Abstract Bacteria have fascinating and diverse social lives. They display coordinated group behaviors regulated by quorum-sensing systems that detect the density of other bacteria around them. A key example of such group behavior is biofilm formation, in which communities of cells attach to a surface and envelope themselves in secreted polymers. Curiously, after reaching high cell density, some bacterial species activate polymer secretion, whereas others terminate polymer secretion. Here, we investigate this striking variation in the first evolutionary model of quorum sensing in biofilms. We use detailed individual-based simulations to investigate evolutionary competitions between strains that differ in their polymer production and quorum-sensing phenotypes. The benefit of activating polymer secretion at high cell density is relatively straightforward: secretion starts upon biofilm formation, allowing strains to push their lineages into nutrient-rich areas and suffocate neighboring cells. But why use quorum sensing to terminate polymer secretion at high cell density? We find that deactivating polymer production in biofilms can yield an advantage by redirecting resources into growth, but that this advantage occurs only in a limited time window. We predict, therefore, that down-regulation of polymer secretion at high cell density will evolve when it can coincide with dispersal events, but it will be disfavored in long-lived (chronic) biofilms with sustained competition among strains. Our model suggests that the observed variation in quorum-sensing behavior can be linked to the differing requirements of bacteria in chronic versus acute biofilm infections. This is well illustrated by the case of Vibrio cholerae, which competes within biofilms by polymer secretion, terminates polymer secretion at high cell density, and induces an acute disease course that ends with mass dispersal from the host. More generally, this work shows that the balance of competition within and among biofilms can be pivotal in the evolution of quorum sensing.

Author Summary Bacteria are increasingly recognized as highly interactive organisms with complex social lives, which are critical to their capacity to cause disease. In particular, many species inhabit dense, surface-bound communities, termed biofilms, within which they communicate and respond to local cell density through a process known as quorum sensing. Enormous effort has been devoted to understanding the genetics and biochemistry of biofilm formation and quorum sensing, but how and why they evolve remain virtually unexplored. Many bacteria use quorum sensing to regulate the secretion of sticky extracellular slime, an integral feature of biofilm life. Intriguingly, however, some pathogenic species turn on slime production at high cell density, whereas others turn it off. Using an individual-based model of biofilm growth, we investigated why different species use quorum sensing to control slime production in opposite ways. The secret underlying this variation appears to reside in the nature of infections. Turning slime on at high cell density can allow one strain to suffocate another when competition is intense, as occurs in long-lived chronic infections. Meanwhile, turning slime secretion off at high cell density can benefit a strain causing an acute infection by allowing rapid growth before departing the host.

Citation: Nadell CD, Xavier JB, Levin SA, Foster KR (2008) The Evolution of Quorum Sensing in Bacterial Biofilms. PLoS Biol 6(1): e14. https://doi.org/10.1371/journal.pbio.0060014 Academic Editor: Nancy A. Moran, University of Arizona, United States of America Received: October 2, 2007; Accepted: December 10, 2007; Published: January 29, 2008 Copyright: © 2008 Nadell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: CDN is supported by a Centennial Fellowship and Robert May Fellowship from Princeton University. KRF and JX are supported by National Institute of General Medical Sciences Center of Excellence Grant 5P50 GM 068763–01. SAL is pleased to acknowledge support from the Defense Advanced Research Projects Agency (DARPA) under grant HR0011-05-1–0057. Competing interests: The authors have declared that no competing interests exist. Abbreviations: EPS, extracellular polymeric substance; EPS+, constitutive extracellular polymeric substance-producing strain; EPS−, non-extracellular polymeric substance-producing strain; QS+, quorum-sensing strain

