Hello everyone, my name is Steve Diggle and I am an Associate Professor in Sociomicrobiology in the School of Life Science at the University of Nottingham in the UK. I started my research career looking at the molecular mechanisms that control cell-to-cell signalling, usually known as quorum sensing, in the opportunistic pathogen Pseudomonas aeruginosa. In 2006 I was awarded a fellowship from the Royal Society to study quorum sensing from a more evolutionary perspective. So, rather than working on ‘how’ quorum sensing works, I started to ask ‘why’ questions such as ‘is quorum sensing really a social behaviour’ and ‘why is it maintained in natural populations’. Such questions are asked in the field of social evolution and so in this lecture, I will tell you about social evolution in microbes, and at the end, discuss why this is important in biofilms. In a microbial biofilm, innumerable cells form complex structures in which each individual cell plays only a small role. Much of the literature traditionally describes a biofilm as a coordinated and cooperative unit where the activity of each individual cell benefits the overall group. Upon closer inspection however, this is not always the case. First, the architectural features such as mushroom like structures, water channels and stratifications may be the result of chemical gradients, simple growth parameters and physiological adaptation to a particular micro-niche. These can arise with no coordination, cooperation or communication between participant cells. Second, when a behaviour is performed that confers a cost to the individual performing the behaviour, and a benefit to the surrounding population, that behaviour is vulnerable to cheating strategies which do not contribute a benefit to the population but benefit individuals. In this lecture I will describe social evolution and social behaviours in microbes and discuss why this might be important in biofilms. Before we talk specifically about biofilms, let us first discuss why we might be interested in studying social evolution in microbes. Social evolution has become an active area of research in microbiology for a number of reasons. For example, microbes display many apparently social or cooperative behaviors, and many of these behaviors seem to be crucial for those microbes to survive in their ecological niches or to become successful pathogens. Biofılm formation could be a prime example of a cooperative social behaviour. In addition, social evolution research also complements mechanistic studies by presenting evolutionary problems for which there must be mechanistic solutions. Next we must talk about what defines a social behavior. There is a huge body of work examining microbial behaviours from a molecular perspective, but comparatively little from an evolutionary standpoint. Social behaviours in microbes have, until relatively recently, been ignored by evolutionary biologists, yet there has been much work done in this area both theoretically and empirically in higher organisms such as insects, birds and mammals. Social behaviours are those which have fitness consequences for both the actor performing the behaviour and the recipient of the behaviour, and can be broadly categorized into four types depending on the fitness consequences for both the actor and the recipient. A behaviour that increases the fitness of the actor is mutually beneficial if the recipient also benefits, and selfish if the recipient suffers a loss. A behaviour that reduces the fitness of the actor is altruistic if the recipient benefits, and spiteful if the recipient suffers a loss. It is important to understand the nature of the interaction between actor and recipient as then we can make very different predictions as to how such a behaviour evolves and is maintained in populations. One of the big challenges for us as microbiologists, is to understand the multitude of behaviours that have been described in a wide range of bacterial species from an evolutionary perspective, which will increase our understanding of the nature of these behaviours, and complement previous and future molecular work. Furthermore, understanding whether a behavior is social can help us understand how bacterial populations interact within infected hosts, which will help explain how virulence and antimicrobial resistance evolves. We could even use some of the ideas of social evolution to help us reduce virulence during infection, and favourably improve the outcome for patients. What are microbial social behaviours? Microbial behaviours might seem simpler than those that have been described in higher organisms, but they share a fundamental similar property: a shared investment in producing a group resource. Many microbial social behaviours come in the form of public goods which are released into the surrounding environment and are costly for individuals to make but provide a benefit for all other individuals in the population. Microbes make a wide variety of public goods including siderophores for iron scavenging, β-lactamases to inactivate antibiotics, exopolysaccarhides, toxins, extracellular proteases, and quorum sensing signal molecules. I am sure that you will be able to think of lots of other examples. However, despite the multitude of cooperative behaviours seen in the natural world, explaining cooperative behaviours such as altruism has been a significant challenge for evolutionary biologists. The problem arises because altruistic behaviours, reduce the fitness of the actor. The question then becomes why help others at a cost to yourself? This appears to conflict with Charles Darwin’s idea of the survival of the fittest, because natural selection favours those individuals with the greatest reproductive success relative to the rest of the population. It is therefore difficult to see why altruistic behaviours, which reduce the fitness of the actor, can be evolutionarily favoured. Because an actor accrues a fitness cost by performing a behaviour, there is the potential of exploitation by cheats or freeloaders who do not cooperate, and who therefore gain a fitness advantage in the population because they do not pay the costs of cooperation. This is sometimes referred to as a ‘tragedy of the commons’, which was originally used to describe human economics and morality, but can also be applied to microbes. The tragedy is that if everyone cooperated this would benefit the population, but cheating, even though these individuals benefit in