13

Social behaviour in microorganisms

Kevin R. Foster

OVERVIEW

Sociobiology has come a long way. We now have Many species do all of this in surface-attached a solid base of evolutionary theory supported communities, known as biofilms, in which the by a myriad of empirical tests. It is perhaps less diversity of species and interactions reaches appreciated, however, that first discussions of bewildering heights. Grouping can even involve social behaviour and evolution in Darwin’s day differentiation and development, as in the spec- drew upon single-celled organisms. Since then, tacular multicellular escape responses of slime microbes have received short shrift, and their moulds and myxobacteria. Like any society, how- full spectrum of sociality has only recently come ever, microbes face conflict, and most groups to light. Almost everything that a microorgan- will involve instances of both cooperation and ism does has social consequences; simply divid- competition among their members. And, as in ing can consume another’s resources. Microbes any society, microbial conflicts are mediated by also secrete a wide range of products that affect three key processes: constraints on rebellion, others, including digestive enzymes, toxins, mol- coercion that enforces compliance, and kinship ecules for communication and DNA that allows whereby cells direct altruistic aid towards clone- genes to mix both within and among species. mates.

13.1 Introduction indeed sociology, microbes have featured in descrip- tions of social life. Prominent among these are the We must be prepared to learn some day, from the students writings of Herbert Spencer, the social philosopher of microscopical pond-life, facts of unconscious mutual sup- who coined the term survival of the fi ttest in the wake port, even from the life of micro-organisms. of Darwin’s Origin . Spencer was widely responsible K r o p o t k i n (1 9 0 2) . for popularising the notion of altruism in Victorian Th e idea of sociality in the mere microbe can be met Britain (Auguste Comte probably fi rst coined the with a raised eyebrow and a smirk. Nevertheless, for term), and importantly used both humans and single- as long as there has been evolutionary biology, and celled life to defi ne altruism’s nature (Dixon 2008 ).

Social Behaviour: Genes, Ecology and Evolution, ed. Tamás Székely, Allen J. Moore and Jan Komdeur. Published by Cambridge University Press. © Cambridge University Press 2010.

331 332 Kevin R. Foster

And it was not long before a near-modern perspec- (Hamilton 1964 , Wilson 1975 ). In the last century the tive emerged at the hands of the eccentric Russian spectacularly social insects, cooperatively breeding explorer and anarchist Peter Kropotkin. In what was vertebrates and, of course, we humans have been arguably the fi rst sociobiology text, Kropotkin ran widely studied and drawn upon to test the core the- the gamut of examples of biological cooperation. ories of social behaviour. Th e colourful chapters that Many were inspired by his wanderings through fro- comprise the majority of this book are a testament zen Siberia, but his imagination wandered further to to this. But there was little word on the microbes. include speculations on microbial life. Until, that is, microbiologists started a revolution Th e concepts of altruism and cooperation in biology, from within and challenged the oft prevailing view therefore, were developed with the appreciation that that the microorganism is a self-absorbed creature, they might be applied to even the smallest of organ- swimming alone in the plankton. Researchers were isms. From there, more familiar organisms took cen- fi nding that many species attach to surfaces, secrete tre stage in the developing fi eld of ethology (Chapter structural polymers, and grow into large and some- 1; Tinbergen 1963 ), which later became sociobiology times diverse communities ( Fig. 13.1 ; biofi lms: Kolter

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Figure 13.1 Microbial groups. (a) Large microbial biofi lm in a Massachusetts river, USA. (b) Colony of the spore-forming bacteria Bacillus subtilis growing on agar. Th e cells secrete structural polymers that contribute to the wrinkly appearance. (c) Biofi lm at the air–water interface in the bacterium Pseudomonas fl uorescens , again formed with the help of extracellular polymer secretion. (d) Flocculation behaviour in the budding yeast Saccharomyces cerevisiae . A slice through a yeast ‘fl oc’ is shown, which forms after the yeast cells have aggregated (Smukalla et al . 2008 ). Th e fl oc has been treated with a toxin (ethanol). Th e dark area shows dead cells on the outside, but these protect the cells on the inside. Photos: (a) by the author, (b) by Hera Vlamakis, (c) by Andrew Spiers, (d) by Kevin Verstrepen. Social behaviour in microorganisms 333

& Greenberg 2006 , Nadell et al. 2 0 0 9) . Th is led to a But what exactly is a social behaviour in a micro- dramatic shift in perspective. Indeed the pendulum organism? First things fi rst; the use of microorganism may have swung too far at fi rst, with some descrip- here will mean a focus on species with individuals tions bordering on the utopian: ‘biofi lms resemble that we cannot see unaided and, in particular, the the tissues formed by eukaryotic cells, in their physio- well-studied bacteria (Box 13.1). Social behaviour logical cooperativity and in the extent to which they simply means a behaviour that aff ects another cell’s are protected from variations in bulk phase condi- evolutionary fi tness ( Table 13.1 ; Hamilton 1964 , tions by a primitive homeostasis provided by the Wilson 1975 ), and the most interesting social behav- biofi lm matrix’ (Costerton et al . 1995 ). But with the iours are the ones that evolved because they aff ect close of the last century, the study of social behav- others (West et al. 2007 ). Only if a behaviour evolved iour in microorganisms bloomed, and most recently because of its social eff ects, or at least partly due to it has come to include sociobiologists, such as myself, them, can it strictly be viewed as a social strategy. who cut their teeth on studies of more classic social Consider a cell that secretes a waste product which organisms (for me, it was the social wasps: Foster & harms another cell. One can probably safely assume Ratnieks 2001 ). And the microbes bring a valuable that the act of secretion is the product of natural new perspective because, for the fi rst time, we can selection on the secreting cell, but the harm caused hope to fi nd the genes that underlie social behaviours to the other cell may not be; it may simply be a by- and watch the emergent dynamics of social evolution product of natural selection to remove waste (Diggle (Foster et al. 2 0 0 7) . et al. 2007a ).

Box 13.1 Some key players in microbial social evolution

Dictyostelium discoideum – Th is slime mould or social amoeba is actually a mem- ber of a little-known kingdom, the Mycetozoa, which is the sister group of animals and fungi. D. discoideum is a bacterial predator that displays impressive multicellu- larity, aggregating upon starvation to form a multicellular slug and then a fruiting body, in which some cells die to hold the others aloft as spores ( Fig. 13.6 ). Bacillus subtilis – A spore-forming soil bacterium that forms tough biofi lms on agar and on the surface of growth medium ( Fig. 13.1b ). Th ese biofi lms show consid- erable diff erentiation, including isolated areas of spores and other areas containing cells that secrete the polymers that bind the group together. Under other lab condi- tions, B. subtilis cells will also diversify into competent and non-competent cells, and into chaining and single cells (see main text). Escherichia coli – Th e classical laboratory species, E. coli is found in the soil and, of course, in the digestive tracts of many healthy animals. E. coli displays evidence of cooperative entry into a dormant state upon starvation, and can carry a plasmid that poisons cells that do not possess it. Myxococcus xanthus – A striking example of convergent evolution with D. discoi- deum, this spore-forming soil bacterium will aggregate upon starvation to form a fruiting body. M. xanthus cells also secrete toxins that kill other cells within the fruit- ing body, other strains, and also species upon which it feeds. Th e latter process is some- times associated with social motility, an example of group predation (Fig. 13.5a). Pseudomonas – P. aeruginosa is a pathogenic bacterium that is sometimes found in soil and often in the clinic ( Fig. 13.2b ). While best known for its ability to cause complications in the lungs of cystic fi brosis patients, it can cause problems in any 334 Kevin R. Foster

Box 13.1 Continued

immunosuppressed patient. P. aeruginosa also forms robust biofi lms ( Fig. 13.4 ), displays quorum sensing, undergoes swarming motility, and secretes many shared products including siderophores that help with the uptake of iron. P. fl uorescens is a generally non-pathogenic soil bacterium that also forms biofi lms, most famously in the form of a mat on the surface of standing cultures in the laboratory ( Fig. 13.1c ). Saccharomyces cerevisiae – Th e workhorse of eukaryotic cell biology, the bud- ding, brewer’s or baker’s yeast has been cultivated by humans for millennia. While laboratory strains are often grown in a manner that limits social behaviours, this species secretes several enzymes, will aggregate with other cells by fl occulation ( Fig. 13.1d ), and can undergo pseudo-hyphal growth whereby growing cells do not separate after division and form long chains. Vibrio – V. cholera is the bacterium that causes cholera through the colonisation of the human intestine and the subsequent secretion of cholera toxin. Growth in the intestine and in the environment is often associated with biofi lm formation and quorum sensing. Th e related species V. fi sheri lives mutualistically in the light organ of the bobtail squid and produces light under quorum-sensing regulation.

