Theoretical Biology, University of Bonn, Kirschallee 1, 53115 Bonn, Germany
Correspondence
Jan-Ulrich Kreft
kreft{at}uni-bonn.de
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ABSTRACT |
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INTRODUCTION |
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Spatial structure is well known to facilitate the evolution of cooperation and altruism (see e.g. Nowak & May, 1992; Sigmund, 1994
; Hofbauer & Sigmund, 1998
; Hauert et al., 2002
). For the altruistic strategy studied here, spatial structure is absolutely necessary since recognition of other individuals and their behaviour, i.e. social structure that could substitute for spatial structure, is beyond the means of bacteria. However, this requirement is easily met, because the spatial structure is automatically generated simply by the asexual division of immotile cells, which leads to clonal clusters. This self-organized structure merely needs to be maintained. Since the earliest forms of life were presumably immotile, asexual cells unable to recognize the individuals with which they interact, our simplest form of altruism could have evolved already at the beginning of life.
An example of the rate versus yield trade-off is the branched catabolism of the anaerobic bacterium Holophaga foetida (Kreft & Schink, 1993). It can double its maximum specific growth rate at the cost of a halved growth yield (Kappler et al., 1997
) by switching catabolism from higher to lower ATP yield. From this example, two growth strategies, high growth rate versus high growth yield, have been abstracted and their fitnesses compared in this study. In H. foetida, the two strategies are followed by one and the same organism, depending on physiological conditions (Kappler et al., 1997
), but for the purpose of comparing and studying these strategies, they have been extracted from measurements with H. foetida (see Table 1
) and assigned to separate virtual organisms each following one heritable, immutable, and pure strategy exclusively. Like H. foetida, the aerobic bacterium Acetobacter methanolicus (Müller & Babel, 1993
) and the yeast Saccharomyces kluyveri (Møller et al., 2002
) can boost their growth rates by shifting catabolic substrate flow into less energy-conserving branches, resulting in lowered biomass yields.
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Non-equilibrium thermodynamics predicts a linear relationship between the rate of a reaction and its driving force (the Gibbs free energy change) near equilibrium (Westerhoff & van Dam, 1987). This prediction agrees reasonably well with measurements. For H. foetida (Kappler et al., 1997
), a doubled growth rate is coupled with a doubled Gibbs free energy dissipation per C-mol biomass formed (Heijnen & van Dijken, 1992
) (
). For the lactate fermenters, the 2·75-fold higher growth rate is coupled with a 3·5-fold higher Gibbs free energy dissipation per C-mol biomass formed, calculated from Seeliger et al. (2002)
. These results argue for a thermodynamic necessity of the trade-off. Thermodynamic necessity combined with the simplicity of the altruistic strategy implies that the evolutionary choice, and conflict, between these selfish and altruistic strategies is as old as life. Microbiologists have tried to understand what sets and limits the specific growth rate [of bacteria] (Koch, 1985
); see also Marr (1991)
. Due to the rate versus yield trade-off, the question for a bacterium is not so much how fast could it grow as how fast should it grow.
Micro-organisms will colonize and grow on almost any available surface, thus forming a biofilm where cells are embedding themselves in a slimy matrix while their metabolism creates substrate and product gradients entailing a very heterogeneously structured microenvironment to which the cells in turn will adapt (Costerton et al., 1995). If this surface happens to be a part of or associated with the human body, such biofilms are of particular concern due to their enhanced resistance to antimicrobials (Costerton et al., 1999
; Gilbert et al., 2002
). Biofilms are teeming with a diversity of bacteria, and life in biofilms has been likened to our human life in cities (Watnick & Kolter, 2000
). The high cell density means that most cells have many neighbours close by. Bacteria may stay in a neighbourhood for prolonged periods of time, punctuated by sudden events such as emigration, dispersal, sloughing, etc.
Under such conditions of high cell density, bacteria could benefit from division of labour, collective actions, and other forms of altruistic behaviour or cooperation with their neighbours, and many examples of such cooperations are known (Bradshaw et al., 1994; Caldwell et al., 1997
; Turner & Chao, 1999
; Velicer et al., 2000
, 2003
; Strassmann et al., 2000
; Crespi, 2001
; Palmer et al., 2001
; Gilbert et al., 2002
). However, the benefits of cooperation can be exploited by selfish individuals or groups not contributing to the costs of cooperation, for example, exoenzyme production or signal production for quorum sensing (Brown & Johnstone, 2001
). Therefore, conflicts of interest arise between cells in a cluster or clusters in different parts of the biofilm, even in single-species biofilms, and a case of general protection against invaders by rhamnolipid surfactant production has been reported (Davey et al., 2003
). Conflicts of interest between individuals and groups over the use of resources exist not only for bacteria but also for humans, where this conflict is known as the tragedy of the commons' (Hardin, 1968
).
