enhanced competition versus earlier mutants (Table 2). These responses to selection for to growth in novel nutrient conditions. genotypes also subsequently gave rise to new R mutants with Relatively less convergent evolution was found at loci other these mutations, indicating that they were globally beneficial and than wsp that affect c-di-GMP metabolism. Despite several re- not specific to a particular niche. Although these mutations may lated mutations in infectious P. aeruginosa (11, 12), only one such simply enhance growth on galactose as a sole exogenous carbon mutation was reported for B. dolosa (10). These mutations were source, adaptation to planktonic growth in the same medium among the first to evolve in our model, so one possibility is that occurred by somewhat different mechanisms (Table S5). Thus, these mutations became common before clinical sampling. these mutations may be specifically adaptive in biofilms by di- Starkey et al. (40) reported that laboratory-derived rugose small- recting resources toward sessile growth. colony variants with increased intracellular levels of c-di-GMP associated with the ability to persist in CF mouse models. As in Iron Competition Engendered Persistence and Evolvability. As de- our model, the earliest adaptive steps to form a biofilm may scribed previously, two independent mutations in the promoter require altered c-di-GMP levels, but the way each population sequence of bacterioferritin led to an ecological revolution that accomplishes this task may be variable. remodeled the community (Figs. 3 and 4). New mutants of S with Perhaps the most compelling example of convergent evolution up-regulated bacterioferritin (Fig. S2) likely succeeded in other in this system and the mutations identified in CF lung isolates are niches (Fig. 3C) because of their enhanced competitive ability the many mutations in genes involving LPS (Table 4). Altered for limiting iron, and the mutations enabling invasion were novel LPS production could influence biofilm formation directly be- wsp alleles (Fig. 3). It is surprising that W mutants with the al- cause of potentially increased surface adherence and cell-to-cell ternative bacterioferritin mutation failed to spread beyond this coherence (26, 27). In this system, complementation of one of niche, either by mutations suppressing the wrinkly phenotype or the key mutants in this pathway adversely affected biofilm pro- by alternative gain of function. Only the S lineage, which bal- duction (Fig S1). Rather than selection for immune avoidance, ances fitness between planktonic and biofilm conditions and altered LPS during chronic infections may underlie biofilm ad- retains motility (4), was able to spawn new R and W types at least aptation. A subset of the 19 mutations found in the B. dolosa seven times. This S lineage may be more “evolvable” because of outbreak restored O-antigen synthesis and may have contributed its larger population size or because of an absence of negative to virulence in the lung (10), but others did not, suggesting that epistasis between its existing mutations and niche-specializing wsp mutations in the same pathway may reflect different selective alleles. Alternatively, the ecological breadth of the S population pressures. One possibility is that these mutants may be adaptive in may have enabled greater access to new mutations that were different niches within the biofilm, as in our B. cenocepacia system. beneficial in a subset of conditions. These alternative explan- ations are central to the debate surrounding evolvability (34–36) Tangled Bank of Biofilms: Perhaps Predictable Dynamics? What do and may be studied tractably in this model biofilm community. we make of this coincidence between the adaptive pathways in our system and the mutated loci occurring in chronic infections, Evolution During Chronic Infections Is, in Part, a Response to the and why might prior experimental models (32, 46) have captured Biofilm Lifestyle. Most mutations from published studies of CF fewer of these mutations? We suggest that both the duration of lung isolates do not strictly overlap with the mutations found in the experiment (6 mo or 1,050 generations) and its requirement this study, as is to be expected. Bacteria face many different to maintain flexible life-history strategies may provide some ex- selective pressures in the CF lung that are absent from our planation. Our model required a daily cycle of surface coloni- model, such as high levels of antibiotic use, oxygen deprivation zation, biofilm growth, dispersal, and then recolonization, i.e., when suspended in mucus, and immune system evasion (5, 10, selection for “reversible stickiness.” Under these conditions, 11, 16, 37, 38). Also, in principle, the fact that Burkholderia and pure biofilm-producing specialists might be disadvantaged in the Pseudomonas are in different classes of Proteobacteria should long run because of their inability to disperse and recolonize the reduce the frequency of convergence. However, most CF isolates next plastic bead. In fact, many early-arising biofilm specialist