Th e heart of this chapter is a review of the social et al . 2007 , Foster 2008 ), and even spite (Foster et al. behaviours of microorganisms, and the possible evo- 2001 , Gardner et al . 2004 ) (see Table 13.1 for defi ni- lutionary benefi ts they carry to the cells that express tions). Finding microbial altruism, we are faced with them ( section 13.2 , Form and function). Th ey range the classic problem of sociobiology (Hamilton 1964 , from the simple eff ects of growth rate through to com- Wilson 1975 ): how can apparently selfl ess traits plex multicellular development, with secretion, com- remain stable in the face of natural selection for con- munication and genetic exchange in between. Some fl ict and cheating ( Chapter 6 )? Section 13.3 , Confl ict behaviours are downright selfi sh, but others have the resolution , is dedicated to this question. appearance of cooperation, whereby the actions of one cell increase the reproductive fi tness of another (Table 13.1; West et al . 2006 ). Identifying when behav- 13.2 Form and function iours are cooperative versus selfi sh – or the associated question of when they are a true social strategy versus 13.2.1 Growth rate a by-product of non-social traits – can be diffi cult for microbes. Th is is due in no small part to our limited A simple way to aff ect your neighbours is to steal understanding of microbial behaviours in nature. For their food. Th is is particularly easy when you share example, when is it good, evolutionarily speaking, to resources like carbon and oxygen as a microbe, and be in a biofi lm? Moreover, what really is a microbial when these become growth-limiting the potential for group? A cell may sit in a massive biofi lm but only ever confl ict is clear (Ratnieks et al. 2006 ). Natural selec- aff ect its very nearest neighbours. So please take all tion can favour cells that hungrily consume resources interpretation herein with such caveats and questions in order to obtain the lion’s share, even though this in mind. may be ineffi cient and give poor future prospects (Kerr Th at said, many behaviours do seem to slow or even et al . 2006 ). At least mathematically, this is analogous prevent a cell’s division, which means a reduction in to the ongoing clashes over fi sheries, and air quality personal fi tness. Keeping focus then not on the strain between laissez-faire capitalists on the one hand and but on the individual cell, these behaviours have the environment-oriented interventionists on the other hallmark of microbial altruism (Hamilton 1964 , West (the tragedy of the commons: Chapter 6, Rankin et al. Social behaviour in microorganisms 335

Table 13.1. Defi nitions of social behaviour based upon the effects on per- sonal lifetime reproduction (direct fi tness)

Eff ect on recipient

Positive Negative

Eff ect on actor Positive Mutual benefi tSelfi shness Negative Altruism Spite Cooperation Competition/confl ict

2007 ). Many microbes have the ability to switch from when a cell bursts (Cascales et al. 2 0 0 7) . B y s e c r e t - aerobic respiration to fermentation, which produces ing products, cells can create an environment that energy more rapidly when oxygen is limited. However, promotes growth – a simple form of niche construc- fermentation is also ineffi cient and yields much less tion (Lehmann 2006 ). For example, when cells of the energy overall (Pfeiff er et al. 2001 , Pfeiff er & Schuster budding yeast Saccharomyces cerevisiae get low on 2005 , Novak et al . 2006 ). Competing cells therefore glucose, they secrete an enzyme called invertase that are expected to more often make use of fermentation hydrolyses sucrose to liberate glucose and fructose (MacLean & Gudelj 2006 ), while cooperative systems, (Greig & Travisano 2004 ). including multicellular organisms, are more likely to Other secreted products bind and scavenge nutri- be altruistically aerobic, whereby each cell slows its ents. Th e pathogen Pseudomonas aeruginosa is a fas- growth to allow other cells to have food in the future cinating but unpleasant bacterium that infects the (Kreft 2004 , Kreft & Bonhoeff er 2005 ). lungs of patients with cystic fi brosis; a genetic dis- High growth rate has many potential knock-on order causing, amongst other symptoms, thickened eff ects. One much discussed is the evolution of viru- airway mucus. P. aeruginosa relies on many secreted lence, where rapid pathogen proliferation can mean products for infection including siderophores (liter- trouble for a patient (Frank 1992 ). However, dividing ally ‘iron carriers’ in Greek; Fig. 13.2 ). Th ese dissolve rapidly will also tend to take resources away from insoluble iron and allow the bacterium to combat cooperative traits like product secretion (Griffi n a common vertebrate response known as localised et al. 2004 ), communication (Sandoz et al. 2007 ) and anaemia that reduces the iron in tissue (Jurado 1997 ). development (Velicer et al . 1998 , Vulic & Kolter 2001 ); But the confl ict lies not only with the patient. Th ere these are discussed below. When these systems are is also the potential for infi ghting: mutants that neg- required for microbes to successfully attack the host, lect secretion are able to cheat those that do (Griffi n et rapid growth can again lower ultimate yield, and lead al. 2004 ), and the emergence of cheating can lead to to reduced, rather than increased, virulence (Brown reduced virulence in animal models (Harrison et al. et al. 2002 , Foster 2005 , Harrison et al. 2 0 0 6) . 2006 ). It is interesting that the genetics underlying both yeast invertase and the siderophores display high variability (Greig & Travisano 2004 , Smith et al. 13.2.2 Secreted products 2005 ). Th is may indicate shifting ecological demands Microbes secrete all manner of substances that (Brockhurst et al . 2007 , Foster & Xavier 2007 ), or even physically and chemically alter their surroundings an evolutionary arms race in which cooperators and their evolutionary fi tness (Fig. 13.2). Th ere are attempt to evolve products that cannot be used by several ways out of a cell, including passive diff u- cheaters (Tuemmler & Cornelis 2005 ). sion (Pearson et al. 1999 ), active pumps (Kostakioti But it is not all darkness and disease. Many et al . 2005 ), packaged in mini-membranes (vesicles: microbes provide metabolic assistance by producing Mashburn-Warren & Whiteley 2006 ) and of course extracellular compounds or resources that their host 336 Kevin R. Foster

a

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Figure 13.2 Secreted products in microbes. (a) Cooperative secreted products and cheating: the paler cells secrete a product that benefi ts all cells, but the darker cells do not, and save energy in the process. Th e darker cells cheat and outgrow the paler cells. (b) Diff erent strains of the bacterium Pseudomonas aeruginosa growing on agar displaying diff erent pigmentations that result, at least in part, from diff erences in pyoverdin (green, left) and pyocyanin (blue, middle) secretion, which both function in iron scavenging. Th e far-right strain also shows the characteristic spotting of viral-driven cell-lysis. Photos by the author.

