BACKGROUND
the production by microorganisms of proteinaceous anticompetitor toxins such as bacteriocins is often cited as a good example of an antagonistic trait  <cit> .
the evolution of antagonistic interactions is difficult to understand because they directly harm both actor and recipient.
at the level of an individual gene, this apparent paradox can be readily resolved using the framework of inclusive fitness  <cit> , which shows that antagonistic interactions can evolve provided they produce a net benefit to actors, even if the act of antagonism itself is costly.
bacteriocin production has the hallmark of a classic antagonistic trait that can evolve through its effects on inclusive fitness.
bacteriocins are produced by nearly all bacteria and are considered the main agents in direct antagonistic interactions between and within bacterial species  <cit> .
the production of bacteriocins is costly, both in terms of the energy diverted away from other functions such as growth and, in gram-negatives at least, because bacteriocin-producing cells release their bacteriocins through lysis and so cause cell death  <cit> .
importantly, cells that are isogenic to the producing strain  are immune to the bacteriocin, usually via an immunity protein, and so gain a benefit from bacteriocin production from clone-mates.
it has also been repeatedly noted that bacteriocins are highly specific in their action, being active primarily against genetically distinct members of the same species or species closely related to the producing strain  <cit> .
we suggest that the mechanism underlying the variation in the antagonistic effects of toxins like bacteriocins is caused by intraspecific resource competition.
we expect that the ability of these toxins to remove competitors, and so free up resources, would evolve to be maximal when resource competition is strongest among genetically distinct individuals.
the logic behind this is straightforward.
toxin production should not be favoured when competing with genetically identical clones because there is no fitness benefit to production.
as genetic distance increases, however, so too does phenotypic and ecological divergence  <cit> , and by extension resource competition.
toxin production is therefore wasted when competing against genetically very divergent strains because there is little resource competition.
in other words, toxin production becomes costly because its benefits are diluted by the fact that the producer and target strain do not compete with each other.
this interpretation leads to the prediction that the strength of antagonism should peak at intermediate genetic distance.
to test this prediction we studied the interaction between two producer strains that produce a multitude of bacteriocins and a range of sensitive 'victim' strains of varying genetic distance to the producers.
specifically, we measured the ability of anticompetitor toxins produced by two laboratory strains of pseudomonas aeruginosa, pa <dig> and pa <dig>  to kill or inhibit  <dig> clinical strains of p. aeruginosa isolated from the lungs of cystic fibrosis  patients.
natural communities of microbes associated with chronic infections such as colonization of the cystic fibrosis lung are often highly diverse  <cit> .
we also measured the degree of ecological similarity among strains, using commercially available biolog plates that contain  <dig> different carbon substrates, and show that ecological similarity can decrease with genetic distance.
this result is consistent with the idea that toxin production is not favoured among genetically divergent strains because of a lack of resource competition.
pyocins and pseudomonas aeruginosa
p. aeruginosa produces a wide variety of toxins and among the most interesting, in part because they are known to be highly specific in their action, are bacteriocins called pyocins.
they are costly to produce because they are released by cell lysis of a fraction of the producer population.
pyocins are proteinaceous compounds that are classified into three groups , with multiple sub-types within each group that attach to different potential receptors in target strains  <cit> .
pa <dig> is known to produce all three pyocins while pa <dig> produces only r- and f-type pyocins  <cit> .
genes coding for production of all pyocins are located on the chromosome and are clustered with genes coding for resistance to the same pyocins.
genomic studies have suggested the presence of more pyocins  <cit> , both from the s- and r-types.
in addition, a recently developed genome-mining tool for bacteriocins has revealed the general existence of yet to be characterized bacteriocins in several bacterial species  <cit> .
other toxins produced by p. aeruginosa include virulence factors such as exotoxin a, pcn and y as well as membrane vesicles  <cit> .
the clinical strains in our study come from a multi-centre canadian study of the epidemiology of chronic p. aeruginosa infections of cf patients  <cit> , see methods.
chronic infection with p. aeruginosa occurs in 60-70% of canadian adults with cf  <cit> .
after confirmation using standard techniques that the isolates were p. aeruginosa , genetic distance among all strains was estimated by comparing banding patterns of a full genome digest using pulsed field gel electrophoresis, pfge  <cit> .
we also confirmed that genetic distance correlates with the degree of overlap in resource use, measured by the ability of strains to metabolize  <dig> different carbon substrates found on commercially available biolog plates.