Introduction Once perceived as organisms that rarely interact, bacteria are now known to lead highly social lives [1–3]. Central to this sociality is an ability to detect local cell density and thereby coordinate group behaviors [4–6]. This ability, termed quorum sensing, functions through the secretion and detection of autoinducer molecules, which accumulate in a cell density-dependent manner. When autoinducer concentrations reach a threshold level, quorum-sensing cells respond, allowing them to modulate behaviors whose efficacy and fitness benefits depend upon the presence, or absence, of other cells. Traits under quorum-sensing control include surface attachment [7], extracellular polymer production [8–10], biosurfactant synthesis [11], sporulation [12], competence [13], bioluminescence [14,15], and the secretion of nutrient-sequestering compounds and virulence factors [16–18]. Quorum sensing is also phylogenetically widespread, which suggests an early origin in bacterial evolution [19]. In addition to sensing and responding to the presence of other cells, many bacteria form multicellular surface-bound aggregates, or biofilms, whose remarkable feats of persistence are the scourge of both medicine and industry [5,6,20–24]. Accordingly, biofilms confer on their members considerable advantages, including the ability to resist challenges from predators, antibiotics, and host immune systems [6,20,25–27]. Quorum sensing and biofilm formation are often closely linked, and it is likely that their interaction is central to the pathogenesis of many bacterial infections [8–10,28–30]. The effects of quorum sensing, however, are highly variable and depend upon both the species under observation and the experimental conditions [28]. Four studies have emphasized how the potential for competition and conflict among strains of bacteria can shape the evolution of quorum sensing [31–34], but none have addressed biofilm formation. An open challenge for microbiology, therefore, is to disentangle the ecological and evolutionary processes that drive quorum sensing and biofilm phenotypes and, in particular, their interaction. A defining feature of many biofilm-forming bacteria is the secretion of extracellular polymeric substances (EPS). These polymers, which consist largely of polysaccharide and smaller amounts of protein and DNA, form a matrix in which bacterial cells are embedded [5,6]. Recent empirical and theoretical work has shown that by secreting EPS, individual bacteria can both help and harm cells in their neighborhood and strongly affect the evolutionary dynamics within biofilms [35–38]. Using an individual-based biofilm simulation framework, Xavier and Foster [36] demonstrated that EPS production can provide an advantage to secreting strains by allowing them to push their descendent cells up into areas of high nutrient availability while suffocating any neighboring cells that do not produce EPS. EPS secretion is under quorum-sensing control in a number of bacterial model systems. Many species, including the pathogen Pseudomonas aeruginosa, activate EPS production at high cell density [8,10]. The evolutionary rationale for this strategy seems clear: it increases the likelihood that polymer secretion will only occur in the biofilm state, where it affords a competitive advantage, and not in the planktonic state, where it is presumably a waste of resources [36]. Unexpectedly, other species behave quite differently. The human enteric pathogen Vibrio cholerae initiates EPS secretion after attaching to a surface and losing flagellar activity [39,40]. Subsequently, in a manner opposite to P. aeruginosa, V. cholerae halts EPS secretion once it reaches its high cell density quorum-sensing threshold [9,39]. Here, we explore evolutionary explanations for this variability in quorum-sensing control of EPS production using an individual-based model of biofilm formation [36]. In particular, we ask why do some species activate the biofilm-specific trait of polymer secretion at high cell density, while others terminate polymer secretion at high cell density?

