Quorum-sensing and cheating in bacterial biofilms

The idea from human societies that self-interest can lead to a breakdown of cooperation at the group level is sometimes termed the public goods dilemma. We tested this idea in the opportunistic bacterial pathogen, Pseudomonas aeruginosa, by examining the influence of putative cheats that do not cooperate via cell-to-cell signalling (quorum-sensing, QS). We found that: (i) QS cheating occurs in biofilm populations owing to exploitation of QS-regulated public goods; (ii) the thickness and density of biofilms was reduced by the presence of non-cooperative cheats; (iii) population growth was reduced by the presence of cheats, and this reduction was greater in biofilms than in planktonic populations; (iv) the susceptibility of biofilms to antibiotics was increased by the presence of cheats; and (v) coercing cooperator cells to increase their level of cooperation decreases the extent to which the presence of cheats reduces population productivity. Our results provide clear support that conflict over public goods reduces population fitness in bacterial biofilms, and that this effect is greater than in planktonic populations. Finally, we discuss the clinical implications that arise from altering the susceptibility to antibiotics.     

DENSITY DEPENDENCE AND COOPERATION: THEORY AND A TEST WITH BACTERIA

Although cooperative systems can persist in nature despite the potential for exploitation by noncooperators, it is often observed that small changes in population demography can tip the balance of selective forces for or against cooperation. Here we consider the role of population density in the context of microbial cooperation. First, we account for conflicting results from recent studies by demonstrating theoretically that: (1) for public goods cooperation, higher densities are relatively unfavorable for cooperation; (2) in contrast, for self-restraint–type cooperation, higher densities can be either favorable or unfavorable for cooperation, depending on the details of the system. We then test our predictions concerning public goods cooperation using strains of the pathogenic bacterium Pseudomonas aeruginosa that produce variable levels of a public good—iron-scavenging siderophore molecules. As predicted, we found that the relative fitness of cheats (under-producers) was greatest at higher population densities. Furthermore, as assumed by theory, we show that this occurs because cheats are better able to exploit the cooperative siderophore production of other cells when they are physically closer to them.     

No effect of intraspecific relatedness on public goods cooperation in a complex community

Many organisms—notably microbes—are embedded within complex communities where cooperative behaviors in the form of excreted public goods can benefit other species. Under such circumstances, intraspecific interactions are likely to be less important in driving the evolution of cooperation. We first illustrate this idea with a simple theoretical model, showing that relatedness—the extent to which individuals with the same cooperative alleles interact with each other—has a reduced impact on the evolution of cooperation when public goods are shared between species. We test this empirically using strain of Pseudomonas aeruginosa that vary in their production of metal‐chelating siderophores in copper contaminated compost (an interspecific public good). We show that nonsiderophore producers grow poorly relative to producers under high relatedness, but this cost can be alleviated by the presence of the isogenic producer (low relatedness) and/or the compost microbial community. Hence, relatedness can become unimportant when public goods provide interspecific benefits.     

The effect of cheats on siderophore diversity in Pseudomonas aeruginosa

Cooperation can be maintained if cooperative behaviours are preferentially directed towards other cooperative individuals. Tag‐based cooperation (greenbeards) – where cooperation benefits individuals with the same tag as the actor – is one way to achieve this. Tag‐based cooperation can be exploited by individuals who maintain the specific tag but do not cooperate, and selection to escape this exploitation can result in the evolution of tag diversity. We tested key predictions crucial for the evolution of cheat‐mediated tag diversity using the production of iron‐scavenging pyoverdine by the opportunistic pathogen, Pseduomonas aeruginosa as a model system. Using two strains that produce different pyoverdine types and their respective cheats, we show that cheats outcompete their homologous pyoverdine producer, but are outcompeted by the heterologous producer in well‐mixed environments. As a consequence, co‐inoculating two types of pyoverdine producer and one type of pyoverdine cheat resulted in the pyoverdine type whose cheat was not present having a large fitness advantage. Theory suggests that in such interactions, cheats can maintain tag diversity in spatially structured environments, but that tag‐based cooperation will be lost in well‐mixed populations, regardless of tag diversity. We saw that when all pyoverdine producers and cheats were co‐inoculated in well‐mixed environments, both types of pyoverdine producers were outcompeted, whereas spatial structure (agar plates and compost microcosms), rather than maintaining diversity, resulted in the domination of one pyoverdine producer. These results suggest cheats may play a more limited role in the evolution of pyoverdine diversity than predicted.     

