Polygenic adaptation, clonal interference, and the evolution of mutators in experimental Pseudomonas aeruginosa populations

In bacterial populations, switches in lifestyle from motile, planktonic growth to surface-associated biofilm is associated with persistence both in the environment and infections. Studies have identified the first steps of adaptation to biofilm growth but have yet to replicate the extensive genetic diversity observed in chronic infections or in the natural environment. We conducted an evolution experiment with Pseudomonas aeruginosa PA14 in growth media that promotes biofilm formation for 90 days in either planktonic culture or in a biofilm bead model. Surprisingly, all populations evolved extensive genetic diversity with hundreds of mutations maintained at intermediate frequencies, while fixation events were rare. Instead of the expected few beneficial mutations rising in frequency through successive sweeps, we observe a remarkable 40 genes with parallel mutations spanning both environments and often on coexisting genotypes within a population. Additionally, the evolution of mutator genotypes (mutS or mutL mutator alleles) that rise to high frequencies in as little as 25 days contribute to the extensive genetic variation and strong clonal interference. Parallelism in several transporters (including pitA, pntB, nosD, and pchF) indicate probable adaptation to the arginine media that becomes highly alkaline during growth, whereas genes involved in signal transduction (including gacS, aer2, bdlA, and PA14_71750) reflect likely adaptations to biofilm-inducing conditions. This experiment shows how extensive genetic and phenotypic diversity can arise and persist in microbial populations despite strong selection that would normally purge diversity. Importance How biodiversity arises and is maintained in clonally reproducing organisms like microbes remains unclear. Many models presume that beneficial genotypes will outgrow others and purge variation via selective sweeps. Environmental structure like biofilms may oppose this process and preserve variation. We tested this hypothesis by evolving P. aeruginosa populations in biofilm-promoting media for three months and found that adaptation and diversification were coupled and mostly uninterrupted by fixation events that eliminate diversity. Genetic variation tended to be greater in lines grown using a bead model of biofilm growth but many lineages also persisted in planktonic lines. Convergent evolution affecting dozens of genes indicates that selection acted on a wide variety of traits to improve fitness, causing many adapting lineages to co-occur and persist. This result demonstrates that some environments may expose a large fraction of the genome to selection and select for many adaptations at once, causing enduring diversity.


Abstract: 11
In bacterial populations, switches in lifestyle from motile, planktonic growth 12 to surface-associated biofilm is associated with persistence both in the environment and 13 infections. Studies have identified the first steps of adaptation to biofilm growth but have 14 yet to replicate the extensive genetic diversity observed in chronic infections or in the 15 natural environment. We conducted an evolution experiment with Pseudomonas 16 aeruginosa PA14 in growth media that promotes biofilm formation for 90 days in either 17 planktonic culture or in a biofilm bead model. Surprisingly, all populations evolved 18 extensive genetic diversity with hundreds of mutations maintained at intermediate 19 frequencies, while fixation events were rare. Instead of the expected few beneficial 20 mutations rising in frequency through successive sweeps, we observe a remarkable 40 21 genes with parallel mutations spanning both environments and often on coexisting 22 genotypes within a population. Additionally, the evolution of mutator genotypes (mutS or 23 mutL mutator alleles) that rise to high frequencies in as little as 25 days contribute to the 24 extensive genetic variation and strong clonal interference. Parallelism in several 25 transporters (including pitA, pntB, nosD, and pchF) indicate probable adaptation to the 26 arginine media that becomes highly alkaline during growth, whereas genes involved in 27 signal transduction (including gacS, aer2, bdlA, and PA14_71750) reflect likely 28 adaptations to biofilm-inducing conditions. This experiment shows how extensive 29 genetic and phenotypic diversity can arise and persist in microbial populations despite 30 strong selection that would normally purge diversity. 31