organisms cannot make. Th is includes our own gut ( Fig. 13.3 ; Noda et al. 2 0 0 3) . Th ese protozoa in turn fauna, and also antifungal bacteria that protect leaf- rely on bacteria, often spirochetes, that provide both cutter ant fungal gardens (Currie et al . 1999 ), light- metabolic assistance (Dolan 2001 ) and even motility producing Vibrio fi sheri in fi sh and squid (Visick et al. (Tamm 1982 , Wenzel et al. 2003 , König et al. 2 0 0 7) . 2000 ), and nitrogen-fi xing bacteria or mycorrhizal Meanwhile, some spirochetes rely on cross-feeding fungi (Helgason & Fitter 2005 ) that provide nutrients from yet other bacteria in the termite gut (Graber and for plants (Denison 2000 ). Cross-feeding among the B r e z n a k 2 0 0 5) . microbes themselves also appears common, whereby Shared products can also be structural. While not the waste product of one species feeds another strictly secreted, the RNA virus φ6 produces proteins (Dejonghe et al . 2003 ), which, in turn, keeps the waste using the cellular machinery of its bacterial host. product at low levels and may improve their com- Th ese proteins package the viral RNA in a protect- bined metabolism (Pfeiff er & Bonhoeff er 2004 ). With ive coat and can be used by all viral RNAs such that many species interacting, there is the potential for cheater virus RNAs, which do not encode the proteins, cross-feeding networks to reach dizzying complexity. can still be successfully packaged (Turner & Chao One nice example occurs inside an insect, itself an 1999 , Brown 2001 ). While viruses coat themselves in exemplar of animal sociality: a termite. Termite soci- rigid protein coats, bacteria instead use slime. More ety rests upon the ability to eat wood, and they are formally known as extracellular polymeric sub- helped along by protozoa that break down cellulose stances (EPS), the slimes found in bacterial biofi lms Social behaviour in microorganisms 337

a b

Figure 13.3 Th e symbioses inside termites, which feed on wood. (a) Th e host: the termite Mastotermes darwiniensis . (b) A symbiont, Mixotricha paradoxa , that helps to break down cellulose. Th e fur on the surface is actually the fl agella of symbiotic spirochete bacteria that help it to swim around (König et al . 2007 ). Photos: (a) by Judith Korb, (b) by Helmut König. are complex mixtures that probably protect the cells cultures in our laboratory. Another interesting toxin– from various stresses (Crespi 2001 , Velicer 2003 , Hall- antitoxin system is found on a plasmid (a small, cir- Stoodley et al . 2004 , Foster 2005 , Keller & Surette 2006 , cular, extrachromosomal piece of DNA) carried by West et al. 2006 ). Slime may even allow one strain to the well-known bacterium Escherichia coli. Th e toxin smother and suff ocate others in the quest for oxygen appears only to be released when some plasmid car- and nutrients, in the manner that a tree trunk allows riers burst, taking non-carrier cells down with them one plant to grow tall and shade another (Fig. 13.4; in dramatic fashion (Cascales et al. 2007 ), an example Foster & Xavier 2007 , Xavier & Foster 2007 ). of evolutionary spite (Gardner et al. 2004 ). But it does Although sinister, a bit of smothering is nothing not always end there, because the success of toxin- compared to the favoured weapon in the microbial producing cells paves the way for a second strain that arsenal: secreted toxins. Th e toxin–antitoxin systems produces only the antitoxin and saves on the cost of carried by many bacteria are probably the closest thing the toxin. Th en this second strain can be replaced that microbes have to aggression (Chapter 7). In their by the original susceptible cell type that produces simplest form, these systems comprise two neigh- neither toxin nor antitoxin (Kerr et al. 2002 ), which bouring genes, one encoding a toxin (bacteriocin) means that toxin producers can reinvade, and so on. that kills other strains, and the other an antitoxin, or Th is non-transitive (rock–paper–scissors) relation immunity protein, that protects the toxin-producing can lead to cyclical dynamics that maintain all three strain. Th e cystic fi brosis pathogen P. aeruginosa types, and which may represent a general principle carries multiple such toxin secretion systems, which for the maintenance of biodiversity (Durrett & Levin evolve rapidly and may again be indicative of arms 1997 , Kerr et al . 2002 , Reichenbach et al. 2 0 0 7) . races (Smith et al. 2 0 0 5) . I f P. aeruginosa is indeed It is tempting to think that toxin–antitoxin sys- involved in an arms race with other species, it appears tems always go hand in hand with confl ict. Perhaps to do well in the rankings: we fi nd it near impossible not; several examples have been instead interpreted to get accidental contamination of P. aeruginosa in terms of cooperation, like that seen between the 338 Kevin R. Foster

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Figure 13.4 Nutrient competition and motility in bacterial biofi lms. (a) An individual-based model of a microbial biofi lm. Th e cells respire and in doing so create oxygen gradients (parallel lines) that slow the growth of cells deep in the biofi lm (the paler cells are growing quickly and the darker ones slowly). (b) A biofi lm of P. aeruginosa in which metabolically active cells have been stained yellow (the palest part of the image). (c) Th e eff ect of polymer secretion on success within a biofi lm. Th e simulation shows cells that secrete a polymer and altruistically push their descendants up into the oxygen, which suff ocates the cells beneath (shown dark) that do not secrete (Xavier & Foster 2007 ). (d) Th e eff ect of motility on success within a biofi lm. Confocal image of a P. aeruginosa biofi lm with a motile strain (pale) that moves towards the top of the biofi lm and a non- motile strain (dark) that does not (Klausen et al . 2003 ). Images (a) and (c) from Joao Xavier, (b) from Phil Stewart, (d) from Tim Tolker-Nielsen.

germ and soma cells of your body. Th e E. coli chromo- can inhibit the spread of viruses to healthy cells some contains the mazEF system (Metzger et al. (Hazan and Engelberg-Kulka 2004 ). However, the 1988 ), where E encodes the antitoxin and F the toxin. benefi ts under other stresses are less clear (Tsilibaris Th is system has a clever feature: the two genes turn et al . 2007 ), and more work is needed to dissect out on and off together (they are an operon) but the toxin the exact evolutionary pressures that drive the degrades more slowly than the antitoxin. Th is means mazEF system and other toxin systems (Magnuson that when the mazEF operon is deactivated, the 2007 ). Critically, if lysis releases the toxin and kills levels of toxin and antitoxin in the cell will steadily non-carriers, like the colicins, then mazEF may also decrease but, importantly, the antitoxin disappears function in competition with other strains and spe- fi rst, leaving behind the toxin, and this kills the cell. cies. Similar questions await the spore-forming bac- What makes this clever is that various environmen- terium Bacillus subtilis (Fig. 13.1b), which actively tal stresses turn off the operon, including heat, DNA secretes a toxin that kills non-expressing cells in the damage and virus attack, so that damaged cells will surroundings (Gonzalez-Pastor et al . 2003 , Dubnau & undergo lysis (Hazan et al. 2 0 0 3) . Th e process looks a Losick 2006 ), and viral-induced cell death in P. aeru- lot like an altruistic behaviour – the weakened com- ginosa biofi lms ( Fig. 13.2b ; Allesen-Holm et al. 2 0 0 6) , mitting suicide to protect the strong – and there is which have both been interpreted as purely coopera- evidence that mazEF -induced lysis of diseased cells tive behaviours. Social behaviour in microorganisms 339