the short term, puts the entire population at a risk of collapse or extinction. To illustrate social evolution in microbes we will look at the well studied social trait of quorum sensing. This is a process where cells communicate with each other via diffusible signal molecules. These signals coordinate a wide range of behaviors at the population or group level. In a range of gram-negative and gram-positive bacterial species, QS signals regulate the production of extracellular “public goods,” including nutrient-scavenging molecules, exoproteases, toxins, and surfactants that aid cellular motility. In the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus, quorum sensing has been shown to be both costly and exploitable by cheats. Using a synthetic growth medium where quorum sensing-dependent exoprotease production is important for growth, wild-type Pseudomonas aeruginosa populations grow well, but populations of quorum sensing mutants grow poorly. Crucially, in mixed cultures, quorum sensing mutants act as social cheats because they have a fitness advantage due to exploitation of the exoprotease production of wild-type cells. Similarly, when mice with burned skin or with chronic wounds are infected with Pseudomonas aeruginosa, quorum sensing mutants act as cheats and invade the bacterial population within days. In waxmoth larvae infections, a similar pattern of sociality for Staphylococcus aureus can be observed. Quorum sensing mutants are less fit than their wild-type counterparts in monoculture infections but demonstrate social cheating when in mixed infection. Such results help us to explain why quorum sensing mutants often arise in clinical infections even though such loss-of-function mutations might appear to be detrimental to fitness. Furthermore, mixed infections of wild type cells with cheats, show a reduced virulence, because cheats do not make tissue damaging toxins. This could be used to our advantage because cheats could be introduced into infections to both reduce virulence and buy time for the immune system to deal with the infection. This has resulted in the term ‘cheatobiotics’, but a lot more research is need in this area. How can cooperation be maintained in the light of cheating? A key question in the social evolution field is how are social behaviours maintained given that cheats enjoy large fitness advantages in mixed populations? One of the most studied ideas is ‘inclusive fitness’. This is the idea that an individual maximises its inclusive fitness, not just by maximising its own reproduction, but the reproduction of its genes even if they happen to be in other individuals. So by helping a relative reproduce, this still increases inclusive fitness. This theory was developed by the evolutionary biologist William Hamilton in the 1960s, and has since been termed ‘kin selection’. A classic example of this can be found in insects. Many social insects such as bees and ants, have sterile worker castes that completely forgo their own reproduction in favour of that of the queen. How is this altruism inherited if the altruists never reproduce? The behaviour can be inherited if the benefits accrue to individuals which share the genes for altruism. This is the case in insects, but how does this manifest in bacterial populations? Selection for cooperative traits in microbes can be maximized when relatedness is kept high but how can this be achieved? One way that kin selection can work is via limited dispersal. Here relatives are kept close together, increasing the probability of interactions occurring amongst relatives, which favours indiscriminate altruism towards neighbours. This does not require complex recognition systems, and therefore is likely to be important in microbes. Biofilms and cooperation Many of the experimental social evolution studies so far, have been performed in well mixed planktonic cultures, where there is ample opportunity for cheats to freely disperse and indiscriminantly exploit public goods production by cooperators. One key way for microbes to limited dispersal, is to grow in a biofilm which is surrounded by an exopolysaccarhide matrix. Such a spatially structured environment has the potential to keep cooperating genotypes together and limit the invasion and spread of cheating genotypes. A possible outcome of this is the general maintenance of social traits within the biofilm. Though it might seem obvious that the formation of a biofilm involves cooperation between cells, the processes involved need not always be cooperative. Inspired by spectacular modern microscopic observations of biofilms, such as the mushroom-like structures of Pseudomonas aeruginosa, researchers have modeled in silico, the growth and spatial patterns of cells growing on a surface with interesting results. Using these computer simulations, it is possible to observe the types of vertical structures typical of in vitro biofilms using only basic growth limitation criteria in the environment and simple growth parameters of the organism. Secondly, in competitive growth simulations between a wild type bacterium and a mutant that does not produce a ‘scaffolding’ polymer, a polymer mutant can be outgrown and covered by the cooperative wild type. Where oxygen gradients are present, an overproducer of the scaffold polymer pushes its cell lineage vertically into favourable oxygen conditions. In this in silico model of growth on a surface, mixing genotypes does not select for cheating due to the local benefit of polymer secretion. However if you introduce a new parameter to the model, the possibility that one genotype can mutate into the other, the result is different. The cheating mutant now gains an advantage if it occurs in an oxygen rich region where it can quickly produce lots of cells. The key point here is that both the biological environment, and the social environment, determine where and how we expect to observe biofilm formation, and why biofilms are maintained. We are only just beginning to study social behaviours in biofilms, and it is now crucial that we perform experimental tests to confirm the predictions that are observed during computer simulations. This will help to inform us why biofilms form, and which traits are social and non-social in biofilms. This is important because it will allow us to test whether targeting specific traits, will result in us being able to better control biofilms, which are becoming increasingly problematic in clinical and industrial settings.