Introduction: 48
Bacterial populations inhabit countless environments along a continuum of 49 spatial structure, ranging from well-mixed liquid to clusters bound to surfaces. Growth 50 on surfaces is associated with biofilm production that in turn generates nutrient and 51 oxygen gradients 1 , but so too does the metabolic activities of neighboring cells. Life in 52 confined spaces results in varied levels of nutrients, wastes, and signaling molecules 53 that may alter selective forces on parts of the population and create novel ecological 54 opportunities 2 . These physical differences are hypothesized to select for genetic 55 differences in biofilm populations distinct from well mixed cultures. Experimental 56 microbial evolution (EME) studies have found that biofilm populations select for different 57 mutations than those seen in planktonic growth, and the greater environmental structure 58 of the biofilm produces increased genetic diversity 3,4,5,6,7, 8,5,8,4,9 . Despite this process of 59 diversification, replicate populations propagated in both biofilm and planktonic 60 conditions exhibit high levels of both phenotypic and genetic parallelism, suggesting 61 some measure of predictability within the same environment 10,7,11,12,13,14,15 . However, we 62 7 populations evolved to become less motile than the ancestor ( Figure S3b). Interestingly, 117 despite selection to attach to a plastic bead each day presumably by producing biofilm, 118 only the B3 population evolved higher biofilm production than the ancestor as measured 119 by the standard crystal violet assay, while four populations, including bead populations 120 B1 and B2, evolved lower biofilm production ( Figure S3c). This result may reflect the 121 high starting biofilm production of PA14 in these conditions being difficult to improve. To 122 merge these three phenotypes, we performed a principal component analysis ( Figure  123 1a) and found that evolved populations were distinguished from the ancestor but not 124 clearly separated by treatment.  A characteristic known to be associated with positive selection is the increase in 150 nonsynonymous (NS) mutations over synonymous (S) mutations 37,27 . In following with 151 this trend, the normalized NS/S ratio for planktonic populations averaged 1.73 and for 152 biofilm populations 2.34. A total of 31 indels were also identified in the six evolved 153 populations (Table S2, Table S3) while, interestingly, no large structural variants were 154 detected. A large number of intergenic mutations were also identified (233 mutations, or 155 27.5%), many in likely promoter or terminator regions. The importance of intergenic 156 mutations still remains unclear, however recent work has implicated intergenic 157 regulatory regions as critical in Pseudomonas aeruginosa evolution in the laboratory 158 and during infections 20,38 . 159 We hypothesized that the structured biofilm environment would maintain greater 160 genetic diversity than mixed liquid culture, but this ecological process is potentially at 161 odds with the diversity-purging effect of selection on beneficial mutations. For example, 162 a mutation that generally improves growth would be expected to sweep and eliminate 163 genetic variation, even though the biofilm phase of growth is predicted to increase the 164 probability that mutations become established within various subpopulations 5,39, 40 . 165 Planktonic populations would also be subject to similar effects of selective sweeps but temporal changes in all mutations supported by three reads on each of both strands. 172 As predicted, the nucleotide diversity of biofilm populations tended to be greater 173 at the end of the experiment than after only 17 transfers (day 17 vs day 90: p = 0.0478, t 174 = 2.821, df = 4 via two tailed t-test). Planktonic populations continued to select on new 175 mutations throughout the experiment, but they did not become more diverse by day 90 176 than at day 17 (p = 0.239, t = 1.384, df = 4 via two tailed t-test), indicating a process 177 where new mutations were displacing older ones. Biofilm populations also tended 178 toward greater diversity than planktonic populations later in the experiment, albeit not 179 significantly (planktonic vs biofilm, day 90, p = 0.0749, t = 2.394, df = 4 via two tailed t-180 test; Figure S4a). These findings are consistent with the greater morphological variation 181 in biofilm populations than planktonic ones that we reported previously 6 . 182 A major cause of the high genetic diversity is the prevalence of mutator alleles, 183 mutations that cause a genome-wide increase in mutation rate, in four populations. In all  (Table S2).

