Convergent evolution of hyperswarming leads to impaired biofilm formation in pathogenic bacteria

Abstract

Most bacteria in nature live in surface-associated communities rather than planktonic populations. Nonetheless, how surface-associated environments shape bacterial evolutionary adaptation remains poorly understood. Here, we show that subjecting Pseudomonas aeruginosa to repeated rounds of swarming, a collective form of surface migration, drives remarkable parallel evolution toward a hyperswarmer phenotype. In all independently evolved hyperswarmers, the reproducible hyperswarming phenotype is caused by parallel point mutations in a flagellar synthesis regulator, FleN, which locks the naturally monoflagellated bacteria in a multiflagellated state and confers a growth rate-independent advantage in swarming. Although hyperswarmers outcompete the ancestral strain in swarming competitions, they are strongly outcompeted in biofilm formation, which is an essential trait for P. aeruginosa in environmental and clinical settings. The finding that evolution in swarming colonies reliably produces evolution of poor biofilm formers supports the existence of an evolutionary trade-off between motility and biofilm formation.

Figure 1. Experimental evolution of swarming motility produces a stable and heritable hyperswarmer phenotype

(A) Three independent lineages (1–3) were subjected to experimental evolution by sequential passages of growth in swarming media. After each 24h-swarming interval, the entire colony was flushed off the plate and a 1/1500 fraction of the recovered population was point-inoculated onto a fresh swarming plate. Lineage #2 acquired a hyperswarming phenotype at day 5, whereas lineages #1 and #3 only did so at day 7. The colonies outlined in color were selected for clonal isolation procedures. The color-coding scheme (cyan for lineage #2 at day 5, magenta for lineage #1 at day 9, yellow for lineage #2 at day 9 and green for lineage #3 at day 9) is maintained throughout the paper. (B) Hyperswarming is stable and heritable. Swarming colonies of the ancestral strain and clones isolated from each of the colonies outlined in panel (A). See also video S1.

Figure 2. Hierarchical clustering of quantitative phenotypic assays suggested the existence of three distinct hyperswarmer clones

(A) Quantitative phenotypic assays performed on the ancestral strain (denoted as ‘anc.’), two non-motile clones (flgK⁻ and pilB⁻) and twelve hyperswarmer clones. Free cells and attached cells in a crystal violet biofilm assay were quantified, together with rhamnolipid secretion (using the sulfuric acid anthrone assay), twitching motility and swimming motility. All measurements were normalized to the ancestral strain. (B) Phenotypic strain grouping using hierarchical clustering. Blue indicates a decrease compared to the ancestral strain and red indicates an increase compared to the ancestral strain (the ancestral strain on the right-hand side is black since all phenotypes are normalized to it). The non-motile mutants cluster separately from hyperswarmers and from the ancestral strain. Within the hyperswarmer clones there are three apparent clusters: clones 1, 3 and 4, clones 2 and 9–12 and clones 5–8. After clustering, clones 1, 2, 4, 5 and 10 were selected for use in the following studies. Clone 5 was indeed different from clones 2 and 10 when looking at cell size (see figure S1).

Figure 3. Hyperswarmers lose competitions In liquid culture against the ancestral strain, but prevail in swarming competitions by segregating at the leading edges of expanding swarming colonies

(A) Growth rates of the ancestral strain and five hyperswarmer clones reveal that hyperswarmers grow slower in liquid culture (see figure S2A for actual determinations). (B–C) Competitions between hyperswarmers and the ancestral strain in liquid media (B) and swarming plates (C). The neutral selection coefficient of 1 is marked with the black line. Each competition was started at a 1:1 ratio of hyperswarmer to ancestral. The error margins of the neutral selection coefficients (gray area) were experimentally determined by competing wild type against itself in the appropriate settings. The selection coefficients represent the ratios of hyperswarmers to ancestral before and after the competition divided over each other. A selection coefficient of >1 means the hyperswarmer wins, while a selection coefficient of <1 means the hyperswarmer loses. (D) Hyperswarmers are enriched at the leading edge of swarms. Fluorescence scans of swarms initiated with a 10:1 ratio of ancestral in green to hyperswarmer in red (top row) or the inverse (bottom row). See figure S2B for repulsion assays. * P < 0.05, *** P < 0.005.

Figure 4. Hyperswarming is caused by point mutations in fleN, which produce polar multi-flagellated cells

(A) 6 distinct fleN point mutations were identified from experimental evolution in swarming motility. FleN(V178G) was found in clones 1, 3 and 4 of the initial experiment and FleN(W253C) was found in clones 2 and 5–12. FleN(V178G) emerged another 11 times and FleN(F176S), FleN(L179Q), FleN(F203L) and FleN(P254L) were all encountered once in independent runs. These mutations are not found in the deposited Pseudomonas spp. genomes. (B) fleN mutations are necessary and sufficient to cause hyperswarming. In cis genetic complementation was performed through allelic replacement. When converting wild type fleN to either FleN(V178G) (top left) or FleN(W253C) (middle left), colony morphology changed from wild type into hyperswarming. Conversely, the reversion of clones back to wild type (right hand column) yielded wild type morphologies. Hyperswarming was not recapitulated by ΔfleN (lower left). (See figure S3A for phenotypic assays of all complemented clones.) (C) Transmission electron microscopy of the ancestral strain, a non-flagellated clone (flgK⁻) and the five hyperswarmer clones shows that hyperswarmers have become multi-flagellated. Scale bar represents 1 μm. Insets show 88,000x magnification of the cell pole. (D) The distribution of flagella in the ancestral strain (anc.) and all five hyperswarmer clones (see figure S3B for the histograms and S3C for fliC expression in liquid culture).

Figure 5. Hyperswarming is not found in clinical or environmental isolates

The shape of swarming colonies was quantified and plotted in 2D using plate coverage and colony roundness (calculated by [4*π*area]/perimeter²). The ancestral strain and a non-flagellated clone (flgK⁻) were included as controls. 47 non-laboratory strains (18 environmental isolates from hydrocarbon-contaminated soil in Canada, shown in blue, and 29 clinical isolates from patients at Memorial Sloan-Kettering Cancer Center, shown in red) were compared to five hyperswarmer clones (color-coded according to their plate of origin) in standard swarming assays. Representative photos of swarming colonies are shown above for illustration. In orange is the clinical isolate that was subjected to experimental evolution (see ancestral and hyperswarmer for this strain in figure S4).

Figure 6. Hyperswarmers lose biofilm competitions against the ancestral strain, indicating an evolutionary tradeoff between motility and biofilm formation

(A) Hyperswarmer clones lose against the ancestral strain already at the phase of initial attachment to a biofilm substratum. Slide biofilms were inoculated at a 1:1 ratio and incubated for 6h before imaging the attached cells (see inset, where ancestral is red and clone 4 is green). The black line indicates the neutral selection coefficient of 1. These experiments were performed in duplicate (also for the neutral selection coefficient). The 95%-confidence intervals (both for neutral and actual selection coefficients) were determined through the probability density functions of the counts. (B) Full grown flow-cell biofilm of clone 4 (green) and ancestral (red). The biofilm was inoculated at a 1:1 ratio and then grown for 24h before imaging. The image is a deconvolved 3D projection of the biofilm. (C) In the ancestral strain, motility and biofilm formation are balanced. The fleN mutations in hyperswarmers, however, cause an imbalance by which the highly motile mutant lacks in biofilm formation. (See figure S5 for all biofilm competition data and Spearman correlation between swarming and biofilm formation in the clinical and environmental isolates.)