13.2.3 Communication transition from a single-celled to a hyphal lifestyle in Microbes also use secretions to communicate with fungi (Hornby et al . 2001 ), DNA uptake and exchange each other. Indeed, killing another cell with a toxin is (Piper et al. 1993 , Magnuson et al. 1994 , Havarstein et a very simple, albeit rude, form of discourse. However, al . 1995 ) and many secreted products such as slime this would not, strictly speaking, be communica- (EPS) (Hammer and Bassler 2003 ), iron-scavenging tion (or signalling) by some evolutionary defi nitions molecules (Stintzi et al . 1998 ) and toxins (van der (Chapter 8), which demand that both producer and Ploeg 2005 ). receiver benefi t from their respective actions (Keller Does quorum sensing always represent active com- & Surette 2006 , Diggle et al. 2007a ). Such cooperative munication from one cell to another? Th is is not yet communication does seem likely in some systems, clear. Some autoinducers may simply be waste prod- including the coordinated development of slime ucts that did not evolve to allow secreting cells to com- moulds discussed below, and perhaps the curious ten- municate (Diggle et al. 2007a ). In some cases, however, dency of S. cerevisiae yeast cells to synchronise their autoinducers have been shown to cost energy, which intracellular physiologies, even though cell division is suggests that they do represent active and evolved not synchronised (Tsuchiya et al. 2 0 0 7) . M i c r o b e s a l s o communication. Th e cost also means that mute cells, display an impressive ability to detect the density of which do not produce the signal, can cheat by listening their own and other species through quorum sensing in without paying the cost of communication, just as (literally, sensing who is around: Fuqua et al. 1 9 9 4) . cells can cheat on a shared enzyme ( Fig. 13.2a , Brown Quorum sensing is found both in bacteria (Miller & & Johnstone 2001 , Diggle et al. 2007b ). Other times Bassler 2001 , Keller & Surette 2006 , Diggle et al. 2007a ) it can pay to play deaf. Some P. aeruginosa strains and in fungi (Hogan 2006 ), and involves a wide var- from cystic fi brosis (CF) patients lack a functional iety of secreted compounds known as autoinducers, copy of the autoinducer sensor protein LasR, which including some packaged in vesicles (Mashburn & is required to respond to the autoinducer. Lacking Whiteley 2005 ). All exploit the same simple principle: the sensor protein stops cells producing cooperative if cells secrete a chemical, then as cell density goes products that may not be needed in the CF lung and, up, so will the local concentration of the chemical. By even if the products are needed, the lasR mutants can responding to the chemical, therefore, the cells can exploit the products of others (D’Argenio et al. 2007 , respond to their own density. Sandoz et al . 2007 ). Finally, strains unable to quorum Quorum sensing was fi rst characterised in V. fi sh- sense in V. cholerae overproduce slime (EPS), which eri, the glow-in-the-dark bacterium that lives in the is thought to inhibit dispersal from biofi lms but can sea, where it exists in a free-living form but also on provide a competitive advantage when it is better to and in squid and fi sh, whom it helps with behav- stay put (Hammer & Bassler 2003 , Nadell et al. 2 0 0 8) . iours like mate or prey attraction (Nealson et al. 1970 , Th ere is also the potential for communication Fuqua et al. 1 9 9 4) . V. fi sheri regulates luminescence among species. One quorum-sensing molecule, with an autoinducer, an N -acylhomoserine lactone, autoinducer-2 (AI-2), is produced by an amazingly such that the cells only turn on at high density in the diverse set of species, which opens the possibility of host, and not when swimming in the sea. Moreover, widespread interspecies communication (Federle & this molecule works through a positive-feedback loop Bassler 2003 ). How often this represents active signal- whereby it induces an increase in its own production. ling on the part of secreting cells is not known (Keller Since then, the name has spread to quorum-sensing & Surette 2006 , Diggle et al . 2007a ), but some species, molecules that do not autoinduce, and quorum sens- including P. aeruginosa, respond to AI-2 even though ing has been found to control a multitude of behav- they do not make it, which is at least consistent with iours, including bacterial spore formation (Hagen et the evolution of eavesdropping (Duan et al. 2003 ) al . 1978 , Gonzalez-Pastor et al. 2003 , Dubnau & Losick ( Chapter 8 ). When multiple species meet, there is 2006 ), biofi lm formation (Davies et al. 1998 ), the also the possibility for devious manipulations (Keller 340 Kevin R. Foster

& Surette 2006 ): E. coli actively takes up AI-2, and some may favour killing-associated competence, thereby prevents Vibrio cholerae from secreting pro- while those that get replaced do not. A human ana- teases that it uses during cholera gut infection (Xavier logy would be the occasions where a conquering & Bassler 2005 ). Meanwhile, the dental bacterium nation incorporates some aspects of a subordinate’s Veillonella atypica secretes a compound that induces culture into its own, such as the Romans adopting Streptococcus gordonii to release lactic acid, which V. Greek gods. atypica then feeds on (Egland et al. 2 0 0 4) . Transport of DNA between cells is even more directed when driven by plasmids (conjugation) and viruses (transduction). Plasmids are the relatively 13.2.4 Genetic exchange small circular pieces of DNA carried by bacteria. Th e social behaviours of microorganisms include sex Despite their small size, plasmids exert a powerful (Chapter 10). Indeed, eukaryotes like yeast are sexual infl uence on cell biology and are often able to engin- creatures that undergo meiosis and mating to make eer their own propagation, by inducing their cell to recombinant progeny (Goddard et al. 2005 ). Although attach to another cell and make a channel through primarily asexual, bacterial cells also perform more which a plasmid copy can pass (Th omas & Nielsen limited DNA exchanges (Th omas & Nielsen 2005 , 2005 ). Many plasmids are benevolent and produce Narra & Ochman 2006 ) via the environment, viruses benefi cial proteins, including antibiotic resistance and plasmids. A cell able to incorporate DNA from genes, but others act like viruses, and spread within the environment into its genome is termed naturally and among cells at a cost to their hosts (Eberhard competent (artifi cial competence means they will 1990 , Smith 2001 , Paulsson 2002 ). And while plasmids do it when forced by a persuasive lab scientist armed show some specifi city, they are often able to trans- with electrodes or chemicals). Sometimes the act of fer their DNA to other species. One example is the DNA uptake itself may not be all that social; the cell tumour-inducing (TI) plasmid that makes the bacter- performing the action may not aff ect the fi tness of ium Agrobacterium tumefaciens into a plant pathogen. others. However, natural competence is often tightly Th is plasmid has amazing abilities. It integrates frag- associated with social traits. ments of its DNA into the plant’s genome and induces V. cholerae becomes naturally competent when the formation of a gall, which protects and feeds the growing in a biofi lm on arthropod exoskeletons bacteria. Th e plasmid also encodes its own quorum- (Meibom et al . 2005 ) and competence in B. subtilis sensing system, and at high quorum it will induce involves both quorum-sensing (Magnuson et al. 1994 ) conjugation and transfer itself to other A. tumefaciens and diff erentiation (below). Perhaps the most interest- cells (Chilton et al. 1980 , Piper et al. 1993 ). Transfers of ing case, however, comes from Streptococcus species, viral and plasmid DNA between species, however, are which upon entering a competent state also activate not perfect and sometimes take host DNA along for the multiple toxin–antitoxin systems (above) that select- ride. Th is can have unintended but important conse- ively lyse surrounding cells that are not competent quences, such as mixing the DNA of prokaryotes and (Claverys et al. 2 0 0 7) . Th e evolution of DNA uptake is eukaryotes, which are otherwise separated by over therefore intertwined with potential confl ict among a billion years of evolution (Gogarten & Townsend strains: the killing factors may both remove compet- 2005 , Th omas & Nielsen 2005 ). ing cells from the environment and also provide DNA for recombination. Th is adaptive story makes a lot of 13.2.5 Group formation sense, but at the same time it has a curiously paradox- ical aspect to it. Why kill another cell only to incorp- Social interactions are most intense when individ- orate its DNA into your own and partially become it? uals live side by side in a group ( Chapter 9 ), which for Th e answer may lie in recognising that there can be many microbes will mean a biofi lm. Biofi lms form diff ering evolutionary interests at diff erent loci, and when cells grow on a surface or attach to each other Social behaviour in microorganisms 341