RESULTS
we measured the level of inhibition by anticompetitor toxins by spotting a dilution series of a cell free extract collected from  <dig> h old p. aeruginosa pa <dig> or pa <dig> culture onto a lawn of one of  <dig> different clinical isolates growing on a solid surface.
the natural isolates differ in their genetic distance to the producing strain; genetic distance is quantified using full genome digests.
the lowest concentration of cell free extract that gave rise to inhibition of the clinical isolate was used to calculate the inhibition score   <cit> .
figure  <dig> depicts the level of inhibition by both pa <dig> and pa <dig> as a function of genetic distance of toxin producing strain to the clinical isolates.
our results lend strong support to the idea that toxins are most effective when active against genotypes of intermediate genetic distance relative to the focal strain.
the relationship between inhibition and genetic distance is unimodal, peaking at intermediate genetic distance for both toxin producers pa <dig> and pa <dig>  this result is confirmed more formally by noting that a quadratic model with an internal maximum is a better descriptor of the data than a linear model , by the lower aic  values for the quadratic models than the linear models  and by an f-ratio test asking if adding the quadratic term provides a significantly better fit than the linear model .
we also tested for the existence of an internal maximum in the data using a mitchell-olds and shaw  test  following mittelbach et al.
<cit> .
this approach tests the null hypothesis that a quadratic function, fitted to the data, has no stationary point  within the range provided.
our results reject this null hypothesis for both pa <dig> and pa <dig> at the p < <dig>  level , the same criterion used in mittelbach et al.
<cit> .
since the sign of the quadratic coefficient for both producer strains was negative, the results of the mos test indicate the presence of a statistically significant 'hump' within the range of genetic distances examined.
the evidence for an internal hump is somewhat weaker for pa <dig> than pa <dig> but we note that our test is conservative, as we have not included data on the effectiveness of either strain at inhibiting itself.
as both of these values are zero , including these values would produce a much more pronounced hump.
to verify that genetic distance correlates with resource use, we measured the metabolic similarity of toxin producing strains to the clinical isolates using biolog plates .
metabolic profiles become more divergent with increasing genetic distance, as expected, reflected in the significantly negative linear relationship observed between jaccard distance and metabolic correlation between pairs of strains .
these results lend support to the idea that genetic distance is linked to ecological divergence.
it is further notable that inhibition score peaked at intermediate metabolic similarities for both pa <dig> and pa <dig> but was statistically significant only for pa <dig> .
it is not immediately obvious why we detected a significant quadratic relationship between inhibition score and metabolic similarity in one strain but not the other.
one possibility is that the biolog plates we used here, which provide profiles on carbon substrate metabolism, represent one of many possible dimensions along which ecological divergence can proceed.
under this interpretation, metabolic divergence in carbon substrate use may reflect a correlated response to divergence in other ecologically important factors such as ability to grow as a biofilm, resistance to various stressors like ph, temperature, or salinity, and possibly even predation, that we did not measure.
thus we would still expect to see some relationship between metabolic similarity and genetic distance, as we did for pa <dig>  even if this is not the sole target of ecological divergence.
there are any number of other differences between pa <dig> and pa <dig> that could be responsible for this difference.
pa <dig> has a slightly larger genome than pa <dig>  and contains a number of unique 'pathogenicity islands' that are thought to be associated with a generally increased level of virulence in most hosts  <cit> .
it also is thought to produce only r- and f-type pyocins, whereas pa <dig> produces all three types   <cit> .
it is notable that s-pyocins differ from both r- and f-pyocins in that they are oligopeptides whereas r- and f-pyocins are both phage-like structures.
