Bacterial lifestyle shapes pangenomes.

Pangenomes vary across bacteria. Some species have fluid pangenomes, with a high proportion of genes varying between individual genomes. Other species have less fluid pangenomes, with different genomes tending to contain the same genes. Two main hypotheses have been suggested to explain this variation: differences in species' bacterial lifestyle and effective population size. However, previous studies have not been able to test between these hypotheses because the different features of lifestyle and effective population size are highly correlated with each other, and phylogenetically conserved, making it hard to disentangle their relative importance. We used phylogeny-based analyses, across 126 bacterial species, to tease apart the causal role of different factors. We found that pangenome fluidity was lower in i) host-associated compared with free-living species and ii) host-associated species that are obligately dependent on a host, live inside cells, and are more pathogenic and less motile. In contrast, we found no support for the competing hypothesis that larger effective population sizes lead to more fluid pangenomes. Effective population size appears to correlate with pangenome variation because it is also driven by bacterial lifestyle, rather than because of a causal relationship.

Kin selection for cooperation in natural bacterial populations.

Bacteria produce a range of molecules that are secreted from the cell and can provide a benefit to the local population of cells. Laboratory experiments have suggested that these "public goods" molecules represent a form of cooperation, favored because they benefit closely related cells (kin selection). However, there is a relative lack of data demonstrating kin selection for cooperation in natural populations of bacteria. We used molecular population genetics to test for signatures of kin selection at the genomic level in natural populations of the opportunistic pathogen Pseudomonas aeruginosa We found consistent evidence from multiple traits that genes controlling putatively cooperative traits have higher polymorphism and greater divergence and are more likely to harbor deleterious mutations relative to genes controlling putatively private traits, which are expressed at similar rates. These patterns suggest that cooperative traits are controlled by kin selection, and we estimate that the relatedness for social interactions in P. aeruginosa is r = 0.84. More generally, our results demonstrate how molecular population genetics can be used to study the evolution of cooperation in natural populations.

Gene transferability and sociality do not correlate with gene connectivity.

The connectivity of a gene, defined as the number of interactions a gene's product has with other genes' products, is a key characteristic of a gene. In prokaryotes, the complexity hypothesis predicts that genes which undergo more frequent horizontal transfer will be less connected than genes which are only very rarely transferred. We tested the role of horizontal gene transfer, and other potentially important factors, by examining the connectivity of chromosomal and plasmid genes, across 134 diverse prokaryotic species. We found that (i) genes on plasmids were less connected than genes on chromosomes; (ii) connectivity of plasmid genes was not correlated with plasmid mobility; and (iii) the sociality of genes (cooperative or private) was not correlated with gene connectivity.

Pleiotropy, cooperation, and the social evolution of genetic architecture.

Pleiotropy has been suggested as a novel mechanism for stabilising cooperation in bacteria and other microbes. The hypothesis is that linking cooperation with a trait that provides a personal (private) benefit can outweigh the cost of cooperation in situations when cooperation would not be favoured by mechanisms such as kin selection. We analysed the theoretical plausibility of this hypothesis, with analytical models and individual-based simulations. We found that (1) pleiotropy does not stabilise cooperation, unless the cooperative and private traits are linked via a genetic architecture that cannot evolve (mutational constraint); (2) if the genetic architecture is constrained in this way, then pleiotropy favours any type of trait and not especially cooperation; (3) if the genetic architecture can evolve, then pleiotropy does not favour cooperation; and (4) there are several alternative explanations for why traits may be linked, and causality can even be predicted in the opposite direction, with cooperation favouring pleiotropy. Our results suggest that pleiotropy could only explain cooperation under restrictive conditions and instead show how social evolution can shape the genetic architecture.

Sociomics: Using Omic Approaches to Understand Social Evolution.

All of life is social, from genes cooperating to form organisms, to animals cooperating to form societies. Omic approaches offer exceptional opportunities to solve major outstanding problems in the study of how sociality evolves. First, omics can be used to clarify the extent and form of sociality in natural populations. This is especially useful in species where it is difficult to study social traits in natural populations, such as bacteria and other microbes. Second, omics can be used to examine the consequences of sociality for genome evolution and gene expression. This is especially useful in cases where there is clear variation in the level of sociality, such as the social insects. Major tasks for the future are to apply these approaches to a wider range of non-model organisms, and to move from exploratory analyses to the testing of evolutionary theory.