to form a living mat ( Fig. 13.1 ) in which cells secrete suggests that the biofi lm state provides considerable all manner of products, including the slimy polymers protection to its inhabitants, which can include resist- that form a structural matrix around the cells ( Fig. ance to antibiotics (Mah et al. 2003 ). Analogously, it 13.4 ; Branda et al . 2005 , Parsek & Greenberg 2005 , has recently been shown in the yeast S. cerevisiae that Nadell et al . 2009 ). Interestingly, some strains turn fl occulation, whereby cells aggregate together at high on the slime at high density while others turn it off . densities, provides protection against stresses and Turning on at high density makes sense: slime will antifungal agents ( Fig. 13.1d ; Smukalla et al. 2 0 0 8) . only be made in the biofi lm and not when swimming Bacterial biofi lms can also be dynamic environ- around at low density, where slime presumably does ments as strains struggle to get the best nutrients, not pay. But why do some species then turn it off again both by altruistically pushing descendant cells with at even higher density? Th e answer may lie in disper- slime (above, Fig. 13.4c ; Xavier & Foster 2007 ) and sal. Negative regulation has been found in V. cholerae, by active cell movement ( Fig. 13.4d ; Klausen et al. which induces a short-lived acute infection that cli- 2003 ). At its height, bacterial movement borders on maxes with dispersal en masse from the gut of the mass exodus when dividing cells engage in a group unfortunate patient. Th is need to disperse may favour migration away from high-density areas, in a process strains that turn off the slime just before they leave, known as swarming. In nature, swarming may often when they will no longer need it (Hammer & Bassler be associated with dispersal from biofi lms, and, like 2003 , Nadell et al. 2 0 0 8) . B i o fi lms often also con- biofi lm formation, swarming is often controlled by tain many species. V. cholerae must compete in the quorum sensing (Daniels et al . 20 0 4 ): a n e x o d u s i s gut with many other species including E. coli, which best when it is really crowded. Furthermore, the cells can interfere with its quorum sensing, as we have appear to cooperate with one another to secrete prod- seen (Xavier & Bassler 2005 ). Th e best studied multi- ucts that ensure things go swimmingly, including species biofi lms, however, must be those growing digestive enzymes (Bindel Connelly et al. 2004 ) and upon your teeth as you read this. Dental plaque con- chemicals that ease movement by reducing surface tains an amazing diversity of species (Kolenbrander tension (biosurfactants: Kohler et al . 2000 ). In swarm- 2000 ), including some that depend upon each other ing sensu stricto , cells propel themselves with beating for attachment and growth (Palmer et al. 2 0 0 1) . fl agella (Harshey 2003 ), but they achieve similar ends Living in a dense biofi lm presents challenges, by pulling themselves along with pili (the molecular including intense nutrient competition that threatens equivalent of grappling hooks), and perhaps even by to slow growth (Fig. 13.4a, b ; Stewart 2003 , Xavier & jetting slime out the back, jet-ski-like (Kaiser 2007 ). A Foster 2007 ). So why make one? Th e answer is likely single species can mix and match its methods: a strain to vary between species (Hall-Stoodley & Stoodley of Myxococcus xanthus that had been mutated to stop 2005 ), but one common benefi t is probably settling in pili-based swarming was able to re-evolve motility a good place to live. A biofi lm at an air–water interface using a diff erent mechanism (Fig. 13.5a; Velicer & Yu has good access to oxygen and light (Fig. 13.1c; Rainey 2003 ). But not all microbes migrate outwards in this & Rainey 2003 , Brockhurst et al . 2007 ), and attach- way. Some actively seek each other out and move ment to solid surfaces can yield similar advantages, together, and it is here that we fi nd the pinnacle of particularly given that cells will often attach revers- microbial sociality, multicellular development. ibly and swim off if they end up in a bad spot (Sauer et al. 2 0 0 2) . B i o fi lm life also allows cells to condition 13.2.6 Differentiation, diversifi cation and the local environment to suit growth and communica- development tion (Parsek & Greenberg 2005 , Keller & Surette 2006 ), and they are tough: biofi lms are the scourge of indus- Not all cells are equal. Many microbes can diff eren- try and medicine, where they clog, contaminate and tiate into distinct physiological states and morpholo- cake machines and people alike (Fux et al. 2 0 0 5) . Th is gies. In the lab, diff erentiation events can be relatively 342 Kevin R. Foster

a b c

Figure 13.5 Swarming behaviours in bacteria. (a) Diff erent swarming genotypes in the spore-forming bacterium Myxococcus xanthus (Velicer & Yu 2003 ). (b) Swarming cells in Salmonella enterica , which become elongated and carry many fl agella for effi cient propulsion (Kim et al . 2003 ). (c) A second strain of Salmonella enterica swarming. Photos: (a) by Greg Velicer, (b) and (c) by Wook Kim.

uniform, such as diff erentiation into swarming cells cells in aggregates of the slime mould Dictyostelium in Salmonella enterica, which become elongated and discoideum are low on food, and as a consequence end carry many fl agella for effi cient propulsion ( Fig. 13.5b , up dead in a stalk rather than becoming reproduct- c ; Kim et al . 2003 ). But this can change in more complex ive spores ( Fig. 13.6 ; David Queller’s profi le; Gomer environments. Cells bathed in oxygen at the surface of & Firtel 1987 ). True randomness is another way to a biofi lm may divide furiously while those below do go. Entry into competence in B. subtilis is driven by little more than provide anchoring ( Fig. 13.4a , b ; Xu a positive-feedback loop; once a cell starts down that et al . 1998 ). Moreover, diversifi cation into multiple road, there is no pulling out. However, entry is not cell types can occur without environmental gradi- guaranteed and appears to be driven by chance fl uc- ents. One champion is B. subtilis, which can form both tuations in expression of the regulatory genes – the individual cells and chains of cells during growth, molecular equivalent of rolling dice (Suel et al. 2 0 0 6) . divide into competent and non-competent cells, and Another possibility in eukaryotes such as yeast is ran- then form some spore cells that kill the others (above; dom acts of gene silencing by modifi cation of DNA, or Dubnau & Losick 2006 ). Another important example the nucleosomes that associate with DNA, which can is the small subpopulations of dormant cells in many suppress transcription (Rando & Verstrepen 2007 ). Or, bacteria groups that are immune to many antibiotics more drastically, cells can undergo genetic changes (persister cells: Balaban et al. 2004 , Keren et al. 2 0 0 4, to diversify their phenotypes. Th is appears to occur in Gardner et al . 2007 ). Yeast diversify too: recent studies P. aeruginosa biofi lms, where multiple growth pheno- have shown bimodal expression of genes involved in types appear from a single-type, and do so dependent phosphate uptake (Wykoff et al . 2007 ), and high vari- upon recA , the gene central to DNA modifi cation and ability in the tendency to become spores (Nachman repair (Boles et al. 2004 ). Finally, the tendency of the S. et al . 2007 ). Finally, we also fi nd microbial diff eren- cerevisiae cells to stick to each other by fl occulation is tiation and diversifi cation in symbioses. Some nitro- controlled by a gene with a large repeat sequence that gen-fi xing bacteria in plant roots diff erentiate into makes fl occulation evolve rapidly and reversibly (Fig. swollen and terminal bacteriods that lose the ability 13.1d ; Verstrepen et al. 2005 , Smukalla et al. 2 0 0 8) . to divide (Mergaert et al. 2006 ), while other cells in the Why would a single strain diversify into multiple surroundings remain viable and may even be altruis- phenotypes? Th ere is no doubt that some of the vari- tically fed by the diff erentiated cells (Denison 2000 ). ability among cells is a simple by-product of unavoid- How do microbial cells diversify without exter- able noise in cellular processes, and this must be kept nal cues? Th e secret of course is internal variability. in mind. But for cases that represent real evolution- Nutritional state is one way to go. Recently divided ary adaptations, there are two major explanations for Social behaviour in microorganisms 343

a b

c d

Figure 13.6 Social life, and strife, in the slime mould, or social amoeba, Dictyostelium discoideum . (a) Cells labelled with fl uorescent beads that are streaming together to form an aggregate, which will then diff erentiate into a migrating slug. (b) Migrating slugs passing left to right through a strip of bacteria. In some hours, cells that were shed by the slugs will consume the bacteria and make new slugs (Kuzdzal-Fick et al . 2007 ), by which time the original slugs will have changed into fruiting bodies containing a stalk of dead cells that holds the rest up as reproductive spores. (c) Birds-eye view of a lawn of D. discoideum fruiting bodies. (d) Development in a D. discoideum cheater mutant. Alone, the mutant is unable to sporulate, but when mixed with the wild-type cells the mutant is competitively superior and produces more spores (Ennis et al. 2000 , Gilbert et al . 2007 ). Photos by the author. functional diversifi cation. One idea is that cells are benefi ts of a division of labour – both in terms of the hedging their bets on the future (Kussell & Leibler cells in your body and in our society. 2005 ). Th at is, if there may or may not be an environ- Th e division of labour in microbial groups is most mental catastrophe on the way, it can pay a strain to clear in development, whereby cells undergo a pre- have some cells growing and others in a protected dictable series of changes over time. Simple develop- survival state as persisters (Balaban et al. 2004 , Keren ment can occur without a division of labour. When et al . 2004 , Gardner et al. 2 0 0 7) . Th e other major over- short of food, for example, E. coli cells simultaneously lapping explanation is a division of labour (Michod slow their growth and transition into a dormant spore- 2006 ). Microbes can divide their labour to perform like state (Vulic & Kolter 2001 ). Th e most convincing a task, which might be bet-hedging or indeed some- examples of development, however, include diversifi - thing else, such as a division between polymer secre- cation and a division among cells between reproduc- tion and spore formation (Vlamakis et al. 2008 ). We, tion and other functions. Th is includes B. subtilis spore of course, are also living breathing testament to the formation, but also some species that have evolved a 344 Kevin R. Foster