why or how the differences in genome content, size, or pyocin identity affects the relationship between inhibition score and metabolic similarity remains an open question, however.
what agents are responsible for killing in our experiments?
bacteriophage were clearly not responsible.
if bacteriophage were causing the inhibition of clinical isolates, they would be able to amplify themselves in an exponential culture of the same clinical isolate.
this was not the case .
three lines of evidence suggest, rather, that toxic compounds such as pyocins or exotoxins excreted by pa <dig> and pa <dig> are the main killing agent.
the first is that pa <dig> and pa <dig> are not killed by their own supernatant.
such a result is consistent with the idea that the toxins are pyocins, as pyocin production involves specific immunity genes that confer resistance by preventing lysis in non-producing kin  <cit> , although it does not rule out the possibility that other toxins with similar immunity properties are also involved.
if killing were associated with a non-specific toxic compound such as some waste product, we would have expected both producer strains to be susceptible to killing and killing would most likely also not depend on genetic or metabolic similarity.
second, repeating the inhibition assay with heat-treated supernatant eliminates killing , providing strong support for the idea that the killing compounds are proteins.
third, and most interestingly, inhibition by pa <dig> is stronger, on average, than that by pa <dig> , a result that is likely due to the fact that pa <dig> produces a larger array of pyocins than pa <dig>  including s-type pyocins  <cit> .
although these three lines of evidence point suggestively to pyocins as being the main killing agent, we have not conducted an explicit test of this hypothesis by, for example, repeating our assays with pyocin knock-out strains.
although it may be possible to conduct such a test by focusing on the prtr/n regulator, which is thought to be a global regulator of known pyocins  <cit> , it is not clear that such a test would be conclusive since a number of the pyocins in both pa <dig> and pa <dig> have yet to be isolated  <cit>  and there may exist other exotoxins that behave in similar ways to pyocins.
note also that knowing the mechanism of killing, while of obvious interest, is in many ways of secondary importance to the observation that the effectiveness of killing depends in a regular way on genetic distance, at least in the strains we have studied here.
our main result is that the strength of antagonistic interactions peak at intermediate genetic distance.
this pattern is strikingly similar to that expected from theoretical  <cit>  and experimental  <cit>  kin selection models for selection using mixed populations of two strains at various ratios to adjust relatedness and considering one bacteriocin and one immunity protein.
these models have emphasized how the cost of bacteriocin production is affected by the social environment: bacteriocin production is not favored when producers are both common, because the majority of competitors are kin and so immune to the bacteriocin, and rare, because there are now too few kin to enjoy the benefits of the extra resources.
this is clearly not an appropriate interpretation of our results because we did not manipulate the frequency of producers and non-producers in our experimental system to adjust relatedness, as inglis et al.
<cit>  have done using degree of kinship as a measure of relatedness.
rather, our results provide some evidence consistent with the idea that ecological divergence may be important in mediating social interactions.
it is notable that the explanation for the ineffectiveness of toxins at inhibiting closely related genotypes  in our experiment is likely similar to that in kin selection models: they share a degree of immunity to each other's toxins.
however, the ineffectiveness of toxins against distantly related genotypes in our system is probably not directly tied to kin selection.
because increasing genetic divergence is accompanied by reduced overlap in resource use, distantly related genotypes are unlikely to compete for similar resources and so the resources liberated through antagonism are therefore unlikely to benefit the producer  <cit> .
the production of antagonistic traits such as bacteriocins in this situation is therefore likely to be costly and so selection should lead to decreased levels of antagonism.
our observation of decreased antagonism among distantly related strains, at least for pa <dig>  is consistent with this interpretation.
this interpretation is in accordance with the idea that the evolution of specific antagonism against conspecifics, as is often observed for bacteriocins, could evolve as a direct response to intraspecific resource competition and is consistent with models describing how fitness varies as a function of the strength of resource competition between types: the strength of resource competition, and so the loss of fitness, is highest when resource overlap is intermediate  <cit> .