Bacteriocin-mediated competition in cystic fibrosis lung infections.

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations,especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that(i) isolates from later infection stages inhibited earlier infecting strains less,but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage overtime; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.

Signatures of kin selection in a natural population of the bacteria Bacillus subtilis.

Laboratory experiments have suggested that bacteria perform a range of cooperative behaviors, which are favored because they are directed toward relatives (kin selection). However, there is a lack of evidence for cooperation and kin selection in natural bacterial populations. Molecular population genetics offers a promising method to study natural populations because the theory predicts that kin selection will lead to relaxed selection, which will result in increased polymorphism and divergence at cooperative genes. Examining a natural population of Bacillus subtilis, we found consistent evidence that putatively cooperative traits have higher polymorphism and greater divergence than putatively private traits expressed at the same rate. In addition, we were able to eliminate alternative explanations for these patterns and found more deleterious mutations in genes controlling putatively cooperative traits. Overall, our results suggest that cooperation is favored by kin selection, with an average relatedness of r = .79 between interacting individuals.

SOCfinder: a genomic tool for identifying social genes in bacteria.

Bacteria cooperate by working collaboratively to defend their colonies, share nutrients, and resist antibiotics. Nevertheless, our understanding of these remarkable behaviours primarily comes from studying a few well-characterized species. Consequently, there is a significant gap in our understanding of microbial social traits, particularly in natural environments. To address this gap, we can use bioinformatic tools to identify genes that control cooperative or otherwise social traits. Existing tools address this challenge through two approaches. One approach is to identify genes that encode extracellular proteins, which can provide benefits to neighbouring cells. An alternative approach is to predict gene function using annotation tools. However, these tools have several limitations. Not all extracellular proteins are cooperative, and not all cooperative behaviours are controlled by extracellular proteins. Furthermore, existing functional annotation methods frequently miss known cooperative genes. We introduce SOCfinder as a new tool to find bacterial genes that control cooperative or otherwise social traits. SOCfinder combines information from several methods, considering if a gene is likely to [1] code for an extracellular protein [2], have a cooperative functional annotation, or [3] be part of the biosynthesis of a cooperative secondary metabolite. We use data on two extensively-studied species (P. aeruginosa and B. subtilis) to show that SOCfinder is better at finding known cooperative genes than existing tools. We also use theory from population genetics to identify a signature of kin selection in SOCfinder cooperative genes, which is lacking in genes identified by existing tools. SOCfinder opens up a number of exciting directions for future research, and is available to download from https://github.com/lauriebelch/SOCfinder.

Long-term evolution of antibiotic tolerance in Pseudomonas aeruginosa lung infections.

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as tolerance. While a variety of resistance mechanisms and underlying genetics are well characterized in vitro and in vivo, an understanding of the evolution of tolerance, and how it interacts with resistance in situ is lacking. We assayed for tolerance and resistance in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to 40 years of evolution, with 3 clinically relevant antibiotics: meropenem, ciprofloxacin, and tobramycin. We present evidence that tolerance is under positive selection in the lung and that it can act as an evolutionary stepping stone to resistance. However, by examining evolutionary patterns across multiple patients in different clone types, a key result is that the potential for an association between the evolution of resistance and tolerance is not inevitable, and difficult to predict.

The evolution of cheating in viruses.

The success of many viruses depends upon cooperative interactions between viral genomes. However, whenever cooperation occurs, there is the potential for 'cheats' to exploit that cooperation. We suggest that: (1) the biology of viruses makes viral cooperation particularly susceptible to cheating; (2) cheats are common across a wide range of viruses, including viral entities that are already well studied, such as defective interfering genomes, and satellite viruses. Consequently, the evolutionary theory of cheating could help us understand and manipulate viral dynamics, while viruses also offer new opportunities to study the evolution of cheating.

Changes in toxin production of environmental Pseudomonas aeruginosa isolates exposed to sub-inhibitory concentrations of three common antibiotics.