Siderophore cooperation of the bacterium Pseudomonas fluorescens in soil

While social interactions play an important role for the evolution of bacterial siderophore production in vitro, the extent to which siderophore production is a social trait in natural populations is less clear. Here, we demonstrate that siderophores act as public goods in a natural physical environment of Pseudomonas fluorescens: soil-based compost. We show that monocultures of siderophore producers grow better than non-producers in soil, but non-producers can exploit others'' siderophores, as shown by non-producers'' ability to invade populations of producers when rare. Despite this rare advantage, non-producers were unable to outcompete producers, suggesting that producers and non-producers may stably coexist in soil. Such coexistence is predicted to arise from the spatial structure associated with soil, and this is supported by increased fitness of non-producers when grown in a shaken soil–water mix. Our results suggest that both producers and non-producers should be observed in soil, as has been observed in marine environments and in clinical populations.     

Antagonistic interactions subdue inter‐species green‐beard cooperation in bacteria

Cooperation can be favoured through the green‐beard mechanism, where a set of linked genes encodes both a cooperative trait and a phenotypic marker (green beard), which allows carriers of the trait to selectively direct cooperative acts to other carriers. In theory, the green‐beard mechanism should favour cooperation even when interacting partners are totally unrelated at the genome level. Here, we explore such an extreme green‐beard scenario between two unrelated bacterial species—Pseudomonas aeruginosa and Burkholderia cenocepacia, which share a cooperative locus encoding the public good pyochelin (an iron‐scavenging siderophore) and its cognate receptor (green beard) required for iron–pyochelin uptake. We show that pyochelin, when provided in cell‐free supernatants, can be mutually exchanged between species and provide fitness benefits under iron limitation. However, in co‐culture we observed that these cooperative benefits vanished and communities were dominated by P. aeruginosa, regardless of strain background and species starting frequencies. Our results further suggest that P. aeruginosa engages in interference competition to suppress B. cenocepacia, indicating that inter‐species conflict arising from dissimilarities at the genome level overrule the aligned cooperative interests at the pyochelin locus. Thus, green‐beard cooperation is subdued by competition, indicating that interspecific siderophore cooperation is difficult to evolve and to be maintained.     

Co‐evolutionary dynamics between public good producers and cheats in the bacterium Pseudomonas aeruginosa

The production of beneficial public goods is common in the microbial world, and so is cheating – the exploitation of public goods by nonproducing mutants. Here, we examine co‐evolutionary dynamics between cooperators and cheats and ask whether cooperators can evolve strategies to reduce the burden of exploitation, and whether cheats in turn can improve their exploitation abilities. We evolved cooperators of the bacterium Pseudomonas aeruginosa, producing the shareable iron‐scavenging siderophore pyoverdine, together with cheats, defective in pyoverdine production but proficient in uptake. We found that cooperators managed to co‐exist with cheats in 56% of all replicates over approximately 150 generations of experimental evolution. Growth and competition assays revealed that co‐existence was fostered by a combination of general adaptions to the media and specific adaptions to the co‐evolving opponent. Phenotypic screening and whole‐genome resequencing of evolved clones confirmed this pattern, and suggest that cooperators became less exploitable by cheats because they significantly reduced their pyoverdine investment. Cheats, meanwhile, improved exploitation efficiency through mutations blocking the costly pyoverdine‐signalling pathway. Moreover, cooperators and cheats evolved reduced motility, a pattern that likely represents adaptation to laboratory conditions, but at the same time also affects social interactions by reducing strain mixing and pyoverdine sharing. Overall, we observed parallel evolution, where co‐existence of cooperators and cheats was enabled by a combination of adaptations to the abiotic and social environment and their interactions.     

Viscous medium promotes cooperation in the pathogenic bacterium Pseudomonas aeruginosa

There has been extensive theoretical debate over whether population viscosity (limited dispersal) can favour cooperation. While limited dispersal increases the probability of interactions occurring between relatives, which can favour cooperation, it can also lead to an increase in competition between relatives and this can reduce or completely negate selection for cooperation. Despite much theoretical attention, there is a lack of empirical research investigating these issues. We cultured Pseudomonas aeruginosa bacteria in medium with different degrees of viscosity and examined the fitness consequences for a cooperative trait—the production of iron-scavenging siderophore molecules. We found that increasing viscosity of the growth medium (i) significantly limited bacterial dispersal and the diffusion of siderophore molecules and (ii) increased the fitness of individuals that produced siderophores relative to mutants that did not. We propose that viscosity favours siderophore-producing individuals in this system, because the benefits of siderophore production are more likely to accrue to relatives (i.e. greater indirect benefits), and, at the same time, bacteria are more likely to gain direct fitness benefits by taking up siderophore molecules produced by themselves (i.e. the trait becomes less cooperative). Our results suggest that viscosity of the microbial growth environment is a crucial factor determining the dynamics of wild-type bacteria and siderophore-deficient mutants in natural habitats, such as the viscous mucus in cystic fibrosis lung.     