to the extent that the opportunity or intensity of resource competition is enhanced through the physical proximity of co-occurring strains in a given habitat  <cit> , such as a cf lung, then this may further promote the evolution of antagonistic interactions such as those mediated by bacteriocins.
it remains to be seen whether our results are specific to the strains we used in this study or whether they apply more broadly to non-cf strains of p. aeruginosa or other species.
this will be an important avenue for future research.
it is not possible with our data to distinguish the specific mechanisms causing variation in toxin susceptibility.
if bacteriocins are indeed responsible for killing, then one possibility is that selection targets the total amount of bacteriocin production or the efficiency with which bacteriocins inhibit or kill their victims.
it is also possible that the target of selection is the number of receptor sites for bacteriocins in target strains.
deciding among these alternatives requires follow-up experiments that focus on finding the evolutionary origin of bacteriocins using direct competition experiments of producer stains and several target strains to ask under what conditions and by what mechanism bacteriocins aid producer populations to invade populations of sensitive strains  <cit> .
these experiments would however be very elaborate since the effect of many complicating factors such as frequency dependence, cross-feeding, the viscosity of the environment and exact costs of producing bacteriocins would have to be determined for the interaction of the producer and each target strain.
it is even possible that the high specificity of bacteriocins results from their having evolved initially as a by-product of selection for fertility-recognition systems such as conjugation that were later co-opted for use as bacteriocidal agents  <cit> .
investigating the relationship between bacteriocin diversity and conjugation frequency or recombination could help shed some light on this issue.
our results have important implications for understanding of the dynamics of infection in clinical settings.
we have firmly established that toxic compounds with high specificity mediate bacterial interactions as antagonistic agents, for instance in structuring pathogen populations in patients with a mixed p. aeruginosa infection  <cit> .
social evolution theory predicts that selection for antagonism among pathogenic strains should be accompanied by reduced virulence to the host.
the consequences of p. aeruginosa infection on patient morbidity and mortality may therefore depend to some extent on the particular strains present.
furthermore, it is notable that recent research on cf patients from ontario suggests that 25% of ontario patients who are infected with p. aeruginosa are infected with one of two predominant epidemic strains.
it may be that the predominance of these epidemic strains is due to the production of specific antagonistic agents such as pyocins  <cit> .
this is an intriguing hypothesis that awaits further testing.
as a start, we have confirmed that three of our clinical isolates produce toxic substances that kill or inhibit other clinical isolates .
thus the antagonistic interactions we have studied here do happen among clinical isolates and are not just the consequence of using strains pa <dig> and pa <dig> as producers in our study  <cit> .
understanding the way toxins such as pyocins kill p. aeruginosa strains, and how this is modulated by genetic relatedness, may also provide insight into the development of novel therapeutic interventions, for example by evolving pyocins specifically against strains that predominate in infections.
they can thus be considered designer drugs  <cit>  and will be a much more direct agent to treatment of the disease than the current practice of using broad spectrum antibiotics against which wide spread resistance exists  <cit> .
interestingly, pyocins are not new in a clinical setting: it has been shown that pyocins slow down the development of several forms of cancer in mammalian cells  <cit> .
also, membrane vesicles produced by p. aeruginosa have been suggested as novel therapeutic agents  <cit> .
however they may be even more effective when used in a targeted way against known infections.
the similarity between strains can then be used as a predictor of the intensity of the antagonistic interaction and thus the effectiveness of the pyocin.
CONCLUSIONS
using clinical and laboratory strains of pseudomonas aeruginosa, we found that the level of antagonism between toxin producing and target strains is maximal at intermediate genetic and metabolic similarity between producer and target strain.
we explained this result in the context of resource competition: resource competition is expected to be maximal for strains that are not your kin but also not completely unrelated since those strains do not share the same need for resources and are less likely to be a competitor.
our results suggest that the importance of antagonism and perhaps other social interactions between bacteria are modulated by the strength of resource competition.