High diversity is a byproduct of clonal interference 208
While mutator alleles are not beneficial themselves 44 and their dominant effect is 209 to introduce neutral or deleterious mutations, they can facilitate the acquisition of 210 combinations of beneficial mutations. A more beneficial genotype that has acquired 211 several beneficial mutations may be able to outcompete less fit lineages with fewer 212 mutations, overcoming competition among adaptive lineages that slows their rate of 213 increase. This dynamic is known as clonal interference or more generally as the Hill-214 Robertson effect 46,47,48 . The ability to acquire many beneficial mutations and escape 215 clonal interference may be particularly advantageous when beneficial genetic variation 216 is limiting or, more likely in these populations, saturating 49,50 . 217 As described above, hundreds of mutations rose to detectable frequencies in 218 these populations within ~200 generations and persisted. We used software recently 219 developed by our lab to group mutations into genotypes on the basis of shared, nested  (Table 1), and two sweeps were 228 linked to mutator alleles in which 24 mutations fixed (Table S3). These results suggest 229 that the biofilm environment selected larger mutational cohorts, which could involve 230 To test the genotype prediction algorithm producing these Muller plots and to 256 identify other mutations undetected due to their low frequencies or sequencing 257 coverage, we sequenced 26 clones from population B1 and 10 from population P1. B1 clones only belonged to two distinct genotypes ( Figure S7). This finding confirms that 265 the biofilm population maintained greater genetic diversity than the planktonic 266 population at the end of the experiment. phenotypes, which included the ability to form more productive biofilm communities in 282 mixture (Table 2). interactions were characterized previously 6 . (B) Ancestry was inferred using LOLIPop 51 . 288 Genotypes of named are shown in blue.

290
For five morphotypes we can predict a genetic cause of the high biofilm formation 291 and the related high c-di-GMP levels. Morphotypes A and H each independently 292 acquired mutations in gacS, a sensor kinase known to be associated with high biofilm 293 production 54 , and which will be discussed more below. The additional three evolved 294 mutations in separate genes that have all been linked to Pseudomonas biofilm 295 production previously: mutant D acquired a mutation in the sensor histidine kinase 296 rcsC 55 , M acquired a mutation in the type III secretion system gene pscQ 56 , and V 297 acquired a mutation in the two component response regulator PA14_52250 57,58 (Table  298 2). While these are not the only mutations that distinguish the morphotypes from one 299 another, these are leading candidates that could produce their varied biofilm 300 phenotypes. 301  Table 2. Unique mutations found in seven clones from an experimentally evolved biofilm 303 populations with distinct ecological roles 6 . 304

Targets of selection 305
Evolution in large populations (>10 8 ) over a few hundred generations greatly 306 empowers selection relative to the effect of drift, even when mutation rates increase. 307 This allows us to predict that genotypes that rise to a detectable frequency in an evolve-308 and-resequence experiment like this one are fitter than their ancestor. Further, parallel, 309 nonsynonymous mutations affecting the same gene provides strong evidence of an 310 adaptation 59 . Across all six populations, 53 mutations reach 100% frequency, as part of 311 the 10 genotype sweeps (Table 1), and each affected a unique gene except for two 312 each in mutS, pitA, and rpoB (Table S3). However, the population dynamics depicted in 313 Figure 3 and the clonal genomes summarized in Figure 5 demonstrate substantial 314 genetic variation that did not fix, so a more appropriate screen for parallelism might 315 involve a lower frequency threshold. At 80% frequency, eight genes were mutated more 316 than once: lysC, mutS, the intergenic region fabI/ppiD, PA14_23670, pitA, PA14_69980, 317 PA14_71750, and rpoB, and all six populations are represented. Mutations affecting 318 these genes were either small indels or nonsynonymous substitutions predicted to alter 319 or eliminate function. Because these genes were mutated in both planktonic and biofilm 320 environments, they likely enable adaptation to the growth medium, temperature, and 321 dilution timing. However, these represent only 17 of the 116 mutations that reach 80% 322 frequency, a remarkably flat distribution of high-frequency genotypes that suggests 323 many loci may provide a benefit under these conditions. 324 We next analyzed the complete set of parallel mutations found at all frequencies 325 for those passing a statistical threshold of expected random co-occurrence and 326 identified 153 genes or intergenic regions. Forty genes were mutated three or more 327 times, more than would be expected by chance, accounting for 179 mutations (21.1% of 328 all mutations; Table 3). Most mutations are nonsynonymous, as expected from positive 329 selection, and many suggest possible adaptive phenotypes. 330 The gene with the most independent mutations, rluB (PA14_23110) with 10 (8 331 nonsynonymous; Table 3, Table S3 populations, and in all cases, mutations rise to greater than 90% in at least one 381 population (Table 3, Table S3). Mutations in rpoB and other RNA polymerase genes 382  genotypes adapted to either biofilm or planktonic growth, we grew 90-day samples of 416 evolved populations B1, B2, P1, and P2 for two days in each of these conditions 417 independently ( Figure 6). We hypothesized that these treatments would enrich 418 genotypes adapted to either condition that we could resolve by resequencing treated 419 populations and correlating their relative frequencies. Mutation frequencies that depart 420 from the diagonal of equivalency could represent genotypes that are adapted to biofilm 421 or planktonic growth. beads were used to obtain the planktonic and biofilm DNA samples, respectively (see 429 methods for more detail).