An experimental test of whether cheating is context dependent

Microbial cells rely on cooperative behaviours that can breakdown as a result of exploitation by cheats. Recent work on cheating in microbes, however, has produced examples of populations benefiting from the presence of cheats and/or cooperative behaviours being maintained despite the presence of cheats. These observations have been presented as evidence for selection favouring cheating at the population level. This apparent contradiction arises when cheating is defined simply by the reduced expression of a cooperative trait and not in terms of the social costs and benefits of the trait under investigation. Here, we use two social traits, quorum sensing and iron‐scavenging siderophore production in Pseudomonas aeruginosa, to illustrate the importance of defining cheating by the social costs and benefits. We show that whether a strain is a cheat depends on the costs and benefits associated with the social and abiotic environment and not the absolute expression of a cooperative trait.     

Social eavesdropping and the evolution of conditional cooperation and cheating strategies

The response of bystanders to information available in their social environment can have a potent influence on the evolution of cooperation and signalling systems. In the presence of bystanders, individuals might be able to increase their payoff by exaggerating signals beyond their means (cheating) or investing to help others despite considerable costs. In doing so, animals can accrue immediate benefits by manipulating (or helping) individuals with whom they are currently interacting and delayed benefits by convincing bystanders that they are more fit or cooperative than perhaps is warranted. In this paper, I provide some illustrative examples of how bystanders could apply added positive selection pressure on both cooperative behaviour and dishonest signalling during courtship or conflict. I also discuss how the presence of bystanders might select for greater flexibility in behavioural strategies (e.g. conditional or condition dependence), which could maintain dishonesty at evolutionarily stable frequencies under some ecological conditions. By recognizing bystanders as a significant selection pressure, we might gain a more realistic approximation of what drives signalling and/or interaction dynamics in social animals.     

Cost of cooperation rules selection for cheats in bacterial metapopulations

Bacteria secrete a large variety of beneficial metabolites into the environment, which can be shared as public goods among producing bacteria, but also be exploited by nonproducing cheats. Here, we focus on cooperative production of iron-chelating molecules (siderophores) in the bacterium Pseudomonas aeruginosa to study how relevant ecological factors influence selection for cheating. We designed patch-structured metapopulations that allowed us introducing among-patch ecological variation. We found that cheating readily evolved in uniform iron-limited environments. This finding is explained by severe iron limitation demanding high siderophore-production efforts, which results in high metabolic costs accruing to cooperators, and thereby facilitates the spread of cheats. In contrast, we observed a significant reduction or even negation of selection for cheating in metapopulations where we introduced patches with increased iron availability and/or opportunities to recycle siderophores. These findings are compatible with the view that cheats are less likely to invade in environments that allow bacteria to reduce siderophore-production efforts, as this lowers the overall metabolic costs accruing to cooperators. Because we increased iron availability and siderophore recycling opportunities moderately, and only in some patches, our findings demonstrate that already-small local variations in ecological conditions as occurring in nature can significantly affect selection for public-goods secretion in microbes. In addition, we found that most (84.6%) of the evolved cheats were partially deficient for siderophore production and not loss-of-function mutants. Genetic considerations indicate that mutations leading to partial deficiency occur more frequent than mutations leading to loss of function, but also suggest that partially deficient mutants might often be the more competitive cheats.     

Cheating on Cheaters Stabilizes Cooperation in Pseudomonas aeruginosa

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Phenotypic plasticity of a cooperative behaviour in bacteria

There is strong evidence that natural selection can favour phenotypic plasticity as a mechanism to maximize fitness in animals. Here, we aim to investigate phenotypic plasticity of a cooperative trait in bacteria – the production of an iron‐scavenging molecule (pyoverdin) by Pseudomonas aeruginosa. Pyoverdin production is metabolically costly to the individual cell, but provides a benefit to the local group and can potentially be exploited by nonpyoverdin‐producing cheats. Here, we subject bacteria to changes in the social environment in media with different iron availabilities and test whether cells can adjust pyoverdin production in response to these changes. We found that pyoverdin production per cell significantly decreased at higher cell densities and increased in the presence of cheats. This phenotypic plasticity significantly influenced the costs and benefits of cooperation. Specifically, the investment of resources into pyoverdin production was reduced in iron‐rich environments and at high cell densities, but increased under iron limitation, and when pyoverdin was exploited by cheats. Our study demonstrates that phenotypic plasticity in a cooperative trait as a response to changes in the environment occurs in even the simplest of organisms, a bacterium.     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