 the production by microorganisms of proteinaceous anticompetitor toxins such as bacteriocins is often cited as a good example of an antagonistic trait. the evolution of antagonistic interactions is difficult to understand because they directly harm both actor and recipient. at the level of an individual gene, this apparent paradox can be readily resolved using the framework of inclusive fitness, which shows that antagonistic interactions can evolve provided they produce a net benefit to actors, even if the act of antagonism itself is costly. bacteriocin production has the hallmark of a classic antagonistic trait that can evolve through its effects on inclusive fitness. bacteriocins are produced by nearly all bacteria and are considered the main agents in direct antagonistic interactions between and within bacterial species. the production of bacteriocins is costly, both in terms of the energy diverted away from other functions such as growth and, in gram-negatives at least, because bacteriocin-producing cells release their bacteriocins through lysis and so cause cell death. importantly, cells that are isogenic to the producing strain are immune to the bacteriocin, usually via an immunity protein, and so gain a benefit from bacteriocin production from clone-mates. it has also been repeatedly noted that bacteriocins are highly specific in their action, being active primarily against genetically distinct members of the same species or species closely related to the producing strain. we suggest that the mechanism underlying the variation in the antagonistic effects of toxins like bacteriocins is caused by intraspecific resource competition. we expect that the ability of these toxins to remove competitors, and so free up resources, would evolve to be maximal when resource competition is strongest among genetically distinct individuals. the logic behind this is straightforward. toxin production should not be favoured when competing with genetically identical clones because there is no fitness benefit to production. as genetic distance increases, however, so too does phenotypic and ecological divergence, and by extension resource competition. toxin production is therefore wasted when competing against genetically very divergent strains because there is little resource competition. in other words, toxin production becomes costly because its benefits are diluted by the fact that the producer and target strain do not compete with each other. this interpretation leads to the prediction that the strength of antagonism should peak at intermediate genetic distance. to test this prediction we studied the interaction between two producer strains that produce a multitude of bacteriocins and a range of sensitive'victim' strains of varying genetic distance to the producers. specifically, we measured the ability of anticompetitor toxins produced by two laboratory strains of pseudomonas aeruginosa, pa and pa to kill or inhibit clinical strains of p. aeruginosa isolated from the lungs of cystic fibrosis patients. natural communities of microbes associated with chronic infections such as colonization of the cystic fibrosis lung are often highly diverse. we also measured the degree of ecological similarity among strains, using commercially available biolog plates that contain different carbon substrates, and show that ecological similarity can decrease with genetic distance. this result is consistent with the idea that toxin production is not favoured among genetically divergent strains because of a lack of resource competition. pyocins and pseudomonas aeruginosa p. aeruginosa produces a wide variety of toxins and among the most interesting, in part because they are known to be highly specific in their action, are bacteriocins called pyocins. they are costly to produce because they are released by cell lysis of a fraction of the producer population. pyocins are proteinaceous compounds that are classified into three groups, with multiple sub-types within each group that attach to different potential receptors in target strains. pa is known to produce all three pyocins while pa produces only r- and f-type pyocins. genes coding for production of all pyocins are located on the chromosome and are clustered with genes coding for resistance to the same pyocins. genomic studies have suggested the presence of more pyocins, both from the s- and r-types. in addition, a recently developed genome-mining tool for bacteriocins has revealed the general existence of yet to be characterized bacteriocins in several bacterial species. other toxins produced by p. aeruginosa include virulence factors such as exotoxin a, pcn and y as well as membrane vesicles. the clinical strains in our study come from a multi-centre canadian study of the epidemiology of chronic p. aeruginosa infections of cf patients, see methods. chronic infection with p. aeruginosa occurs in 60-70% of canadian adults with cf. after confirmation using standard techniques that the isolates were p. aeruginosa, genetic distance among all strains was estimated by comparing banding patterns of a full genome digest using pulsed field gel electrophoresis, pfge. we also confirmed that genetic distance correlates with the degree of overlap in resource use, measured by the ability of strains to metabolize different carbon substrates found on commercially available biolog plates.  