Rapid radiation and the evolution of collective spreading

We recently isolated a mucoid strain in colonies of Pseudomonas fluorescens Pf0-1 (ref. 30), which we call ‘M’ here that gets its mucoidy from a glucose-based polymer (Supplementary Table 1). M harbours a single-nucleotide frameshift mutation in the rsmE gene, which encodes a repressor of multiple secretions30 (Supplementary Table 2). When plated clonally, we observe that M evolves and repeatedly generates a new phenotype that enables the colonies to spread outwards (Fig. 1a, see Methods for details). Outward fan-like growth is common at the edge of ageing bacterial colonies. However, these fans are typically thought to be a clonal population of a mutant that has acquired the ability to grow or move faster as compared with the parent cells31. In contrast, we found that our spreading fans consistently comprised a mixture of two strains: both the original strain M and a new morphotype that we call ‘D’ here because of its dry and wrinkly colony morphology. The spreading phenotype is reproduced whenever M and D are mixed, but only appears after a significant delay in pure colonies because, as we will show, it rests upon mutations to generate the other variant (Fig. 1a). The spreading phenotype allows colonies to both gain territory and to produce more cells in way that neither strain achieves on its own (Fig. 1b). These observations were consistent with the de novo evolution of a division of labour where distinct types generate a shared phenotype—rapid spreading—that is not possible on their own. We therefore subjected our experimental system to detailed analyses in order to understand how an apparent division of labour could so rapidly evolve from a simple bacterial colony.

Figure 1: Emergence of the spreading phenotype and comparison of the population size of single and mixed morphotype colonies. (a) Spreading fans emerge from a spotted mucoid M colony (top), comprising M and a genetic variant, D, exhibiting a dry colony morphology. The same phenotype emerges from D (middle), and is reproduced by mixing M and D (bottom). Scale bar, 5 mm. (b) Estimates of the total population size in colony-forming units (CFU) of single or mixed morphotype colonies on day 8. One-way analysis of variance (ANOVA; P=0.000957). Tukey’s honest significant difference (HSD): M versus D (P>0.05); M versus M+D (P<0.01) and D versus M+D (P<0.01). (c) Ratio of strains in mixed colonies, as indicated on the x axis, on day 8. Each strain was engineered to be either kanamycin (K) or streptomycin resistant (S). One-way ANOVA (P=0.002824). Tukey’s HSD: MK/MS versus DK/DS (P>0.05); MK/MS versus MK/DS (P<0.05); MK/MS versus MS/DK (P<0.05); DK/DS versus MK/DS (P<0.05); DK/DS versus MS/DK (P<0.05) and MK/DS versus MS/DK (P>0.05). All mixed colonies were initiated at a 1:1 ratio. For all experiments, plotted are the means from three independent colonies, and the error bars represent the s.d. Full size image

M and D robustly self-organize in space

To probe the robustness of the phenotype, we started the experiment across an extremely wide range of initial frequencies of M and D that covers several orders of magnitude. Not only did all conditions generate the spreading phenotype, we were surprised to find that the two strains always approach a characteristic ratio over time, ∼10% M to 90% D, irrespective of the starting frequencies (Figs 1c and 2a). Accordingly, we detect strong negative frequency-dependent selection in the system where each strain can outcompete the other when rare (Fig. 2b). The stability in the frequency of the two types is further suggestive of a well-organized collective phenotype. To understand this better, we directly imaged the mixed colonies using epifluorescence and confocal laser scanning microscopy using a metallurgic objective, which allows us to image without using a coverslip that would distort the colony structure. This revealed that the two strains segregate strongly in space: M mostly localizes at the centre, whereas D dominates the spreading bulk of the colony (Fig. 3a) where it sits atop a thin layer of M (Fig. 3b).