Pseudomonas aeruginosa is an environmental pathogen that can cause severe infections in immunocompromised patients. P. aeruginosa infections are typically treated with multiple antibiotics including tobramycin, ciprofloxacin, and meropenem. However, antibiotics do not always entirely clear the bacteria from the infection site, where they may remain virulent. This is because the effective antibiotic concentration and diffusion in vitro may differ from the in vivo environment in patients. Therefore, it is important to understand the effect of non-lethal sub-inhibitory antibiotic concentrations on bacterial phenotype. Here, we investigate if sub-inhibitory antimicrobial concentrations cause alterations in bacterial virulence factor production using pyocyanin as a model toxin. We tested this using the aforementioned antibiotics on 10 environmental P. aeruginosa strains. Using on-the-spot electrochemical screening, we were able to directly quantify changes in production of pyocyanin in a measurement time of 17 seconds. Upon selecting 3 representative strains to further test the effects of sub-minimum inhibitory concentration (MICs), we found that pyocyanin production changed significantly when the bacteria were exposed to 10-fold MIC of the 3 antibiotics tested, and this was strain specific. A series of biologically relevant measured pyocyanin concentrations were also used to assess the effects of increased virulence on a culture of epithelial cells. We found a decreased viability of the epithelial cells when incubated with biologically relevant pyocyanin concentrations. This suggests that the antibiotic-induced virulence also is a value worth being enclosed in regular testing of pathogens.

Ten recent insights for our understanding of cooperation.

Since Hamilton published his seminal papers in 1964, our understanding of the importance of cooperation for life on Earth has evolved beyond recognition. Early research was focused on altruism in the social insects, where the problem of cooperation was easy to see. In more recent years, research into cooperation has expanded across the entire tree of life, and has been revolutionized by advances in genetic, microbiological and analytical techniques. We highlight ten insights that have arisen from these advances, which have illuminated generalizations across different taxa, making the world simpler to explain. Furthermore, progress in these areas has opened up numerous new problems to solve, suggesting exciting directions for future research.

Plasmids do not consistently stabilize cooperation across bacteria but may promote broad pathogen host-range.

Horizontal gene transfer via plasmids could favour cooperation in bacteria, because transfer of a cooperative gene turns non-cooperative cheats into cooperators. This hypothesis has received support from theoretical, genomic and experimental analyses. By contrast, we show here, with a comparative analysis across 51 diverse species, that genes for extracellular proteins, which are likely to act as cooperative 'public goods', were not more likely to be carried on either: (1) plasmids compared to chromosomes; or (2) plasmids that transfer at higher rates. Our results were supported by theoretical modelling which showed that, while horizontal gene transfer can help cooperative genes initially invade a population, it has less influence on the longer-term maintenance of cooperation. Instead, we found that genes for extracellular proteins were more likely to be on plasmids when they coded for pathogenic virulence traits, in pathogenic bacteria with a broad host-range.

Conflict within cooperation.

Stu West and Melanie Ghoul introduce the special issue on conflict and cooperation.

Privatisation rescues function following loss of cooperation.

A single cheating mutant can lead to the invasion and eventual eradication of cooperation from a population. Consequently, cheat invasion is often considered equal to extinction in empirical and theoretical studies of cooperator-cheat dynamics. But does cheat invasion necessarily equate extinction in nature? By following the social dynamics of iron metabolism in Pseudomonas aeruginosa during cystic fibrosis lung infection, we observed that individuals evolved to replace cooperation with a 'private' behaviour. Phenotypic assays showed that cooperative iron acquisition frequently was upregulated early in infection, which, however, increased the risk of cheat invasion. With whole-genome sequencing we showed that if, and only if, cooperative iron acquisition is lost from the population, a private system was upregulated. The benefit of upregulation depended on iron availability. These findings highlight the importance of social dynamics of natural populations and emphasizes the potential impact of past social interaction on the evolution of private traits.

Diversity, Prevalence, and Longitudinal Occurrence of Type II Toxin-Antitoxin Systems of Pseudomonas aeruginosa Infecting Cystic Fibrosis Lungs.

