D A V I D T. M U L D E R & B R I A N K . C O O M B E S

Bacterial pathogens promote their sur-vival by deploying virulence factors that modify the host environment.

But these virulence proteins are energetically costly to produce, and so avirulent defec-tor mutants that grow more quickly than the wild-type population can compromise the success of an infection. On page 353, Diard et al.1 report a remarkable strategy used by the gastro intestinal pathogen Salmonella enterica serovar Typhimurium (hereafter termed Salmonella typhimurium) to evade this threat, while at the same time safeguarding its geno-type — it produces a subpopulation of equally fast-growing bacteria that are rendered pheno-typically avirulent through regulation of gene expression rather than mutation.

When studying host–pathogen interactions, a common misconception is that the bacterial population that initiates the infection remains

as a genetically identical collective of cells that retains its initial characteristics, or phenotype. In fact, most in vivo data on virulence-factor function and bacterial fitness are inferred from population-level studies, which do not consider the fate of individual bacteria. However, stud-ies of antibiotic-resistant persister cells and the social evolution of cooperative traits in bacteria2 have reinforced the idea that regulation of gene expression is a significant source of phenotypic variation within a population. For example, in a process called bistability, a single bacterial popu lation can bifurcate into two subpopula-tions that are genetically identical, but pheno-typically different. Bistability is epigenetic in the sense that the subpopulations arise without hereditary changes to the DNA sequence, but instead through changes to gene expression. The concept is not new3,4, but the evolutionary significance of bistability for pathogenic bac-teria during infection of a host has remained unclear, until now.

A well-known example of bistability is the expression of virulence factors by S. typhi-murium. These bacteria deploy an extensive suite of virulence proteins that generates an inflammatory response in the infected host and allows the bacteria to invade the epithelial cells that line the gut (Fig. 1). The inflamma-tion also kills off some of the non-pathogenic commensal bacteria that normally reside in the gut, creating an expanded niche in which S. typhimurium can grow5. Virulence factors are known to be expressed in a bistable way by the S. typhimurium population6, which gener-ates subpopulations of slow-growing virulent cells that perform ‘public good’ functions that benefit the whole population, and fast-growing cells that do not express virulence factors but are beneficiaries of this public good7. This provides a fitness advantage to avirulent, non-cooperating bacteria, but it also raises the question of whether the infecting popu-lation is vulnerable to invasion by avirulent mutant defectors. Although the susceptibil-ity that results from this social structure has already been firmly established8,9, Diard and colleagues have provided greater clarity by analysing the genetics of the within-host evo-lution that takes place during infection.

To better understand the altruistic coopera-tive behaviour of the virulent subpopulation, the authors modelled the effect of various infec-tion ratios of cooperating bacteria and mutant cheats, and tested these scenarios in vivo in

but this form of cultivation is not possible for most abundant bacteria in nature. So, no host, no virus. Even when a method of culturing P. ubique was developed, finding viruses that might infect it was not straightforward because this SAR11 representative grows only in liquid media. In addition, the sequencing approaches that are used to identify bacteria do not work for viruses because they lack a name-tag gene such as the 16S rRNA gene.

To solve this identification problem, Zhao et  al. sequenced four pelagiphages and com-pared their genomes with those of known and unidentified viruses. The sequence data, as well as the morphology of the viruses, indicated that pelagiphages belong to the same families as the viruses that attack cyano bacteria. The authors then estimated the relative abun-dance of SAR11 viruses by combining the pelagi phage genomic data with viral meta-genomic data from several oceans, including the Pacific Ocean. (Metagenomic data are taken directly from all targeted organisms or viruses in a sample without prior culti-vation.) The analyses suggest that SAR11 viruses are more numerous than viruses known to attack marine bacteria from three other abundant groups: Roseo bacter, Pro-chlorococcus and Syn echococcus. Zhao et al. successfully defend their pelagiphage num-bers by considering methodological problems,

including ‘greedy’ recruitment. One unsolved issue is that the authors’ calculations depend on data from viruses that infect just a few bacterial types — a puny number compared with the huge diversity of marine microbial communities. Still, they have identified a good chunk of the even larger genomic diversity carried by marine viruses.

Zhao et al. conclude by considering what implications their data have for the popular ‘kill-the-winner’ hypothesis — which asserts that as a bacterial type increases in abundance, it should attract more viruses, preventing it from becoming dominant6. The discovery of pelagiphages suggests this is not the case for SAR11, but the existence of SAR11 viruses should not be a surprise. SAR11 could still be a defence specialist, albeit an imperfect one. Indeed, Zhao et al. discuss signs in the P. ubique genome of defences against viruses. The ‘defence specialist’ label may fit if SAR11 grows as slowly in the oceans as P. ubique does in the lab. However, although some studies suggest slower than average growth rates for SAR11 (ref. 7), others have found faster rates8. The latter seems consistent with the superior competitive traits of SAR11 suggested by P. ubique strains and their genomes.