Figure 2: The frequency of M and D morphotypes approach a characteristic ratio independent of the initial frequency. (a) The ratio of kanamycin-resistant M (MK) compared with streptomycin-resistant D (DS) in mixed colonies on day 8 (blue) and day 13 (red) plotted against the initial ratio. Shown below are corresponding images of mixed colonies on day 8. Scale bars, 5 mm. (b) The data in a plotted as relative fitness of MK compared with DS. The dotted line represents equal fitness. All error bars represent the s.d. of the mean from three independent colonies. Full size image

Figure 3: M and D self-organize into characteristic spatial patterns. (a) Epifluorescence image of a mixed colony of fluorescently tagged M (green) and D (red) on day 8. (b) Corresponding confocal laser scanning microscopy image of the edge. The white arrow points at the edge of the colony. (c,d) Confocal images of the edge on day 2 (c) and day 22 (d). The two images within each panel are identical except the red channel has been removed from the bottom images. (e,f) Day 8 epifluorescence image of a mixed colony disturbed daily from day 3 (e) or only once on day 3 (f). All mixed colonies were initiated at a 1:1 ratio. Scale bars, 2 mm or 20 μm in epifluorescence and confocal images, respectively. All confocal images are three-dimensional renderings. Full size image

The ability of D to sit on top of M is seen before spreading starts and likely explains its ability to initially increase in frequency as cells at the top of a colony gain an advantage by having the best access to oxygen (Fig. 3c)21,30. However, segregation is most striking at the edge of the colony where the spreading is actually occurring. Here, D again dominates except at the very edge where there is a continuous strip of M, measuring ∼100 μm, that forms the circumference of the entire colony. This thin strip forms within the first 2 days of mixing (Fig. 3c) and remains stable for as long as we run the experiments (22 days, Fig. 3d). The relative positioning of the two strains is crucial. Daily disruption of the mixed colony inhibits spreading (Fig. 3e). When the mixed colony is disrupted only once, however, the two strains self-organize back into their relative positions and spreading ensues (Fig. 3f). This robustness also helps to explain why the strains are co-selected during spreading and the convergence of the two morphotypes on a characteristic ratio (Fig. 2). A specific spatial structuring involving both strains is necessary for both the commencement and maintenance of the collective phenotype.

D and M carry out distinct tasks to spread collectively

How are the two strains interacting to enable spreading? One possibility is that D is passively supplying a factor—a signal or metabolite—that promotes the motility of M cells. However, time-lapse confocal microscopy suggests instead that D pushes M along at the edge of the colony (Supplementary Movie 1), because narrow tracks of stationary M cells are seen underneath the moving D cells akin to scraped earth beneath the base of a moving glacier (Supplementary Movie 2). Furthermore, pure D colonies exhibit a wrinkly morphology (Fig. 1a), which has been shown to be associated with the buildup of compressive forces that accumulate through individual cell division events within colonies that produce adhesive extracellular polymers32. Consistent with their adhesive nature, the dry-wrinkly D colonies expand slower in comparison to the smooth M colonies (Fig. 1a). Moreover, this wrinkly morphology is greatly reduced in the mixed morphotype colonies (Fig. 1a), suggesting that the forces generated by D are released in the form of pushing when interacting with M.

To further support the importance of D pushing M, we sought an experimental test of this role of D. We reasoned that if D is pushing M and the edge of the colony outwards, then physically removing the cells between the edge and the centre of the colony, where D occupies the bulk of the space (Fig. 3, ), will slow colony spreading. We performed this test and found that spreading is indeed inhibited. However, the same treatment does not deform the shape of a colony without the spreading phenotype (Supplementary Fig. 1a). This demonstrates that the treatment does not impact the basal level of cell division-driven colony expansion. In sum, multiple lines of evidence support the model that D pushes the edge along, and we provide further evidence below.