we measured the level of inhibition by anticompetitor toxins by spotting a dilution series of a cell free extract collected from h old p. aeruginosa pa or pa culture onto a lawn of one of different clinical isolates growing on a solid surface. the natural isolates differ in their genetic distance to the producing strain; genetic distance is quantified using full genome digests. the lowest concentration of cell free extract that gave rise to inhibition of the clinical isolate was used to calculate the inhibition score. figure depicts the level of inhibition by both pa and pa as a function of genetic distance of toxin producing strain to the clinical isolates. our results lend strong support to the idea that toxins are most effective when active against genotypes of intermediate genetic distance relative to the focal strain. the relationship between inhibition and genetic distance is unimodal, peaking at intermediate genetic distance for both toxin producers pa and pa this result is confirmed more formally by noting that a quadratic model with an internal maximum is a better descriptor of the data than a linear model, by the lower aic values for the quadratic models than the linear models and by an f-ratio test asking if adding the quadratic term provides a significantly better fit than the linear model. we also tested for the existence of an internal maximum in the data using a mitchell-olds and shaw test following mittelbach et al.. this approach tests the null hypothesis that a quadratic function, fitted to the data, has no stationary point within the range provided. our results reject this null hypothesis for both pa and pa at the p < level, the same criterion used in mittelbach et al.. since the sign of the quadratic coefficient for both producer strains was negative, the results of the mos test indicate the presence of a statistically significant'hump' within the range of genetic distances examined. the evidence for an internal hump is somewhat weaker for pa than pa but we note that our test is conservative, as we have not included data on the effectiveness of either strain at inhibiting itself. as both of these values are zero, including these values would produce a much more pronounced hump. to verify that genetic distance correlates with resource use, we measured the metabolic similarity of toxin producing strains to the clinical isolates using biolog plates. metabolic profiles become more divergent with increasing genetic distance, as expected, reflected in the significantly negative linear relationship observed between jaccard distance and metabolic correlation between pairs of strains. these results lend support to the idea that genetic distance is linked to ecological divergence. it is further notable that inhibition score peaked at intermediate metabolic similarities for both pa and pa but was statistically significant only for pa. it is not immediately obvious why we detected a significant quadratic relationship between inhibition score and metabolic similarity in one strain but not the other. one possibility is that the biolog plates we used here, which provide profiles on carbon substrate metabolism, represent one of many possible dimensions along which ecological divergence can proceed. under this interpretation, metabolic divergence in carbon substrate use may reflect a correlated response to divergence in other ecologically important factors such as ability to grow as a biofilm, resistance to various stressors like ph, temperature, or salinity, and possibly even predation, that we did not measure. thus we would still expect to see some relationship between metabolic similarity and genetic distance, as we did for pa even if this is not the sole target of ecological divergence. there are any number of other differences between pa and pa that could be responsible for this difference. pa has a slightly larger genome than pa and contains a number of unique'pathogenicity islands' that are thought to be associated with a generally increased level of virulence in most hosts. it also is thought to produce only r- and f-type pyocins, whereas pa produces all three types. it is notable that s-pyocins differ from both r- and f-pyocins in that they are oligopeptides whereas r- and f-pyocins are both phage-like structures. why or how the differences in genome content, size, or pyocin identity affects the relationship between inhibition score and metabolic similarity remains an open question, however. what agents are responsible for killing in our experiments? bacteriophage were clearly not responsible. if bacteriophage were causing the inhibition of clinical isolates, they would be able to amplify themselves in an exponential culture of the same clinical isolate. this was not the case. three lines of evidence suggest, rather, that toxic compounds such as pyocins or exotoxins excreted by pa and pa are the main killing agent. the first is that pa and pa are not killed by their own supernatant. such a result is consistent with the idea that the toxins are pyocins, as pyocin production involves specific immunity genes that confer resistance by preventing lysis in non-producing kin, although it does not rule out the possibility that other toxins with similar immunity properties are also involved. if killing were associated with a non-specific toxic compound such as some waste product, we would have expected both producer strains to be susceptible to killing and killing would most likely also not depend on genetic or metabolic similarity. second, repeating the