dramatic way of getting out of a tight spot. When short (Brockhurst et al. 2007 , Foster & Xavier 2007 ), broadly of its bacterial prey, cells of the soil-dwelling slime speaking these are the benefi ts that favour social mould D. discoideum (Kessin 2001 , Shaulsky & Kessin life. But, as we have seen, group-level benefi ts alone 2007 ) secrete a signalling molecule (cyclic AMP) that may not be enough, and the potential for confl ict induces the cells to stream together and diff erentiate looms large in the microbial world. But it is not all- into a multicellular slug that migrates towards heat out war, and mechanisms that limit confl icts must and light (Fig. 13.6a). Th e slug sheds cells as it goes that and do exist. Th e next sections review these mecha- colonise nearby patches (Fig. 13.6b; Kuzdzal-Fick et al. nisms, dividing them up into direct eff ects – eff ects on 2007 ) and near the soil surface transforms into a fruit- the reproduction of the cell that performs the social ing body, in which around a quarter of the cells die action – and indirect eff ects – eff ects on cells that share in a stalk that holds the other cells aloft as dispersing genes with the focal cell (the structure is based upon spores (Fig. 13.6c). Analogously, cells of the bacterium Ratnieks et al. 2006 , Gardner & Foster 2008 and refer- Myxococcus xanthus , also a bacterial predator, will ences therein). aggregate upon food shortage to form a fruiting body containing spores (Julien et al. 2000 ). Again, many cells die in the process (Wireman & Dworkin 1977 ). 13.3.1 Direct effects: constraints As with all social behaviours in microorganisms, and coercion with diversifi cation and development comes potential N o n - e n f o r c e d evolutionary confl ict. Indeed, whenever two neigh- bouring cells have a diff erent phenotype, the chances As for herding wildebeest, if it is good to be in a group, are that one is doing better reproductively than the then joining with other cells can simultaneously other. Natural selection, therefore, can favour cells benefi t the joiner and the joinees, which may mean that become the best type all the time, even though limited confl ict ( Chapter 9 ). Increasing the size of a this may mess things up for everyone. Examples are microbial group may often in this way confer benefi ts seen in the slime moulds and myxobacteria, where on all inhabitants: a large biofi lm can provide better single mutations can produce cheaters that over-pro- protection from the environment, and larger slime- duce spores in mixtures with other strains ( Fig. 13.6d ; mould slugs migrate further (Foster et al. 2 0 0 2) . O n c e Ennis et al. 2000 , Velicer et al. 2000 , Santorelli et al. in a group, though, there is the potential for things to 2008 ). But it also applies to the more simple cases, like turn nasty – a slime-mould cell may well benefi t from the evolution of persister cells, which are dormant other cells joining the slug ( Fig. 13.6b ), but not if they cells that suff er poor reproductive prospects, at least then force it to make a stalk (Santorelli et al. 2 0 0 8) . in the short term (Gardner et al. 2007 ). However, the Mechanisms such as enforcement or genetic related- fact that slime moulds still fruit and persisters persist ness may then be needed to keep the peace (below). tells us that disruptive mutants do not always win out. However, there are also reasons to believe that micro- Why not? Th at comes next. bial groups, and indeed all social species, will tend to have intrinsic properties that moderate confl icts (Travisano & Velicer 2004 ). 13.3 Confl ict resolution If cooperative systems that avoid cheating persist longer than those that do not, over time there will be Th e social behaviours of microorganisms carry a var- enrichment for systems with pre-existing constraints iety of benefi ts, from regulating growth to maximise on the origin of cheaters (Foster et al. 2007 , Rankin et yield, through niche construction and communi- al. 2007a ). Th ere are several mechanisms that might cation, to the formation of dense protective group- cause these constraints, including reduced muta- ings. Although the ability to cash in on these benefi ts tion rate (below; Harrison & Buckling 2005 , 2007 ) will ultimately depend upon ecological conditions and genetic redundancy, whereby a cooperative trait Social behaviour in microorganisms 345

is expressed by multiple genes (Foster et al. 2 0 0 7) . a saturated environment where all niches are fi lled, Another way is through the ubiquitous phenomenon which makes it more diffi cult for cheaters to arise, as of pleiotropy (each gene aff ecting multiple traits: has been seen in bacterial biofi lms (Brockhurst et al. Foster et al . 2004 ). For example, the group-wide entry 2006 ). In addition, if two species no longer compete, into a dormant spore-like state in starving E. coli the way is open for evolution of between-species cultures (above) is sometime hijacked by the GASP cooperation. Th is occurs when species can exchange mutant (growth at stationary phase: Vulic & Kolter resources that are cheap for the donor but expen- 2001 ). Like a cancer, the mutant cells keep on growing sive for the recipient (Schwartz & Hoeksema 1998 ), under low nutrient conditions and replace the normal e.g. a photosynthesiser exchanging organic carbon cells. However, this comes at a pleiotropic cost: long for inorganic carbon with a heterotroph (Kuhl et al. term the cheaters are compromised and probably 1996 ). And a recent simulation suggested that nat- doomed owing to poor acid tolerance, which prevents ural selection operating on communities of microbes the cooperative trait being lost. can promote such positive interactions (Williams & Th e gradual enrichment of constraints on cheater Lenton 2008 ). It requires that associations are fairly mutations, through pleiotropy or otherwise, may also stable, however, so that the benefi ts from investing in be driven by shorter-term processes than trait or spe- the other species feed back on the investing cell or its cies persistence. As we will see below, some bacterial descendants (partner-fi delity feedback: Sachs et al. strains always have some cells that mutate into cheat- 2004 , Foster & Wenseleers 2006 ). ers. When this harms the strain’s prospects for found- ing new biofi lms, selection will favour the evolution E n f o r c e d of genetic protection against cheater mutations, in the same way that multicellular organisms gain con- In many societies, including our own, confl ict is straints on cancer mutations (Nunney 1999 , Michod & reduced by coercion that forces individuals to comply Roze 2001 ). In this vein, natural selection for cooper- (Wenseleers & Ratnieks 2006 ). We understand rela- ation has been shown to lead to reduced global muta- tively little of mechanisms of coercion in microbes, but tion rate in P. aeruginosa (below; Harrison & Buckling there are some interesting candidates. One example is 2007 ). Constraints on cheating can also arise through conjugation. If a cooperative secretion is encoded on an arms-race-like process in which a cheater strain a plasmid, then plasmid transfer will force other cells paves the way for a new strain that can resist the to secrete (Smith 2001 ). Th ere is also toxin secretion cheater and others like it (Foster 2006 ). An experi- in the bacteria B. subtilis (Gonzalez-Pastor et al. 2003 ) ment that mixed cheater strains with natural strains and M. xanthus (Wireman & Dworkin 1977 ) that kills in the spore-forming bacterium M. xanthus caused some cells and provides nutrients for spore formation. some lab populations to go extinct (Fiegna et al. 2 0 0 6) . One must be careful here, however, as toxin secretion Th e cheater strains would rise to dominance, but this can obviously also signify raw confl ict, and not its drove their own demise because they are impotent resolution. We need more data before we can tell if, in without the natural strains. However, in one case, a nature, the secretion of these toxins really promotes new mutant arose that could both outcompete the the total reproduction of the bacterial group (confl ict cheaters and form spores on its own: the system had resolution: Ratnieks et al . 2006 ) or simply allows one evolved to constrain the cheaters. strain to monopolise the resources of another (plain Another process that can align evolutionary old confl ict). interests within a social group is niche separation. Th e role of enforcement is perhaps clearest in social When strains or species compete, natural selection behaviours that occur between species. Th ere are can favour individuals that specialise on a diff erent many examples from symbioses, where a microbe resource than the competing species. Th is can have lives in or on a host species and the host is able to two knock-on eff ects. First, diversifi cation may create select the microbial cells that help it the most (partner 346 Kevin R. Foster