Differential selection according to the degree of cheating in a status signal

The maintenance of honesty in a badge-of-status system is not fully understood, despite numerous empirical and theoretical studies. Our experiment examined the relationship between a status signal and winter survival, and the long-term costs of cheating, by manipulating badge size in male house sparrows, Passer domesticus. The effect of badge-size manipulation on survival was complex owing to the significant interactions between the treatments and original (natural) badge size, and between the treatments and age classes (yearlings and older birds). Nevertheless, in the experimental (badge-enlargement) group, males with originally large badges had increased winter survival, while males with originally small badges had decreased survival. This indicates that differential selection can act on a trait according to the degree of cheating.     

Bacterial cheating drives the population dynamics of cooperative antibiotic resistance plasmids

Inactivation of β ‐lactam antibiotics by resistant bacteria is a ‘cooperative’ behavior that may allow sensitive bacteria to survive antibiotic treatment. However, the factors that determine the fraction of resistant cells in the bacterial population remain unclear, indicating a fundamental gap in our understanding of how antibiotic resistance evolves. Here, we experimentally track the spread of a plasmid that encodes a β ‐lactamase enzyme through the bacterial population. We find that independent of the initial fraction of resistant cells, the population settles to an equilibrium fraction proportional to the antibiotic concentration divided by the cell density. A simple model explains this behavior, successfully predicting a data collapse over two orders of magnitude in antibiotic concentration. This model also successfully predicts that adding a commonly used β ‐lactamase inhibitor will lead to the spread of resistance, highlighting the need to incorporate social dynamics into the study of antibiotic resistance.     

Multilevel selection analysis of a microbial social trait

The study of microbial communities often leads to arguments for the evolution of cooperation due to group benefits. However, multilevel selection models caution against the uncritical assumption that group benefits will lead to the evolution of cooperation. We analyze a microbial social trait to precisely define the conditions favoring cooperation. We combine the multilevel partition of the Price equation with a laboratory model system: swarming in Pseudomonas aeruginosa. We parameterize a population dynamics model using competition experiments where we manipulate expression, and therefore the cost‐to‐benefit ratio of swarming cooperation. Our analysis shows that multilevel selection can favor costly swarming cooperation because it causes population expansion. However, due to high costs and diminishing returns constitutive cooperation can only be favored by natural selection when relatedness is high. Regulated expression of cooperative genes is a more robust strategy because it provides the benefits of swarming expansion without the high cost or the diminishing returns. Our analysis supports the key prediction that strong group selection does not necessarily mean that microbial cooperation will always emerge.     

Characterization of a novel Spirillum-like bacterium that degrades ferrioxamine-type siderophores

A novel Gram-negative Spirillum-like bacterium (ASP-1) was isolated from lake water by enrichment culture on desferrioxamine B as sole source of carbon and energy. ASP-1 was able to degrade the siderophores desferrioxamine B and E. The property of siderophore degradation was inducible in the presence of desferrioxamine B. The ferric complexes, however, were not measurably degraded but served as an iron source. Degradation of desferrioxamines in culture was followed by measuring the residual ferrioxamines colorimetrically at 430 nm after addition of iron. Degradation in cell-free assays was followed quantitatively by HPLC on a reversed-phase column measuring the time-dependent disappearance of the desferrioxamines B and E. Cell-free assays also revealed that degradation of the cyclic desferrioxamine E was rapid and complete, whereas degradation of the linear desferrioxamine B yielded two intermediate iron-binding metabolites of shorter chain length. Preparative isolation by HPLC and mass spectrometric analysis of the metabolites revealed masses at 361 and 419 a.m.u., respectively, suggesting a splitting at the two amide bonds. ASP-1 is a nitrogen fixing Spirillum bacterium which could also use ammonium and glucose or several organic acids as a carbon source but grew poorly with amino acids. Physiological comparisons with Aquaspirillum and Azospirillum failed to assign ASP-1 to any of the presently known Spirillum species. Based on 16S rDNA sequence analysis the strain could be placed within the radiation of the Azospirillum/Rhodocista group. The closest relative was Azospirillum irakense, showing 98.8% similarity.