Figure 4: The mucoid polymer produced by M is required for both spreading and spatial structuring. (a) A schematic of the gene cluster associated with mucoid polymer production in M. The numbers represent the annotated Pfl01_ORF designation, and the red vertical bars indicate the sites of transposon insertions that result in the loss of the mucoid phenotype in M. (b) Colony morphology of non-mucoid M* (MΔPfl01_3834) on its own and in mixture with D or M on day 8. Scale bars, 5 mm. (c) Estimates of the total population size of various mixed colonies on day 8. One-way analysis of variance (ANOVA; P=0.000167). Tukey’s honest significant difference (HSD): M+D versus M*+D (P<0.01); M+D versus M*+M (P<0.01) and M*+D versus M*+M (P>0.05). (d) Day 8 strain ratio in mixed colonies as indicated on the x axis. D is streptomycin-resistant and M and M* are kanamycin-resistant. One-way ANOVA (P<0.0001). Tukey’s HSD: D/M versus D/M* (P<0.01); D/M versus M/M* (P<0.01) and D/M* versus M/M* (P>0.05). (e) Epifluorescence images of mixed colonies of fluorescently tagged M (green, MG) or M* (green, M*G) and D (red, DR) on day 8. (f) Mucoidy in M is needed for characteristic spatial structuring of the two morphotypes. Three-dimensional rendering of confocal images of the edge of mixed colonies after spotting (0 h) and 24 h later. For all experiments, plotted are the means from three independent colonies, and the error bars represent the s.d. All mixed colonies were initiated at a 1:1 ratio. Scale bars, 2 mm or 20 μm in epifluorescence and confocal images, respectively. Full size image

Why is M required for the spreading phenotype? On their own, M colonies expand faster than D colonies, but this is dramatically enhanced when D and M are mixed together (Fig. 1a). Based on the mucoid appearance of M, we predicted that a mucoid polymer secreted by M could function as a lubricant. More formally, we hypothesised that that the polymer acts as a wetting agent that hydrates the colony and reduces the resistance to movement (viscous flow), which allows D to push M along from behind. To address this, M was subjected to random transposon mutagenesis to disrupt the production of the polymer. This identified three independent transposon insertions within a cluster with homologies to extracellular polysaccharide biosynthetic genes (Fig. 4a). Deleting one of the targeted genes in the M background (M*) also removed mucoidy (Fig. 4b). Moreover, the M* strain that lacks the polymer does not evolve the spreading phenotype (Fig. 4b), and pre-mixed colonies of M* and D do not show spreading (Fig. 4b,e). The M*+D mixture increases in population size at a rate that is comparable to the pure cultures and other mixtures that do not exhibit the spreading phenotype (Figs 1b and 4c). The loss of spreading is also associated with a loss of the characteristic spatial structuring. Without the mucoidy in M, the two strains remain relatively well mixed (Supplementary Fig. 2) and D fails to reach the colony edge and dominate the colony (Fig. 4f). D also reaches a much lower population size when mixed with M* relative to being mixed with M (Fig. 4d).

We have shown that the mucoid polymer of M is central to the spatial organization of D and M, which ultimately allows D to drive the spreading phenotype (Figs 3 and 4). But does mucoidy also have a lubricating role as we first hypothesized? To examine this, we artificially created the spatial structure that emerges by self-organization of D and M in mixed colonies (Fig. 3). We did this by spotting either M or M* cells at the edge of pure D colonies. Although the correct spatial structure is present whether M or M* is added, D is able to spread when M is provided at the edge but not when M* is added that lacks the polymer (Supplementary Fig. 1b). This suggests that a lubrication effect of the polymer may indeed be important. However, as the spatial structure we created using pure cultures is crude compared with the one that emerges naturally in mixed colonies, we carried out an additional manipulation. This experiment has a more complex design but arguably provides the strongest evidence for both lubrication by M and pushing by D in one go. Here, we prepared mixed colonies of fluorescently labelled M+D cells that, as always, proceeded to naturally self-organize and establish their characteristic spatial structure with M at the front and D behind. On day 3, we then added a droplet of unlabelled cells of either M, M* or D at the edge of the expanding colony (Fig. 5a) in order to test if the colony could continue spreading in the face of this potential barrier. The results of this experiment are clear: only when M is placed at the edge can the colony continue to spread unhindered (Fig. 5b). Moreover, epifluorescence imaging shows that it is the D cells from the original colony that stream into the M droplet and re-establish the spreading phenotype with the unlabelled M cells from the added droplet (Fig. 5c and Supplementary Fig. 3). The observation that the D cells, and not the M cells, continuously stream into the droplet lends further support to the role of D as the strain that generates the pushing force for spreading.