In this study, I will show how the clustered growth and substrate gradients in biofilms promote altruism, and make the following predictions on biofilm characteristics: (1) biofilms formed by altruists have a higher surface area coverage, (2) biofilms are predominated by altruists, (3) such altruists have to grow and stay in clusters and this is why clusters are the main unit of biofilm architecture, and (4) such clusters have to propagate, from time to time, by breaking up into single cells rather than staying as a unit. The study ends with conclusions on the mechanisms maintaining biodiversity as well as the selection of fast-growing microbes by enrichment cultures and how this has biased mainstream microbiological research, and finally asks why biofilms have not evolved into multicellular organisms.
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THE BIOFILM MODEL |
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Since substrate diffuses down through the boundary layer into the biofilm, cells at the top will receive more substrate than cells further down or inside the biofilm (Fig. 2) and therefore grow faster, producing more offspring. The offspring in turn will be even higher, like the new leaves of a tree already above the canopy. This positive feedback loop is the cause of finger formation in biofilms and fractal colony edges (Picioreanu et al., 1998
). This can also be seen as lateral inhibition because those cells which are higher than their neighbours divert substrate flux away from these neighbours, thereby inhibiting their growth. Once a certain finger is higher than the neighbours (see the YS clusters which are higher than their neighbouring RS clusters in Fig. 4c
3), it will win the competition due to this lateral inhibition. As soon as lateral inhibition suppresses the growth rate of the neighbouring clusters below that of the topmost or leading cluster, the neighbouring clusters no longer have any chance to overgrow the leading cluster, and the outcome of competition is decided. The later this decision time, the more the long-term advantage of YS will pay off. This is a case of truncation selection (Sober & Wilson, 1998
), as also found in the desert leaf-cutting ant Acromyrmex versicolor, where the first colony to produce workers as offspring will win the competition because these workers will first raid the neighbouring colonies (Sober & Wilson, 1998
).
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To obtain quantitative results, the generic, abstract strategies RS and YS were applied to the concrete example of an ammonia-oxidizing bacterium. This matters insofar as ammonia oxidizers grow slowly and their small substrates oxygen and ammonia diffuse rapidly. For the sake of simplicity, only one substrate (oxygen) was assumed to be limiting growth, substrate inhibition by ammonia was assumed to be absent, and the maintenance rate was set to zero. Model parameters were as described by Kreft et al. (2001). Most importantly, the oxygen concentration was 1 mg l1, the simulated domain was 200x200x2 µm wide, high and deep, respectively, and the height of the concentration boundary layer was 40 µm. The growth parameters for YS were those given for the ammonia oxidizer (Kreft et al., 2001
), and the growth parameters of RS were chosen relative to the values for YS (Table 1
). The source code and Java program of the model are available from my web site (http://www.theobio.uni-bonn.de/people/jan_kreft).
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RESULTS AND DISCUSSION |
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Competition in biofilms
In biofilms, cells grow in clusters (microcolonies); this creates a substrate gradient into and within the clusters. Such clustered growth, plus its inevitable combination with substrate gradients, is necessary but not sufficient to change the outcome of competition in favour of YS (Figs 3 and 4). Comparing pure biofilms of RS and YS strategists, it is obvious that the latter's higher yield allows more biomass to accumulate in the long run despite an initial advantage of RS (Fig. 3
). In direct side-by-side competition (Figs 3 and 4
), qualitatively the same picture emerges, independent of the initial density (150 cells each, data shown for 10 cells each). When the arrangement of clusters is alternating, RS will win the competition only if the density of clusters is above a threshold that depends on growth parameters and environmental conditions such as substrate transport rate (Figs 3 and 4
). However, at even higher densities, the self-organization into clusters (clustering effect) that results from the cell divisions of immotile cells gives YS the chance to spread out of a dominantly RS biofilm in the shape of wedges or fans (Figs 4b
10, 4e10 and 5c10; Movie 1, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org). Wedge-like growth patterns have long been known in dental plaque (Lai et al., 1975
; Listgarten, 2000
). Clustering has also been reported in P. aeruginosa biofilms (Davey et al., 2003
). Clustering can also result from polymer production, which effectively binds cells and their evolutionary interests together (Rainey & Rainey, 2003
; Velicer & Yu, 2003
).
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In principle, the same results as above were obtained at higher substrate concentrations or thinner boundary layers (data not shown), where the short-term initial advantage of RS becomes more pronounced. Therefore, for the alternating arrangement, the switch from YS winning to RS winning occurs at a lower density.