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1207025110 Traverse et al. Downloaded by guest on September 26, 2021 lineages ultimately were displaced by derivatives of the more extracted using standard methods. Libraries for Illumina sequencing were PNAS PLUS flexible S lineage (Fig. 2). Such mutants, successful only in the prepared following the manufacturer’s procedures and were sequenced by short term, may resemble those types found in prior studies (31, the Joint Genome Institute (http://jgi.doe.gov) with an Illumina GAII using 32), but the longer-term cyclical requirement for persistence, paired-end 36-bp reads. The average read depth for each metagenome was 189×, 110×, and 141× for the early (315 generations), intermediate (735 dispersal, and recolonization may better resemble the dynamic generations), and late (1050 generations) time points, respectively. In addi- of bacterial populations during chronic infections, because life- tion, representative single clones of S, R, and W types were isolated at 315 style flexibility will be needed in a lung environment with diverse and 1,050 generations and sequenced at >100× coverage (SI Methods). conditions (e.g., host immune response, oxygen deprivation, and Subsequently, early and late metagenomes were sequenced from five other continuous biofilm establishment and migration). replicate biofilm populations (B2–B6) and from a control, planktonically Phenotypic diversity is known to be common among CF lung evolved population P2 (Table S7, SI Methods, and ref. 4). isolates of B. cepacia, Burkholderia multivorans,andB. cenocepacia, The complete approach for identifying mutations and estimating allele particularly for biofilm formation and EPS production (5, 13, 47). frequencies is described in SI Methods. Mapping of short reads to the B. Seemingly identical genetic relatives isolated from the same lung at cenocepacia HI2424 reference sequence (51) was performed using maq-0.7.1 (52) and later was confirmed using BWA (53). Sequences of single clones the same time may display distinct differences in the ability to form fl fi were analyzed as having two haplotypes to ag regions of poor alignment, bio lms and may occur within multiple patients (5). Variation and sequences of mixed population metagenomes were analyzed at hap- in EPS production was associated with different abilities to lotypes = 20, using clones from these populations as controls for false pos- initiate biofilm formation and form mature biofilms (47). One itives. The high GC content of this genome produced many initial false possible interpretation of these findings is that the Burkholderia positives and anomalously low coverage in some genome regions. Thus, all isolates within those patients inhabited distinct niches and putative mutations were examined manually Table S8 (SI Methods) and most functioned as a community, even among many other bacterial were confirmed by Sanger sequencing. species in the CF lung (48). fi We also explored the high degree of competition both within Phylogeny and Haplotype Identi cation. To associate mutations in the metagenome with both clones and ecotypes, single colonies were obtained by niches and among ecotypes in these biofilms and suggest that growing population samples from 525 and 1,050 generations in the selective such forces are more intense than in well-mixed environments environment by plating as described. Ten clones of the three morphotypes at (49). This interference has the effect of maintaining diversity each time point were chosen randomly and frozen for reference. DNA from over longer time scales and could enhance coevolutionary forces. each clone was isolated, and the loci containing each of the putative Thus, evolutionary dynamics in biofilms can be considered ex- mutations were amplified by PCR and sequenced using standard methods

ceptional, because greater variation is maintained and multiple (SI Methods). MICROBIOLOGY adaptive mutations are required for any mutant to exclude others The estimated frequencies of mutations identified in sequenced clones, in (Table 2) (23). However, these dynamics also may be expected metagenomes, and by manual screening of variant loci from many clones were integrated to produce a phylogeny of how population B1 evolved and and may even become predictable. We offer the hypothesis that fi the tangled bank of biofilm adaptation (18), in which adaptive diversi ed. For instance, mutations detected in the metagenome were not yet associated with a clone or ecotype but enabled estimates of their radiations fuel a pattern of ecological succession, ultimately changing frequencies over time, which allowed us to determine their likely drives evolution along proscribed paths. If valid, such a model order of occurrence. Sequencing multiple isolates at several loci confirmed offers