choice: Sachs et al. 2004 , Foster & Wenseleers 2006 ). like the cells in your body. Th is means that selection Th is includes fungus-growing ants that remove para- can lead to dramatically altruistic traits, like cell sitic and foreign fungi (Currie & Stuart 2001 , Mueller death, when this allows a cell to pass on the genes that et al. 2004 , Poulsen & Boomsma 2005 ) with the help of they carry in common with other individuals (indir- a streptomycete bacterium that secretes antifungals ect genetic benefi ts: Chapter 6). It does not matter, (Currie et al. 1999 ), and leguminous plants that dir- evolutionarily speaking, which cells in a clonal group ect resources to the roots with the most industrious get to reproduce, so long as the group performs well nitrogen-fi xing bacteria (Kiers et al. 2003 , Simms et al. overall. 2006 ). Even your intestine possesses mechanisms, Th e key theoretical corollary is that anything that some more subtle than others, which presumably tend breaks the genetic correlation among group members to favour more cooperative bacteria and get rid of the will promote competition and confl ict (Hamilton less favourable ones. Th e subtle mechanisms include 1964 ). Th is holds, whether the group is the bounded the still poorly understood eff ects of the immune sys- aggregation of a slime mould or a more nebulous bio- tem upon the microbial fl ora (Backhed et al. 2005 , Ryu fi lm, so long as grouping is defi ned by the scale of et al. 2008 ), and the less subtle mechanisms have been social interactions (Grafen 2007 ). Th e importance of experienced by anyone who has travelled to a foreign genetic relatedness among cells is supported by sev- country with a very diff erent diet. eral studies that have mixed up microbes. Th ese have Successful coercion, however, requires that the shown that mixing strains promotes rapid waste- recipient cannot easily escape its eff ects. Just as plei- ful growth in bacterial viruses (Kerr et al . 2006 ) and otropy can internally constrain against cheating, it the success of cheater mutants, which has been seen can also prevent escape from coercion. High-nutrient in many contexts including yeast enzyme secretion slime-mould cells (Fig. 13.6) induce low-nutrient cells (Greig & Travisano 2004 ), P. aeruginosa iron scav- to become stalk cells by secreting a chemical DIF-1, enging (Griffi n et al. 2 0 0 4) , P. aeruginosa quorum and avoiding DIF-1 is not an option: mutant cells that sensing (Diggle et al . 2007b ), and the development do not respond to DIF-1 also fail to enter the spore of M. xanthus (Velicer et al . 2000 ) and D. discoi- head to become spores (Foster et al. 2 0 0 4) . Th ere are deum , where a myriad cheater mutants have now host–symbiont examples as well: the bobtail squid been found (Ennis et al. 2000 , Santorelli et al. 2 0 0 8) . that hosts V. fi sheri bacteria in its light organ creates But why does a cheater do better at low relatedness? an environment that enables fl uorescent strains to Consider the fate of a rare cheater mutant that has outcompete non-fl uorescent ones (Visick et al . 2000 ). just arisen in a natural population of slime moulds (Fig. 13.6; Gilbert et al . 2007 ). If many strains of D. dis- coideum mix together (low relatedness), the cheater 13.3.2 Indirect effects: kinship cells will mostly meet cooperator cells and do well. But when aggregates form from a single strain, as S t r a i n m i x i n g they often do, the cheaters will have no one to exploit Personal benefi ts are an easy way to promote cooper- and will do badly: cheating does not pay when you ation and make microbes behave as a unifi ed group. are surrounded by relatives. Similar principles apply However, just as important are benefi ts to related to multi-species cooperation, only now one needs cells, or kin. Relatedness here means not genealogical not only low mixing within species, but also between relatedness, as in the statement ‘rats and bats are species, because this allows enough time for invest- related, they are both mammals’, but rather related- ments in a partner species to provide feedback ben- ness among individuals within a species, as in ‘I am efi ts (Foster & Wenseleers 2006 ). related to my sister Gillian’. Simply put, if a group of One does not have to wait for evolutionary time microbes are recently derived from a single progeni- scales to see strain mixing aff ecting microbial groups. tor, their evolutionary interests are perfectly aligned, Th is is particularly true when cells can pinpoint Social behaviour in microorganisms 347

non-relatives, such as spitefully secreting a toxin to motility, attachment and, as we have seen, quorum kill them (Gardner & West 2004 , Gardner et al. 2 0 0 4) . sensing (D’Argenio et al . 2007 , Sandoz et al. 2 0 0 7) . With toxins, mixing immediately leads to problems: But mutation-driven evolution in microbes does not spore production is often decreased when M. xanthus need years. Bacteria sitting in a fl ask on a bench will strains are mixed (Fiegna & Velicer 2005 ) and mixing evolve cooperation and lose it again to cheating in a two bacteriocin- producing bacteria limits their abil- matter of days. One of the more friendly pseudomon- ity to infect a host (Massey et al. 2 0 0 4) . Th e harmful ads, Pseudomonas fl uorescens , has a solitary smooth eff ects of mixing can also be more subtle: slugs con- strain that swims around in the broth but will rapidly taining multiple strains of D. discoideum migrate mutate to generate a second wrinkly strain, so-called poorly (Foster et al. 2002 , Castillo et al. 2 0 0 5) . because it secretes sticky stuff that makes its colonies If mixing is costly, then why do it? For some, the ben- look, well, wrinkly (Fig. 13.1b shows a similar case efi ts of a large group will outweigh the costs, but others from another species). Th e wrinklies cooperate with limit their mixing. A sister species of D. discoideum, each other and form a mat that suff ocates the smooth D. purpureum , preferentially aggregates with cells of the cells below them (Fig. 13.1c), only to have new mutant same strain (Mehdiabadi et al . 2006 ), and strains of the smooth cells appear in their midst that sink the whole bacterium Proteus mirabilis create an inhibition zone lot (Paul B. Rainey’s profi le; Rainey & Rainey 2003 , with other strains when swarming (Gibbs et al. 2 0 0 8) . Brockhurst et al. 2007 ) in a turbid tragedy of the com- In addition, so-called green-beard genes (Dawkins mons (Rankin et al. 2007b ). 1976 ), which code for cell-to-cell adhesion proteins, Global mutation rate is also important. If mutation allow microbial cells that express the adhesion gene towards cheating occurs more often than towards to preferentially associate with other expressing cells, cooperation, high mutation rate will tend to pro- while excluding non-expressers from aggregations. mote confl ict: Pseudomonas aeruginosa strains that Such genes are likely to have promoted kinship in the mutate rapidly (Oliver et al. 2000 ) produce more early evolution of D. discoideum aggregation (Queller cheaters (Harrison & Buckling 2005 ). A high mutation et al. 2003 ), and remain important today in S. cerevisiae, rate then can reduce genetic relatedness in a group where only some strains express its key adhesion gene and lead to the emergence of cheaters and confl ict. in natural populations (Smukalla et al. 2 0 0 8) . Q u o r u m Moreover, the starting levels of genetic relatedness sensing can achieve similar eff ects. As we have seen, in a microbial group can interact with this process many benefi cial secretions are only secreted when cells and aff ect the evolution of mutation rates. Growing have grown to reach high density, which in biofi lms strains of P. aeruginosa in isolation (high related- may make a good proxy for being surrounded by rela- ness) favours a low mutation rate, because strains are tives (Xavier & Foster 2007 ). And B. subtilis has taken less likely to produce cheater mutants that harm the this one step further by developing strain-specifi c quo- group (Harrison & Buckling 2007 ). By contrast, grow- rum sensing (Tortosa et al . 2001 , Ansaldi et al. 2 0 0 2) . ing mixtures of strains (low relatedness) can, at least temporarily, favour a higher mutation rate, as now the rise of cheater mutants harms not only the source M u t a t i o n strain but also its competitor. A bacterial group with a One fundamental diff erence between microbes and low relatedness among cells is prone to processes that more familiar social organisms such as insects, birds drive relatedness even lower. and mammals is probably the relative importance Mutation in key genes then can simultaneously of mutation within groups (West et al. 2 0 0 6) . Th is is reduce relatedness and produce cheaters that exploit exemplifi ed by the evolution of the bacterium P. aeru- others, even though cells may be genetically identical ginosa in the cystic fi brosis lung (Smith et al. 2 0 0 6) . at every other locus. Th is brings us to an important Over the years, strains arise that lose function in many point: when using relatedness to make evolutionary genes important for normal social life, including predictions about cooperation, one must focus on the 348 Kevin R. Foster