These results demonstrate how both strategies can originate in an environment dominated by the other strategy, albeit in different ways. The YS strategy can arise in a spatially structured environment dominated by RS because of (a) more intense competition among RS clusters themselves, (b) the clustering effect, or (c) the overall density of cells being low enough due to a scarcity of resources. The RS strategy can arise in well-mixed environments such as the planktonic phase, and in patchy environments if the short-term advantage of RS (see Fig. 3) dominates over the long-term advantage of YS. In summary, YS wins the competition in the long run if it has not been overgrown by RS during the initial phase of biofilm growth.
Invasibility by individuals and groups
For a given strategy to survive in the long term, at least some conditions under which the strategy can arise de novo must first of all exist, and the preceding section has demonstrated that suitable conditions for the origin of both strategies can easily be found. Secondly, once a strategy has emerged, it must be able to be maintained against competing strategies, and this is studied in this section, employing the usual invasibility criterion. A strategy is called an evolutionarily stable strategy (ESS) if a population of individuals using that strategy cannot be invaded by a rare mutant adopting a different strategy (Maynard Smith, 1982). If group-level selection plays a dominant role, the invasibility criterion should be applied on the individual and group levels of selection separately.
To test for invasibility by individuals, a single cell at the top or in the middle of a developing, fairly even and uniform biofilm was mutated into one with an alternative strategy (data not shown). As expected from the Monod kinetics of the two strategies (Fig. 1), initially the RS strategist always had a higher growth rate than the one a YS strategist would have had in the same place. In real biofilms, this mutant is more likely to be an immigrant or arise from phase variation (phenotypic switching; Drenkard & Ausubel, 2002
).
To test for invasibility by groups, vertical strips of cells (5100 µm) were changed into cells of the other strategy. RS clusters could not invade YS biofilms, but YS clusters could invade RS biofilms (Fig. 5). The growth of a strip of YS strategists invading an RS biofilm, relative to a same-sized strip of RS strategists invading a YS biofilm, showed a trend of increasing advantage for the YS clusters with increasing cluster size (data not shown). Comparing the growth of pure RS and YS biofilms with a randomly mixed biofilm (same number of cells for RS and YS to begin with) initially shows the expected results (data not shown): pure YS biofilms grow much better than pure RS biofilms, and the mixed biofilm is in between, with RS strategists growing slightly better than the YS strategists in the mixed biofilm. However, YS strategists overtake in the long run because of the clustering effect (Fig. 5c
3, c10; Movie 2, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org).
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Within each cluster (neighbourhood), the proportion of RS strategists will tend to increase because of their unconditional growth rate advantage (Fig. 1), giving the RS strategy a higher relative fitness in individual-level selection. But clusters will grow better and produce more offspring the higher the fraction of YS strategists is, under the same conditions, giving the YS strategy a higher relative fitness in group-level selection. This may lead to a global increase of the number of YS strategists although the proportion of YS strategists decreases in all clusters, a counter-intuitive result that is known as Simpson's Paradox (Sober & Wilson, 1998
).
Despite major differences with respect to substrate transport, cell growth, cell motility, and biomass spreading, the results of this study are qualitatively similar to those of Bonhoeffer and coworkers (Pfeiffer et al., 2001; Pfeiffer & Bonhoeffer, 2003
), suggesting that these results are very robust.
Predictions on biofilm characteristics
Based on these findings, a set of predictions on the characteristics of biofilm life follows, which are then compared with the empirical evidence when available.
(1) Higher substratum coverage of YS biofilms.
YS strategists should form biofilms with higher surface area coverage than RS strategists, due to the weaker competition between neighbouring clusters.
(2) Dominance of YS in biofilms.
Altruism can evolve in natural biofilm communities and biofilms should be dominated by YS strategists. In contrast, planktonic bacteria that are primarily growing as single cells should be RS strategists. Planktonic cells of P. aeruginosa grow twice as fast as P. aeruginosa cells after attaching to a surface (Rice et al., 2000), suggesting that they switch strategy from RS to YS upon surface attachment.
(3) Clustering of YS.
YS strategists should have a tendency to literally stick to themselves and thereby facilitate and maintain clustering. RS strategists should try to avoid self and mix, e.g. by surface-bound twitching motility. Bacteria from natural river biofilms show either a tendency to avoid self, called spreading, rolling, or shedding maneuvers, or to cluster, called packing maneuver (Lawrence & Caldwell, 1987). Biofilms as a whole should resemble an ensemble of distinct, clonal and identifiable microcolonies with a very limited amount of mixing between these clusters. The most direct studies of mixing within biofilms involve cells of the same strain tagged with different colours. Movement of Pseudomonas putida cells between and within microcolonies was frequently observed apart from flagellum-less mutants; however, the extent of mixing was too limited to change the overall composition or structure of the microcolonies (Tolker-Nielsen et al., 2000
). Recently, P. aeruginosa biofilm structure was shown to depend on the carbon source, and an initial phase of clonal growth was followed by a phase of twitching-motility-driven mixing (Klausen et al., 2003
). Similar studies of dual-species biofilms show the same trend: mixing is surprisingly limited even for commensalistic associations where satellite microcolonies are formed, growing on the products of a big cluster in the centre (Nielsen et al., 2000
; Tolker-Nielsen & Molin, 2000
; Christensen et al., 2002
). Formation of P. aeruginosa microcolonies has been proposed to occur by aggregation (O'Toole et al., 2000
), but the evidence is conflicting among various laboratories (Chiang & Burrows, 2003
; Klausen et al., 2003
). The typically limited mixing explains why microcolonies are the basic structural unit of biofilms (Costerton et al., 1995
; Tolker-Nielsen et al., 2000
).