hope for the development of novel therapeutics that this order and also allowed us to assemble haplotypes and to associate target these pathways. More worrisome, however, is the evolu- mutations with ecotypes, i.e., the mutations associated with the colony tion of progressively greater synergy within our model, in which morphotypes at each stage. This approach also captured multiple putatively late-stage–related ecotypes are particularly robust (4). Identi- adaptive alleles that were undetected in the metagenome. Further details are fying which community members are most central to biofilm given in SI Methods. resilience therefore becomes paramount, with the caution that the key community member may re-evolve once targeted ther- Fitness Assays. After overnight growth from clonal freezer stocks and 24 h of solitary growth in the selection environment, competitors were removed from apy has ended. the plastic bead, vortexed in 1 mL of GMM, and standardized by optical density. Competitions of evolved isolates versus the ancestor were started by in- Methods oculating 4 mL of GMM with 100 μL of evolved culture and 900 μLofthe Strains and Growth Conditions. The experimental evolution is described in ref. ancestor; this ratio enabled more accurate assays because of the relatively low 4. Briefly, a Burkholderia cenocepacia HI2424 isolate was marked with fitness of the ancestor. Competitions of earlier S clones versus the M3–M7 + aTn7::lacZ construct to enable blue/white screening (50). A single lac clone haplotype were conducted by using lacZ+ and lacZ− competitors (Table S9). was inoculated into M9 salts supplemented with 3% (171 mM) galactose Cultures for competitions were started as described for the ancestral com- (GMM) containing a polystyrene bead (American Educational Products). petitions, except that 4 mL of GMM was inoculated with 500 μL of earlier S Every 24 h, the bead and any adherent bacteria were transferred to a fresh isolates and 500 μLofM3–M7. Initial ratios were enumerated, and pop- tube of GMM containing an oppositely marked (white or black) polystyrene ulations were incubated at 37 °C in a New Brunswick TC-7 model roller drum bead. Cultures were enumerated and characterized by plating on half-con- at 50 rpm. After 24 h, the bead was removed, vortexed in 1 mL of PBS, diluted, centration agar plates that were incubated at 37 °C for 24 h and at room and plated. Competitions between S clones were enumerated on tryptic soy temperature for 48 h. Cultures for fitness assays were grown under identical plates supplemented with X-Gal. Selective values were calculated as: conditions except that strains were grown for 24 h in tryptic soy broth (Fisher) from frozen stocks and then were transferred to the selection en- Et E0 ln − ln vironment. Biofilm production was quantified using standard methods (3) A A t ¼ t 0 with sixfold replication of the sequenced S, R, and W clones isolated from lnð1 þ sÞ the end of the experiment. Mixed-population biofilm production was quantified from an aliquot of the final evolved community. Expected biofilm where t = time (generations), E = frequency of evolved, A = frequency of the production (Fig. 1) was calculated by multiplying biofilm production by the ancestor, and s = selective value. Selection rates in the metagenome were fraction of the community associated with each ecotype and then summing calculated using the same formula, substituting mutation frequencies at these values; contributions of each morph to the observed biofilm produced known times in the numerator. Selection rates of ecotype-specificmutations are estimates based on the frequency of plated morphotypes. were calculated by adjusting their frequency to that of the particular ecotype (e.g., a W-specific mutation should never exceed 10% of the total community). Metagenomic Sequencing and Analysis. Population samples from 43, 71, and Additional methods are described in SI Methods. 143 d, reflecting ∼315, 735, and 1,050 generations, were grown in the selective environment for 24 h. [These cell-division estimates differ from those ACKNOWLEDGMENTS. We thank Laura Benton, Gabrielle Bergeron, Wendy reported in ref. 4 and reflect more precise measurements of the growth Carlson, Zhang Qian, Marcus Dillon, Crystal Ellis, Kenneth Flynn, Megan cycle (SI Methods)]. The bead was vortexed in PBS, and genomic DNA was McLaughlin, and Christopher Waters for helpful discussions and technical

Traverse et al. PNAS Early Edition | 9of10 Downloaded by guest on September 26, 2021 support and thank three anonymous reviewers for their comments. This re- work conducted by the U.S. Department of Energy Joint Genome Institute search was supported by National Institutes of Health Grant 1R15AI082528 was supported by the Ofﬁce of Science of the US Department of Energy and National Science Foundation Career Award DEB-0845851. Collaborative under Contract DE-AC02-05CH11231.

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