Discussion Biofilm formation and quorum sensing are central and often interconnected features of bacterial social life [4–6,15,28,29]. Our evolutionary analysis is the first to address both of these major classes of bacterial social behavior, and it suggests that quorum sensing enables bacteria to turn on and off the secretion of extracellular polymeric substances (EPS) so as to increase their competitive ability against other species and strains within biofilms. This result builds upon the conclusions of Xavier and Foster [36], who predicted that EPS secretion affords an advantage to secreting strains in competition with nonsecreting strains. It is important to note that this view contrasts with the conventional wisdom that EPS is a public good that simply binds the biofilm together and protects it against external threats [52,53], although a combination of these perspectives is also a realistic possibility. We modeled both positive and negative quorum-sensing regulation of EPS production. Though explicit simulations of the planktonic phase were omitted, it seems clear that secreting EPS at high cell density allows cells to selectively activate EPS synthesis in biofilms and avoid the cost of EPS production in the planktonic phase [36]. However, we also find potential benefits for down-regulating EPS at high cell density, which allows cells to redirect energy from EPS production into growth and cell division prior to a dispersal event (Figure 1). Such a quorum-sensing phenotype will only be favored if detachment events are predictable, due to consistent extrinsic disturbance, or if dispersal is induced by the bacteria themselves. Our findings are consistent with the known biology of V. cholerae, which exhibits negatively quorum-sensing–regulated EPS secretion. The environments that V. cholerae occupies appear to present opportunities for EPS-mediated competition within biofilms. Although we do not yet know how often different V. cholerae strains compete within human hosts, it is clear that infections always involve multiple species. These include other pathogenic genera, such as Pseudomonas, Salmonella, and Campylobacter [54,55], and there is compelling evidence that V. cholerae must compete with native intestinal microbial fauna in order to become established [56]. Furthermore, EPS secretion appears to be important for within-biofilm competition: quorum-sensing–deficient V. cholerae mutants that overproduce EPS take over biofilm cultures coinoculated with wild-type bacteria [9]. The ecology of pathogenic V. cholerae is characterized by cycles of rapid growth followed by massive dispersal events; the bacteria effect a stereotypical disease progression from initial infection, through the formation of biofilm-like aggregates [57], to release from the intestinal tract after enormous toxin-induced fluid release. This suggests that quorum sensing in V. cholera can be tuned to coincide with purging from the gut. Interestingly, quorum-sensing mutants that overproduce EPS suffer a greatly decreased ability to escape from biofilms [58–60], which indicates that, in addition to saving energy, reducing EPS secretion also actively assists dispersal. Moreover, on reaching a quorum, V. cholerae produces a protease whose putative function is to facilitate detachment [9,39,58,59]. By secreting a “detachase” and down-regulating EPS production at high cell density, V. cholerae appears to be inducing a growth burst coincident with efficient dispersal. The cycle of growth and detachment may also play a role in the initial colonization of the host: cells in a biofilm formed early in an infection can, upon detecting a threshold autoinducer concentration, halt EPS secretion, detach, and seed other areas of the intestine. Whereas V. cholerae terminates EPS secretion at high cell density, many other species, including the opportunistic human pathogen P. aeruginosa, activate EPS secretion at high cell density. Hammer and Bassler [9] suggested that the explanation for this stark contrast in quorum-sensing behavior may lie in different infection strategies. Our results support this argument and, furthermore, suggest that this divergence hinges upon the evolutionary tradeoff between within-biofilm competition on the one hand and dispersal ability on the other. In particular, chronic infections are less likely to involve discrete and predictable moments of detachment that would favor a clear cutoff point for polymer secretion. Instead, dispersal is likely to occur through many small events over a long, but indeterminate, period of time. In such conditions, strains that can dominate locally, thereby maximizing their chances of detachment over an interval of uncertain length, will attain an evolutionary advantage. We therefore predict that up-regulation of EPS secretion at high cell density, which focuses resource investment into sustained local competitive ability, is more likely to be favored. This is precisely the pattern exhibited by P. aeruginosa, which is notorious for the chronic, and often terminal, infections it establishes in the lungs of cystic fibrosis patients. Interestingly, populations of P. aeruginosa sampled from the cystic fibrosis lung often also contain quorum-sensing mutants that are fixed in a high cell-density state [61] and a low cell-density state [33,62], although the link between these results and the EPS secretion phenotype, if any, is not yet clear. The biofilm simulations performed in this study highlight several hypotheses amenable to experimental testing. We anticipate that EPS production by V. cholerae is at least partially a competitive behavior in the human intestinal tract, as it is in lab biofilm assays [9]. Although we lack a direct test of this prediction, Nielsen et al. [60] found that V. cholerae mutants unable to produce EPS are just as effective at colonizing rabbit intestine as wild-type cells, which shows that EPS is not secreted simply to aid surface colonization. The same study found that rpoS, which encodes an important stationary-phase regulator, is necessary for escape from the intestinal wall, implying that the detection of nutrient starvation also regulates dispersal [60]. A comparison of different V. cholerae strains offers additional opportunities to test our conclusions. Natural isolates show considerable variation in quorum-sensing ability, with strains fixed in either low or high cell-density states [63]. Our simulations raise the possibility that variation in quorum-sensing state within V. cholerae is linked to different dispersal requirements across the bacterium's diverse ecology. V. cholerae strains are known to form biofilms on both biotic and abiotic surfaces in marine environments [64–66], and not all cause disease [63]. Specifically, we predict that pathogenic strains selected for rapid colonization of, and efficient dispersal from, human hosts or other temporary environments will exhibit negatively quorum-sensing–regulated EPS production. The Classical V. cholerae biotype, which was responsible for the first six global cholera pandemics, has a nonfunctional copy of a key regulatory protein, HapR, involved in the quorum-sensing response. However, in line with our predictions, it was recently discovered that these strains are capable of HapR-independent quorum sensing and may still repress EPS expression in response to high cell density [67]. The associated prediction is that strains that occupy single locations for long periods should accumulate mutations that enable constitutive EPS production in biofilms, regardless of local population density. In support of this, standing cultures of EPS− V. cholerae cells are reliably taken over by spontaneous, constitutive EPS+ mutants [9]. Cooperation, competition, and communication are all intertwined in microbial communities, and we are only beginning to unravel the processes that drive this rich interaction [1,2,68,69]. Although our simulations inevitably miss many biological details of any one species or strain, a familiar principle of sociobiology emerges. A full understanding of quorum sensing in bacterial biofilms will require consideration of evolutionary competition within and among these social groups.