(4) Biofilms are not units of proliferation.
Maintenance of YS requires clusters to disperse. Even an originally pure YS cluster will not stay pure forever, due to mutation, phase variation and immigration. In a mixed cluster, RS strategists will grow faster than their cluster neighbours, thereby increasing the fraction of RS strategists within the cluster. A purification step is required for YS strategists to survive: clusters must at least occasionally be broken up into single cells that leave the cluster to colonize another surface. Supporting this view, biofilms shed not only clusters but also single cells in proportion to the total biofilm mass (Stoodley et al., 2001). If the biofilm community were the unit of proliferation (Caldwell et al., 1997
), the YS strategists would become extinct.
Conclusions
Two conditions are necessary and sufficient for the origin and maintenance of simple altruistic strategies without direct and recognition-based interactions: (a) spatial structure (clustering), and (b) dissociation of clusters into individuals before the RS strategists have taken over the cluster. (A third condition, resource limitation, is a necessary consequence of clustering.) These conditions are clearly met in biofilms.
Economy of resource use has probably been overlooked as a form of altruism in biofilms (Caldwell et al., 1997) because it does not involve the specific, direct interactions that would catch one's attention, yet here I argue that it is the earliest form of altruism, widespread since life began and having a profound impact on biofilm structure and function ever since.
The range of possible strategies from selfish to altruistic, whether pure or mixed, obligate or facultative, opens up a neglected dimension for the diversification of life. There are a plethora of mechanisms and effects that help maintain biodiversity (Chesson, 2000). While resources, predators, time and space are commonly viewed as axes of niche space (Chesson, 2000
), cooperation should be added as the fifth major axis.
The study of fast-growing bacteria in liquid culture has formed the mainstream of microbiological research since Beijerinck and Winogradsky established enrichment cultures about a century ago (Brock, 1998). Unfortunately, enrichment cultures tend to select RS strategists, thereby ensuring that most laboratory studies have been carried out with RS strategists: growth of bacteria was found, using mosaic non-equilibrium thermodynamics, to be optimized for maximization of growth rate while keeping efficiency as high as possible (Westerhoff & van Dam, 1987
). Also, the organization of the Escherichia coli metabolic network was found to be optimized to maximize growth (Edwards et al., 2001
). However, these results may not hold for YS strategists, which probably predominate in nature. Isolating bacteria by dilution culture (dilution of the inoculum prior to isolation) rather than enrichment culture will pick the most abundant species able to grow under the given conditions. Using dilution culture of samples from spatially structured habitats should favour isolation of YS strategists. It may be expected that the more frequent use of dilution cultures will allow a fraction of so-called unculturables to become cultured. A case in point is the isolation of H. foetida (Bak et al., 1992
) from which the growth parameters of this study were derived and other members of the class Acidobacteria (Joseph et al., 2003
).
Given that biofilms promote the evolution of clusters with group-beneficial behaviour, why did biofilms not develop into multicellular organisms? After all, multicellular organisms have evolved mechanisms to counter selfish defection of individual cells from altruistic behaviour (cancer). Yet bacteria have, by and large, not evolved into multicellular organisms over more than 3 billion years, despite the ease with which cooperating groups of bacteria evolve (Rainey & Rainey, 2003; Velicer & Yu, 2003
). Two main reasons for this can be envisaged. (1) The architecture of prokaryotic cells with haploid genomes constrains evolution. (2) The main ecological advantage of bacteria would be lost upon transition to obligate multicellularity. There are many links in the carbon, nitrogen and sulphur cycles provided by the unique metabolic capabilities of bacteria, whereas eukaryotes are metabolically far less diverse. If metabolic versatility is the foundation for the evolutionary success of bacteria, it seems likely that the flexibility in time and space of the metabolic capacities of bacterial communities requires a modular construction which would be lost if the single bacterial cells (the modules) were to integrate into defined and permanent larger units. The relationship between community-level metabolic flexibility and performance on the one hand and modularity of organization on the other is the topic of a future study.
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ACKNOWLEDGEMENTS |
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Received 13 October 2003;
revised 28 April 2004;
accepted 7 May 2004.
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