Figure 5: M functions as a lubricant allowing D to push from behind. (a) Schematic of the spatial structure construction experiment. Fluorescently labelled M (green) and D (red) cells are mixed 1:1 and spotted. M and D self-organize and spatially segregate, establishing the characteristic spatial structure. Droplets of unlabelled M, M* (no mucoid polymer) or D cells are then placed near the edge of the spreading front on day 3 and visualized on days 4 and 8. (b) Spreading continues when the mixed colony encounters a droplet of M, but not M* or D. Main images were captured on day 8 and the inset images show the spatial arrangements on day 4. Scale bars, 5 mm. (c) The red-labelled D strain streams into the droplet of cells and pushes the colony edge along, but only when the droplet cells are the mucoid M strain. Epifluorescence images on day 4 (top; 0.5 mm scale) and day 8 (bottom; 2 mm scale). D continues to push through M, but not M* or D. These images correspond to the bottom droplets shown in b. See also Supplementary Fig. 3 for more detail on the key treatment where D streams into mucoid M. Full size image

We observe then that the mucoid polymer does indeed act like a lubricant because the spreading colony is blocked by the addition of a droplet that lacks the polymer. An alternative model is that the mucoid polymer of M acts as a signalling or nutrient source to induce spreading in D cells. However, this model would predict that D cells are still fed (or signaled to) by the accompanying M cells as they encounter the M* or D droplets. In this case, therefore, we should expect that the labelled D cells enter all droplets but, in the absence of the mucoid polymer, they would gradually slow down and stop moving somewhere within the droplet. However, this is not observed. Instead, both M* and D droplets appear to create an immediate physical barrier that generates a sharp border against the incoming M+D population (Fig. 5c). In sum, these experiments explain why M and D are both necessary for the spreading phenotype. M makes a polymer that is important for both the emergence of spatial structure and as a lubricant, and D cells are needed to push the edge of the colony outwards.

Genetic basis of the evolution of the D morphotype

Our experiments document the evolution of a phenotype that is generated by two morphotypes working together and performing distinct roles. This shows that rapid diversification in bacteria can indeed allow them to make use of the division of labour. Rapid evolution, however, typically rests upon very few mutations. How then can bacteria evolve a well-organized, and robust, collective phenotype with only the time for a handful of mutations? In order to answer this question, we sequenced the genome of the D strain, which revealed a two-nucleotide deletion at the tail end of the wspC gene (Supplementary Table 2). This mutation places the downstream wspD gene within the same reading frame as wspC. Wsp proteins function together as a signal transduction system that responds to growth on surfaces (Fig. 6a)20,33,34,35: methylation of WspA triggers a phosphorelay to activate WspR, a diguanylate cyclase, which catalyses the formation of cyclic di-3′,5′-guanylate (c-di-GMP) from two molecules of GTP. c-di-GMP is a universal secondary messenger molecule in bacteria, which impacts diverse physiological processes36. The Wsp system has been demonstrated to modulate c-di-GMP production in P. fluorescens37, and specifically in our strain background38. Wsp mutants are well known to emerge in P. fluorescens in liquid cultures where the phenotype allows cells to stick to the edge of glass culture vessel and form a mat across the liquid surface20,21. Although the functional outcome is different to our system, the prior observation of wsp mutants in experimental evolution made them a particularly promising general candidate for our D morphotype.