Supporting Information Figure S1. Summary of Simple Competition Involving the QS* Strain, Which Up-Regulates Polymer Secretion at High Density (A) Competition between the QS* strain and the constitutive EPS-secreting strain (EPS+). (B) Competition between the QS* strain and the non–EPS-secreting strain (EPS−). These simulations differ from those carried out for Figure 2 (main text); here, the QS* strain produces no EPS at low cell density and initiates EPS secretion only after autoinducer concentration exceeds the threshold value. Each competition was repeated 50 times, and plotted lines represent mean QS* frequency time series from each set of simulations, shown with shaded 95% confidence intervals. https://doi.org/10.1371/journal.pbio.0060014.sg001 (1.0 MB EPS) Figure S2. An Evolutionary Stability Analysis for Investment into EPS (f) Each box-and-whisker plot summarizes the results of 20 replicate simulations. (A) Invasion analysis (see Equation 1, main text) of EPS+ strains with slightly higher f values than the rest of the population (f − Δf) yields f* = 0.52. (B) Invasion analysis of EPS+ strains with slightly lower f values than the rest of the population (f + Δf) yields f* = 0.45. Together, these two analyses demonstrate that the evolutionarily stable strategy for EPS investment, f*, lies between 0.45 and 0.52, and f = 0.5 was used for the simulations in our main text. The value of Δf used for this evolutionary stability analysis was 0.1. Focal biofilms were initiated with an equal number of cells of each type (average relatedness of 0.5), and invasiveness was calculated using t end = 14 d (see main text). https://doi.org/10.1371/journal.pbio.0060014.sg002 (502 KB EPS) Text S1. Simulation of a Bacterial Strain that Up-Regulates EPS Production (QS*) at High Cell Density in Competition with Constitutive EPS Producers (EPS+) and Non-Producers (EPS−), and an Evolutionary Stability Analysis for Investment into EPS Secretion https://doi.org/10.1371/journal.pbio.0060014.sd001 (44 KB DOC) Video S1. Movie File for the Simulation Shown in Figure 1 Also available for download at: http://sysbio.harvard.edu/csb/foster/joao/QSposVsEPSpos_alpha8e-3_seed1.mov. https://doi.org/10.1371/journal.pbio.0060014.sv001 (3.6 MB MOV)

Acknowledgments We are very grateful to Bonnie Bassler, Katharina Ribbeck, Ned Wingreen, Karina Xavier, Adrian de Froment, Jonathan Dushoff, Andy Gardner, and two anonymous reviewers for comments on this manuscript. We also thank Brian Hammer for invaluable discussions that helped to motivate this project, and Iain Couzin for organizing the meeting that led to our collaboration.

Author Contributions CDN, JBX, and KRF designed simulations. CDN and JBX performed simulations. CDN, JBX, and KRF analyzed data. JBX contributed analytical tools. CDN, JBX, SAL, and KRF wrote the paper.