inhibition assay with heat-treated supernatant eliminates killing, providing strong support for the idea that the killing compounds are proteins. third, and most interestingly, inhibition by pa is stronger, on average, than that by pa, a result that is likely due to the fact that pa produces a larger array of pyocins than pa including s-type pyocins. although these three lines of evidence point suggestively to pyocins as being the main killing agent, we have not conducted an explicit test of this hypothesis by, for example, repeating our assays with pyocin knock-out strains. although it may be possible to conduct such a test by focusing on the prtr/n regulator, which is thought to be a global regulator of known pyocins, it is not clear that such a test would be conclusive since a number of the pyocins in both pa and pa have yet to be isolated and there may exist other exotoxins that behave in similar ways to pyocins. note also that knowing the mechanism of killing, while of obvious interest, is in many ways of secondary importance to the observation that the effectiveness of killing depends in a regular way on genetic distance, at least in the strains we have studied here. our main result is that the strength of antagonistic interactions peak at intermediate genetic distance. this pattern is strikingly similar to that expected from theoretical and experimental kin selection models for selection using mixed populations of two strains at various ratios to adjust relatedness and considering one bacteriocin and one immunity protein. these models have emphasized how the cost of bacteriocin production is affected by the social environment: bacteriocin production is not favored when producers are both common, because the majority of competitors are kin and so immune to the bacteriocin, and rare, because there are now too few kin to enjoy the benefits of the extra resources. this is clearly not an appropriate interpretation of our results because we did not manipulate the frequency of producers and non-producers in our experimental system to adjust relatedness, as inglis et al. have done using degree of kinship as a measure of relatedness. rather, our results provide some evidence consistent with the idea that ecological divergence may be important in mediating social interactions. it is notable that the explanation for the ineffectiveness of toxins at inhibiting closely related genotypes in our experiment is likely similar to that in kin selection models: they share a degree of immunity to each other's toxins. however, the ineffectiveness of toxins against distantly related genotypes in our system is probably not directly tied to kin selection. because increasing genetic divergence is accompanied by reduced overlap in resource use, distantly related genotypes are unlikely to compete for similar resources and so the resources liberated through antagonism are therefore unlikely to benefit the producer. the production of antagonistic traits such as bacteriocins in this situation is therefore likely to be costly and so selection should lead to decreased levels of antagonism. our observation of decreased antagonism among distantly related strains, at least for pa is consistent with this interpretation. this interpretation is in accordance with the idea that the evolution of specific antagonism against conspecifics, as is often observed for bacteriocins, could evolve as a direct response to intraspecific resource competition and is consistent with models describing how fitness varies as a function of the strength of resource competition between types: the strength of resource competition, and so the loss of fitness, is highest when resource overlap is intermediate. to the extent that the opportunity or intensity of resource competition is enhanced through the physical proximity of co-occurring strains in a given habitat, such as a cf lung, then this may further promote the evolution of antagonistic interactions such as those mediated by bacteriocins. it remains to be seen whether our results are specific to the strains we used in this study or whether they apply more broadly to non-cf strains of p. aeruginosa or other species. this will be an important avenue for future research. it is not possible with our data to distinguish the specific mechanisms causing variation in toxin susceptibility. if bacteriocins are indeed responsible for killing, then one possibility is that selection targets the total amount of bacteriocin production or the efficiency with which bacteriocins inhibit or kill their victims. it is also possible that the target of selection is the number of receptor sites for bacteriocins in target strains. deciding among these alternatives requires follow-up experiments that focus on finding the evolutionary origin of bacteriocins using direct competition experiments of producer stains and several target strains to ask under what conditions and by what mechanism bacteriocins aid producer populations to invade populations of sensitive strains. these experiments would however be very elaborate since the effect of many complicating factors such as frequency dependence, cross-feeding, the viscosity of the environment and exact costs of producing bacteriocins would have to be determined for the interaction of the producer and each target strain. it is even possible that the high specificity of bacteriocins results from their having evolved initially as a by-product of selection for