locus or loci that cause the social action ( Chapter 6 ). taking both known and unknown species and sim- Th is is less of an issue in animal societies because sex- ply documenting their social behaviours. Here I ual recombination means that, on average, related- highlight three key questions that can be asked of ness is identical across the whole genome: any locus a new microbial group, with the important caveat is a proxy for the loci that really matter. But in a mutat- that any detailed study will always require that one ing asexual microbe, the relatedness among cells can can fi rst get cells to grow in the laboratory setting. diff er sharply at diff erent loci, which suggests that the Th ese questions are borrowed from a recent review loci may also diff er systematically in their evolution- on biofi lms (Nadell et al. 2 0 0 9) . ary interests, creating within-genome confl icts (Burt Function: what are the constituent genes and & Trivers 2006 , Helantera & Bargum 2007 ). phenotypes that drive microbial social traits? A candidate worthy of a little speculation in this Identifying the genes that underlie social traits regard is the reliable production of multiple growth allows one to dissect complex group traits – like phenotypes by P. aeruginosa within biofi lms ( Fig. 13.4 ; biofi lms – into their constituent behaviours. Th is Boles et al. 2 0 0 4) . Th is appears to be genetically deter- enables the systematic study of the costs and ben- mined, and diversifi cation makes biofi lms tougher, efi ts of a given behaviour (below) and an assess- suggesting that the majority of the loci in the genome, ment of the evolutionary forces acting upon traits which do not alter, will benefi t. However, if there are by comparing DNA sequence variation within loci that must mutate to cause diversifi cation, there and between species (Smith et al. 2 0 0 5) . is the potential for confl ict among the resulting alle- Cooperation: what are the costs and benefi ts of les, and also between these alleles and the rest of the microbial social behaviours? In order to under- genome. Examples such as this suggest that microbes stand the evolution of microbial behaviours, we may simultaneously indulge in the benefi ts of high need to identify which cells are aff ected by each relatedness at some loci (cooperation), while at the behaviour. In particular, following the logic of same time benefi ting from variability at others (social Chapter 6, the two key questions are (1) what heterosis: Nonacs & Kapheim 2007 ). are the costs and benefi ts of a behaviour to the cell that expresses it, and (2) what are the costs and benefi ts of a behaviour to cells other than 13.4 Conclusions and future directions the individual that expresses it? A key experi- ment in this regard is to mix mutants that do Recent years have seen the development of a host not express a social trait with cells that do, in of so-called ‘culture-independent’ technologies for order to evaluate the potential for cheating (e.g. assessing microbial diversity. Th ese detect a species Gilbert et al . 2007 ). Only by asking such ques- directly from its RNA or DNA and remove the require- tions of a whole range of microbial behaviours ment that – in order to be detected – a species must will we get an idea of how many involve true grow in the laboratory setting. Th e new technologies altruism, whereby one cell helps another at a fi t- have led to the realisation that many species were ness cost to itself ( Table 13.1 ). missed by traditional techniques and that natural Ecology: which strains and species are in natural microbial communities frequently contain hundreds microbial groups, and how are they arranged? of species living in close proximity. In many ways, We need a much better knowledge of microbial therefore, the study of microbial behaviour and social ecology in order to understand how costs and interaction are only now beginning. benefi ts measured in the laboratory translate Th e newly recognised species diversity in micro- into real fi tness eff ects in the wild. An important bial communities hints at an equally broad range start, which is well under way, is to identify the of behavioural diversity that remains to be uncov- species and strains within natural communities. ered. Th ere is therefore considerable scope for But to answer questions of social evolution one Social behaviour in microorganisms 349

must go further still and assess the microscopic Th e simplest beings habitually multiply by spontaneous fi s- distribution of the diff erent genotypes. While sion. Physical altruism of the lowest kind, diff erentiating from challenging, such fi ne-scale assessments in nat- physical egoism, may in this case be considered as not yet inde- ural groups will provide estimates of genetic pendent of it. For since the two halves which before fi ssion con- stituted the individual, do not on dividing disappear, we must relatedness and, importantly, the spatial scale say that though the individuality of the parent infusorium or at which one can expect cells to act as a unifi ed other protozoon is lost in ceasing to be single, yet the old indi- group of common interest ( Fig. 13.2 ). vidual continues to exist in each of the new individuals. As you next stroll through a sun-dappled wood- Herbert Spencer, Th e Data of Ethics (1879 ) land, consider the humble microorganism. For they thrive all around us, be it on the surface of a water droplet, in the depths of the soil, or nestled inside the Acknowledgements tiny pores of a leaf. Wherever they live, they are often packed together with many other species and strains. Many thanks to Anne Foster, John Koschwanez, Here social interaction is rife: cells jostle for position, Katharina Ribbeck, Th omas Dixon, Carey Nadell, grab, secrete, poison and even shape-shift as the need Joao Xavier, Allan Drummond, Wook Kim, Beverly arises. Only now is the true extent of the microbe’s Neugeboren, Freya Harrison and two anonymous social sophistication becoming clear, and with it referees for comments and discussions. Th ank you comes a rich opportunity to unravel the evolutionary also to Helmut König, Greg Velicer, Kevin Versterpen, paths that brought them there. But she who studies Judith Korb, Hera Vlamakis, Phil Stewart, Tim Tolker- their societies faces a bewildering complexity, not Nielsen, Mike Brockhurst and Andrew Spiers for only in terms of the diversity of those that interact, but images. KRF is supported by National Institute of also in the potential for rapid evolutionary responses General Medical Sciences Center of Excellence Grant when they do. Th ere is some solace, however, in the 5P50 GM 068763–01. knowledge that familiar principles of sociobiology are emerging from these microbial melees. Kinship, Suggested readings coercion and constraints are all important, and all- important. And in the end we know that each cell has C r e s p i, B . J . (2 0 0 1) Th e evolution of social behavior in micro- its own interests at heart, and will act to safeguard organisms. Trends in Ecology and Evolution, 16, 178 –183. the reproductive interest of itself and its clone-mates. K e l l e r, L . & S u r e t t e, M . G . (2 0 0 6) C o m m u n i c a t i o n i n b a c - Th ere can be no doubt that microbial communities teria: an ecological and evolutionary perspective . Nature Reviews Microbiology, 4, 249 –258. are shaped by the individual struggles that face all N a d e l l, C . D ., X a v i e r, J . & F o s t e r, K . R . (2 0 0 9) Th e sociobiology organisms, but there is room for some romanticism of biofi lms . FEMS Microbiology Reviews, 33, 206 –224. too. For with any struggle comes the benefi t of alli- W e s t, S . A ., G r i ffi n, A . S ., G a r d n e r, A . & D i g g l e, S . P. (2 0 0 6) ances, familial or otherwise, that invest in a shared Social evolution theory for microorganisms. Nature common good. Some microbes will even die for the Reviews Microbiology, 4, 597 –607. cause, and rupturing to secrete a toxin can be sim- ultaneously altruistic to those that are immune and References spiteful to those that are not. In applying terms like altruism to microbes, however, we are met with A l l e s e n - H o l m, M ., B a r k e n, K . B ., Y a n g, L . et al. (2006 ) A characterization of DNA release in Pseudomonas aerugi- something of a paradox. As Spencer realised long nosa cultures and biofi lms . Molecular Microbiology, 59, ago, the individual microorganism seems destined 1114 –1128. to be both selfi sh and altruistic because, in its eager- A n s a l d i, M ., M a r o l t, D ., S t e b e, T ., M a n d i c - M u l e c, I . & ness to divide, it faces the ultimate metaphysical sac- D u b n a u, D . (2 0 0 2) S p e c i fi c activation of the Bacillus quo- rifi ce: a loss of self. rum-sensing systems by isoprenylated pheromone vari- ants. Molecular Microbiology, 44, 1 5 6 1– 1 5 7 3 . 350 Kevin R. Foster

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