Figure 6: Genetic analyses of the D morphotypes and the spreading phenotype. (a) Schematic of the wsp operon (top). Red vertical bars indicate the site of individual mutations found among the D morphotypes. A simplified model of the corresponding Wsp signalling system modulating c-di-GMP production (bottom). (b) Genetic analysis suggests that the original D strain has a mutation that hybridizes the WspC and WspD proteins. Comparison of the colony morphology of the engineered strains to M and D on day 3. Only the wspC:D hybrid mutant (that is, the same mutation found in D) exhibits the same dry morphology as D. (c) Comparison of the spreading phenotype of the engineered strains mixed at a 1:1 ratio with either M or D on day 8. Scale bars, 5 mm. Full size image

Introduction of the same two-nucleotide deletion in M (wspC:D) reproduces the same D morphology. However, we found that deleting each gene, or both, fails to create the D morphology (Fig. 6b). Furthermore, the wspC:D hybrid mutant reproduces the spreading phenotype when mixed with M, whereas the other mutants require the presence of D to spread (Fig. 6c). The D morphotype then appears to be the product of the function of the WspCD hybrid rather than that of loss of function of either or both proteins. Given that WspC is a constitutive activator and WspD is a scaffolding protein that binds to WspA (Fig. 6a), we predict that the WspCD hydbrid increases methylation of WspA and drives up the production of c-di-GMP. In addition to the wrinkly colony morphology, which results from the production of structurally rigid extracellular polymers in P. fluorescens SBW25 and many other species36,39, motility is also a strongly conserved phenotypic indicator of c-di-GMP production36,40. Increased c-di-GMP production has been explicitly demonstrated to reduce motility in our specific strain background38, and flagella synthesis has been shown to be repressed directly by c-di-GMP in P. aeruginosa41. Further consistent with the role of c-di-GMP in our phenotypes, both the original D strain and wspC:D hybrid mutant display the wrinkly colony morphology (Fig. 6b) and impaired motility (Supplementary Fig. 4). In sum, we observe a mutation in the Wsp system, which is predicted to increase c-di-GMP production, and the resulting wrinkly morphology and reduced motility phenotypes are both the expected effect of increased c-di-GMP.

Individual spreading fans that emerge from M colonies always contain a variant of a single dry-wrinkly morphology in addition to M. Do the wsp loci then serve as a common mutational target for the evolution of the D morphotype? Sequencing the wsp operon in several independently evolved D morphotypes revealed that all had a single mutation in a wsp locus (Figs 6a and 7a, Supplementary Table 2, see Methods). This was again associated with both the wrinkly colony morphology (Fig. 7a) and reduced motility phenotype (Supplementary Fig. 4), suggesting increased c-di-GMP production in all strains that we tested. The most frequent example is a missense mutation in wspE (WspED648G), which encodes a phosphorelay protein that activates WspR (Figs 6a and 7a). WspE harbours both histidine kinase and receiver domains. The receiver domain is highly conserved in bacteria and work from the homologous CheY of Escherichia coli shows that the specific D648G mutation that we observe can activate the protein in a phosphorylation-independent manner when accompanied by two additional missense mutations42,43. Moreover, recent studies show that the diguanylate cyclase activity of WspR in P. aeruginosa is also not exclusively dependent on phosphorylation34,44. This suggests that WspED648G may stimulate WspR or other diguanylate cyclases through another mechanism that does not depend on phosphorylation.