431
In all four populations tested we found significant enrichment in both biofilm and 432 planktonic environments ( Figure S8). As expected, the two biofilm populations tested 433 had more biofilm enriched mutations than planktonic enriched (37 and 55 biofilm 434   Table S4). Upon closer examination, 437 the B2 population appears to contain a set of 36 mutations belonging to 4 genotypes 438 enriched for biofilm fitness (Table S4). These genotypes also rose in frequency between 439 days 75 to 90 in the evolution environment and appear to be generally adaptive. 440 However, these experiments did not generally cause large (>10%) shifts in genotype 441 frequency, which suggests that they correspond to adaptations to the overall 442 experimental conditions and not just these growth phases.  (Table S3). We confirmed this finding 459 through clonal sequencing, finding clones H and A to each contain a gacS mutation in 460 their distinguishing genotypes (Table 2), and further, these clones were previously 461 shown to compete for a common niche 6 . While clonal interference likely contributes to 462 the high genetic variation observed in these populations, we also predict that multiple 463 niches exist within each population as our previous study indicated 6 . As further 464 evidence, most cases of gene-level parallel mutations reached only 20-30% frequency 465 within their respective populations (Table 3). We hypothesize that this results from 466 multiple coexisting ecotypes within each population, a phenomenon that has been 467 shown in previous evolution studies, particularly with biofilms 5 , 50 , 77 . 468 To identify selected traits during evolution in a minimal medium containing 469 arginine as sole energy source, we focused on the 40 loci mutated most frequently 470 (Table 2). This medium was selected because it had previously been shown to induce with only ten across all six replicate populations over 600 generations. Further, one of 495 the most commonly mutated pathways was DNA mismatch repair, which increased 496 mutation rates of linked genotypes in the four populations in which they evolved. We 497 hypothesize these mutator genotypes provided the means to escape clonal interference 498 because they had a higher probability of producing multiple, linked beneficial mutations 499 that could outcompete other genotypes. 500 Our previous study of these populations predicted that the different colony 501 morphotypes represented lineages that had evolved to occupy distinct ecological niches 502 within a biofilm population and interact with one another 6 . This genomic study confirms 503 that these genotypes indeed represent different long-lived lineages within the B1 504 population. Each clone possesses different mutations that are candidates for the causes 505 of their varied fitness versus the ancestral PA14 strain, levels of cyclic-di-GMP, and 506 biofilm-associated traits 6 . However, considerably more variation was detected by 507 population sequencing than these lineages could explain, and likewise, sequencing 508 additional clones led to an ever-increasing census of new mutations (Figure 4). These 509 results reinforce the polygenic model of adaptation to the biofilm-inducing arginine 510 medium and the diversity of genotypes that can generally improve growth and survival 511 or inhabit the niches illustrated in our prior study 80,6 . More generally, this work has 512 implications for understanding processes underpinning the evolution and maintenance 513 of high levels of genetic diversity, including how different nutrient sources influence this 514 process. Although differences in the mode of growth have been shown to influence 515 genetic diversity 81,82,9,83 , the metabolic environment may be equally or even more