fertility-recognition systems such as conjugation that were later co-opted for use as bacteriocidal agents. investigating the relationship between bacteriocin diversity and conjugation frequency or recombination could help shed some light on this issue. our results have important implications for understanding of the dynamics of infection in clinical settings. we have firmly established that toxic compounds with high specificity mediate bacterial interactions as antagonistic agents, for instance in structuring pathogen populations in patients with a mixed p. aeruginosa infection. social evolution theory predicts that selection for antagonism among pathogenic strains should be accompanied by reduced virulence to the host. the consequences of p. aeruginosa infection on patient morbidity and mortality may therefore depend to some extent on the particular strains present. furthermore, it is notable that recent research on cf patients from ontario suggests that 25% of ontario patients who are infected with p. aeruginosa are infected with one of two predominant epidemic strains. it may be that the predominance of these epidemic strains is due to the production of specific antagonistic agents such as pyocins. this is an intriguing hypothesis that awaits further testing. as a start, we have confirmed that three of our clinical isolates produce toxic substances that kill or inhibit other clinical isolates. thus the antagonistic interactions we have studied here do happen among clinical isolates and are not just the consequence of using strains pa and pa as producers in our study. understanding the way toxins such as pyocins kill p. aeruginosa strains, and how this is modulated by genetic relatedness, may also provide insight into the development of novel therapeutic interventions, for example by evolving pyocins specifically against strains that predominate in infections. they can thus be considered designer drugs and will be a much more direct agent to treatment of the disease than the current practice of using broad spectrum antibiotics against which wide spread resistance exists. interestingly, pyocins are not new in a clinical setting: it has been shown that pyocins slow down the development of several forms of cancer in mammalian cells. also, membrane vesicles produced by p. aeruginosa have been suggested as novel therapeutic agents. however they may be even more effective when used in a targeted way against known infections. the similarity between strains can then be used as a predictor of the intensity of the antagonistic interaction and thus the effectiveness of the pyocin.  using clinical and laboratory strains of pseudomonas aeruginosa, we found that the level of antagonism between toxin producing and target strains is maximal at intermediate genetic and metabolic similarity between producer and target strain. we explained this result in the context of resource competition: resource competition is expected to be maximal for strains that are not your kin but also not completely unrelated since those strains do not share the same need for resources and are less likely to be a competitor. our results suggest that the importance of antagonism and perhaps other social interactions between bacteria are modulated by the strength of resource competition. 
we found that the strength of antagonism was maximal among genotypes of intermediate genetic distance and we show that genetic distance and resource use are linked. we studied the ability of laboratory strains of pseudomonas aeruginosa to prevent growth of different clinical p. aeruginosa isolates derived from cystic fibrosis patients. our results suggest that the importance of social interactions like antagonism may be modulated by the strength of resource competition. the strength of antagonism is therefore expected to peak at intermediate genetic distance. genetic distance was determined using genetic fingerprints. the evolution of such antagonistic interactions between individuals is expected to depend on both the social environment and the strength of resource competition. antagonism is expected to be weak among highly similar genotypes because most individuals are immune to antagonistic agents and among dissimilar genotypes because these are unlikely to be competing for the same resources and antagonism should not yield much benefit.
 bacteria excrete costly toxins to defend their ecological niche. the evolution of such antagonistic interactions between individuals is expected to depend on both the social environment and the strength of resource competition. antagonism is expected to be weak among highly similar genotypes because most individuals are immune to antagonistic agents and among dissimilar genotypes because these are unlikely to be competing for the same resources and antagonism should not yield much benefit. the strength of antagonism is therefore expected to peak at intermediate genetic distance.  we studied the ability of laboratory strains of pseudomonas aeruginosa to prevent growth of different clinical p. aeruginosa isolates derived from cystic fibrosis patients. genetic distance was determined using genetic fingerprints. we found that the strength of antagonism was maximal among genotypes of intermediate genetic distance and we show that genetic distance and resource use are linked.  our results suggest that the importance of social interactions like antagonism may be modulated by the strength of resource competition. 