Figure 7: Multiple D genotypes evolve and bidirectional evolution generates M from D and vice versa. (a) Phenotypes of additional D morphotypes individually isolated from a single spreading fan emerging from discrete M colonies (day 3, top). Each variant reproduces the spreading phenotype when mixed with M (day 8, bottom). (b) Mucoid colony morphology of the wspE# and wspE::Tn revertants (day 3) and their spreading phenotype when mixed with D (day 8). (c) Emergence of spreading fans from various mutants captured on day 9. Each starting strain harbours a mutation predicted to terminally shut down the Wsp signalling pathway, suggesting that additional c-di-GMP production pathways are involved in the transitions between the M and D morphotypes. (d) Summary of the bidirectional evolution of M and D morphotypes. All strains of the D morphotype exhibit reduced motility, indicative of reduction in c-di-GMP production. All mutations were naturally selected with the exception of wspE::Tn (black outline). All mixed colonies were initiated at a 1:1 ratio. Scale bars, 5 mm. Full size image

The remaining adaptation events occurred through a mutation in wspA: an in-frame deletion which removes 28 amino acids or a missense mutation (WspAA381V; Fig. 7a and Supplementary Table 2). Methylation of WspA stimulates the histidine kinase activity of WspE, which in turn activates WspR (Fig. 6a)35. This suggests that these WspA mutations stimulate WspE and ultimately WspR to amplify the production of c-di-GMP. All mutations generate a similar colony morphology as the original D strain, with small variations, and each of the independently derived dry morphotypes reproduce the spreading phenotype when mixed with M (Fig. 7a). Moreover, none of the other mutations are predicted to result in loss-of-function of the encoding protein. Instead, they all indicate that the evolution of the D genotype is associated with the activation of the Wsp system and an increase in production of c-di-GMP; the Wsp system is in a low-activity state in M, and the mutations are selected to stimulate it (Supplementary Fig. 4).

Bidirectional selection of D and M morphotypes

Natural selection repeatedly targets the wsp operon and generates the spreading phenotype in M colonies. In addition, the spreading phenotype also repeatedly evolves from pure D colonies (Fig. 1a). Here, the spreading fan always comprises both D and a new morphotype that appears identical to M, and mixing the two strains reproduces the spreading phenotype. Given that specific mutations within the wsp operon could act to either stimulate or dampen the signalling pathway (Fig. 6a), we hypothesized that subsequent mutations in wsp loci are responsible for the bidirectional selection of D and M morphotypes. We found this to be the case. Resequencing the wsp operon in one of the M revertants (wspE#) revealed a nonsense mutation in wspE that is predicted to shut down the Wsp system (Figs 6a and 7b and Supplementary Table 2). In addition, we undertook a transposon mutagenesis screen in the D background to isolate mutants that produce the M morphology, which isolated a single mucoid mutant (wspE::Tn) where the transposon had inserted into the wspE gene (Fig. 7b and Supplementary Table 2). As with the original M strain, mixing the M-revertant or the mucoid transposon mutant with D reproduces the spreading phenotype (Fig. 7b). Furthermore, in addition to the loss of the wrinkly colony morphology, both strains regained motility (Supplementary Fig. 4), in accordance with the predicted reduction in c-di-GMP production.

Mutations that first activate and then suppress the Wsp system allow the strain to evolve first from M to D and then back to M (Fig. 7d). However, the last class of mutations work by inactivating the Wsp system and make its reactivation extremely unlikely because this would rest upon reversion and/or restoration of function in the affected proteins (Fig. 6a). This begs the question of whether these strains would be capable of generating the spreading phenotype by again evolving from M to D. To test this, we started colonies using a number of evolved, or engineered, M strains that have a dysfunctional Wsp system. We find that the spreading phenotype re-evolves in each case, where a new D morphotype is once again co-selected (Fig. 7c). This suggests that natural selection for the D morphotype has extended beyond the function of the Wsp system. Consistent with this, previous work has identified at least 38 proteins in our strain background that may harbour a diguanylate cyclase activity38, which provides a large set of mutational targets to evolve D from M, and vice versa. Accordingly, every new D morphotype exhibits reduced motility (Supplementary Fig. 5), suggesting c-di-GMP production is changed once again in the evolutionary sequence from D to M, then back to D. Although the underlying genetic effects are diverse, therefore, we observe robust bidirectional evolution that reliably generates whichever partner is missing for collective spreading.