Phenotypic characterization of evolved populations 548
All populations were revived from freezer stocks for characterization by adding 549 50 µl of freezer stock to a culture of 4:1 M63 media to LB media, with trace elements to 550 recreate previous water mineral content (110.764 g/L CaCl2, 0.824 g/L MnSO4, 33.48 551 g/L KBr, 132.737 g/L Na2SiO3, 123.485 ZnSO4, 0.178 g/L CoCl2 *6H2O, 1.458 g/L 552 CuSO4). This media combination was used to minimize selection effects of growing in a 553 fully complex medium, while providing enough nutrients for the frozen cells to revive. 554 Resurrected populations were grown for 24 hr at 37 °C on a roller drum before being 555 vortexed for 10 seconds, twice, to ensure biofilm growth was disrupted from the sides of 556 the glass tube. 557 Growth curves were measured for resurrected populations in 96 well plates, at 37 558 °C. All growth curves were started at an OD600 of 0.01 and growth was measured every 559 10 min following 9 min of shaking for 24 hr in M63 media with trace elements. Maximum 560 growth rate was determined for the average of all replicates, by using the following 561 equation: 562 = ∆log ( 600) ∆ 563 Census population size was calculated by diluting resurrected populations 1:100 564 in 5 mL M63 media with trace elements. One 7 mm polystyrene bead was added to 565 each biofilm culture at time zero. After 24 hr growth, all populations were vortexed for 30 566 seconds and then diluted and plated in triplicate on tryptic soy agar and grown at 37 °C 567 for 48 hrs. Average colony counts of the three technical replicates were reported for 568 each population. Additionally, populations plated for census population size were also 569 used for pH measurements as they were complete replications of experimental 570 conditions. Average pH of triplicate readings are reported. 571 Biofilm assays were performed on resurrected populations diluted to an OD600 of 572 0.01 in M63 media and grown in a 96 well plate under static conditions for 24 hr at 37 573 °C. Media was discarded and the plate was washed twice with deionized H2O, which 574 was also discarded. Wells were then stained with the addition of 250 µl 0.1% crystal 575 violet solution for 15 minutes. Plates were rinsed to remove excess dye and the plate 576 was allowed to dry for 24 hours. A de-stain solution (95% ethanol, 4.95% dH2O, and 577 0.05% Triton X-100 (fisher bioreagents)) was added to wells (250 µl per well), and, after 578 a 15-minute incubation, was transferred to a new 96 well plate. Crystal violet 579 absorbance readings were measured at 590 nm. Reported values are the average of 21 580 replicates, done across three plates (7 technical replicates per plate). Data were 581 analyzed with a one-way ANOVA with post hoc Tukey test. 582 Motility assays were performed as described previously (Ha, et al 2014). Briefly, 583 pipet tips were dipped into resurrected populations and stabbed into motility agar (0.3% 584 agar, 6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 0.2% glucose, 0.5% casamino acids, 585 1 mM MgSO4). Increased or decreased motility was determined for all day 90 evolved 586 populations as the diameter of bacterial growth after 24 hr growth at 37 °C compared to 587 the clonal ancestor. Reported values are the average of 5 replicates. Results were 588 analyzed by one-way ANOVA with post hoc Tukey test. 589

Mutation rate estimate 590
Isogenic mutants of biofilm evolved mutator alleles mutS T112P and mutL 591 D467G were used as reported previously 6 . Briefly, the two mutator strains and the PA14 592 ancestor were revived from freezer stocks by streaking on ½ trypic soy agar. After 24 593 hrs growth individual colonies were used to start 30 replicate 5-ml cultures for each 594