Type II toxin-antitoxin (TA) systems are most commonly composed of two genes encoding a stable toxin, which harms the cell, and an unstable antitoxin that can inactivate it. TA systems were initially characterized as selfish elements, but have recently gained attention for regulating general stress responses responsible for pathogen virulence, formation of drug-tolerant persister cells and biofilms-all implicated in causing recalcitrant chronic infections. We use a bioinformatics approach to explore the distribution and evolution of type II TA loci of the opportunistic pathogen, Pseudomonas aeruginosa, across longitudinally sampled isolates from cystic fibrosis lungs. We identify their location in the genome, mutations, and gain/loss during infection to elucidate their function(s) in stabilizing selfish elements and pathogenesis. We found (1) 26 distinct TA systems, where all isolates harbor four in their core genome and a variable number of the remaining 22 on genomic islands; (2) limited mutations in core genome TA loci, suggesting they are not under negative selection; (3) no evidence for horizontal transmission of elements with TA systems between clone types within patients, despite their ability to mobilize; (4) no gain and limited loss of TA-bearing genomic islands, and of those elements partially lost, the remnant regions carry the TA systems supporting their role in genomic stabilization; (5) no significant correlation between frequency of TA systems and strain ability to establish as chronic infection, but those with a particular TA, are more successful in establishing a chronic infection.

The Ecology and Evolution of Microbial Competition.

Microbes are typically surrounded by different strains and species with whom they compete for scarce nutrients and limited space. Given such challenging living conditions, microbes have evolved many phenotypes with which they can outcompete and displace their neighbours: secretions to harvest resources, loss of costly genes whose products can be obtained from others, stabbing and poisoning neighbouring cells, or colonising spaces while preventing others from doing so. These competitive phenotypes appear to be common, although evidence suggests that, over time, competition dies down locally, often leading to stable coexistence of genetically distinct lineages. Nevertheless, the selective forces acting on competition and the resulting evolutionary fates of the different players depend on ecological conditions in a way that is not yet well understood. Here, we highlight open questions and theoretical predictions of the long-term dynamics of competition that remain to be tested. Establishing a clearer understanding of microbial competition will allow us to better predict the behaviour of microbes, and to control and manipulate microbial communities for industrial, environmental, and medical purposes.

Toward an evolutionary definition of cheating.

The term "cheating" is used in the evolutionary and ecological literature to describe a wide range of exploitative or deceitful traits. Although many find this a useful short hand, others have suggested that it implies cognitive intent in a misleading way, and is used inconsistently. We provide a formal justification of the use of the term "cheat" from the perspective of an individual as a maximizing agent. We provide a definition for cheating that can be applied widely, and show that cheats can be broadly classified on the basis of four distinctions: (i) whether cooperation is an option; (ii) whether deception is involved; (iii) whether members of the same or different species are cheated; and (iv) whether the cheat is facultative or obligate. Our formal definition and classification provide a framework that allow us to resolve and clarify a number of issues, regarding the detection and evolutionary consequences of cheating, as well as illuminating common principles and similarities in the underlying selection pressures.

Loss of social behaviours in populations of Pseudomonas aeruginosa infecting lungs of patients with cystic fibrosis.

Pseudomonas aeruginosa, is an opportunistic, bacterial pathogen causing persistent and frequently fatal infections of the lung in patients with cystic fibrosis. Isolates from chronic infections differ from laboratory and environmental strains in a range of traits and this is widely interpreted as the result of adaptation to the lung environment. Typically, chronic strains carry mutations in global regulation factors that could effect reduced expression of social traits, raising the possibility that competitive dynamics between cooperative and selfish, cheating strains could also drive changes in P. aeruginosa infections. We compared the expression of cooperative traits - biofilm formation, secretion of exo-products and quorum sensing (QS) - in P. aeruginosa isolates that were estimated to have spent different lengths of time in the lung based on clinical information. All three exo-products involved in nutrient acquisition were produced in significantly smaller quantities with increased duration of infection, and patterns across four QS signal molecules were consistent with accumulation over time of mutations in lasR, which are known to disrupt the ability of cells to respond to QS signal. Pyocyanin production, and the proportion of cells in biofilm relative to motile, free-living cells in liquid culture, did not change. Overall, our results confirm that the loss of social behaviour is a consistent trend with time spent in the lung and suggest that social dynamics are potentially relevant to understanding the behaviour of P. aeruginosa in lung infections.