Determining whether SAR11 is a defence specialist or a superior competitor is impor-tant. The answer would help to explain

SAR11’s population dynamics and be a notable start in determining the bacteria’s significance in the carbon cycle and other biogeochemical processes. A slow-growing defence specialist may be on the sidelines of the biogeochemi-cal action carried out by faster-growing com-petitors. Zhao et al. touch on these issues, but more work is needed. Nevertheless, by uniting two stories that started more than 20 years ago, this report is a notable chapter in understanding the most abundant viruses and bacteria in the oceans. ■

David L. Kirchman is in the School of Marine Science and Policy, University of Delaware, Lewes, Delaware 19958, USA.e-mail: kirchman@udel.edu

1. Bergh, Ø., Børsheim, K. Y., Bratbak, G. & Heldal, M. Nature 340, 467–468 (1989).

2. Giovannoni, S. J., Britschgi, T. B., Moyer, C. L. & Field, K. G. Nature 345, 60–63 (1990).

3. Zhao, Y. et al. Nature 494, 357–360 (2013).4. Bahr, M., Hobbie, J. E. & Sogin, M. L. Aquat. Microb.

Ecol. 11, 271–277 (1996).5. Rappé, M. S., Connon, S. A., Vergin, K. L. &

Giovannoni, S. J. Nature 418, 630–633 (2002).6. Thingstad, T. F. & Lignell, R. Aquat. Microb. Ecol. 13,

19–27 (1997).7. Campbell, B. J., Yu, L., Heidelberg, J. F. &

Kirchman, D. L. Proc. Natl Acad. Sci. USA 108, 12776–12781 (2011).

8. Malmstrom, R. R., Cottrell, M. T., Elifantz, H. & Kirchman, D. L. Appl. Environ. Microbiol. 71, 2979–2986 (2005).

I N F E C T I O N B I O L O G Y

Cheats never prosperFast-growing ‘defector mutants’ can threaten the success of a bacterial infection. But one bacterial species prevails over these cheats by forming a subpopulation that has shut down expression of virulence genes. See Letter p.353

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Commensalbacterium

VirulentS. typhimuriumAvirulent

S. typhimurium

Lum

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Mucus layer

Epith

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Day 0 Day 2 Day 10 Day 0 Day 2 Day 10

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mice. They predicted that avirulent mutant bacteria would be unable to establish an infec-tion on their own, but that if avirulent mutants arose spontaneously from a population of wild-type bacteria that had already conditioned the host environment, then the mutants would prosper. This was indeed the case. In mice infected with wild-type bacteria, the authors found a small population of defector bacteria by day 2. This population rapidly expanded to dominate the infection (Fig. 1a). Interestingly, all the mutants had a mutation in hilD, the ‘master regulator’ gene for one particular viru-lence system in S. typhimurium10. Re inforcing their predictions, this mutant type was in capable of establishing infection on its own, but it rapidly dominated during co-infections with wild-type bacteria.

Interestingly, infections that were initiated by a mixture of wild-type and hilD mutants were short-lived and were cleared more quickly by the host, demonstrating that mutant defectors undermine the ultimate survival of the popula-tion. This is in agreement with a previous obser-vation2 that high relatedness in a population is essential for evolutionary stability of coopera-tive behaviour. Thus, the fitness advantage of accruing mutations that lead to fast-growing avirulent cells is due to social exploitation because this advantage is only apparent in the presence of wild-type cells that can elicit the beneficial inflammatory response from the host.

So how is it that cooperative virulence is maintained when the spontaneous generation of mutant defectors during infection is favoured by selection? Diard and colleagues predicted that a bistable population of genetically identical bacteria that differ in virulence-factor expres-sion might hold a clue. When they modelled a bistable population, the researchers found that

maintaining a high proportion of fast-growing phenotypically avirulent cells kept the rise of mutant defectors in check, whereas lowering the proportion of phenotypically avirulent cells accelerated the emergence of such mutants. To validate this model in vivo, the authors mani-pulated the bistable population distribution by disrupting the protein HilE, a negative regulator of HilD (ref. 11). In these experiments, lowering the proportion of phenotypically avirulent bac-teria did make the population more susceptible to mutant defectors, and caused early cessation of inflammation and loss of S. typhimurium from the gut (Fig. 1b).

The finding that genetically identical yet phenotypically distinct subpopulations control the rise of avirulent cheats provides fascinating insight into the evolution of virulence. How-ever, questions remain. For any pathogen, the key to evolutionary success lies in its ability to transmit from host to host12, but it is unclear from the current work whether S. typhi-murium transmission is actually compromised in the presence of defectors. If it is, this would strengthen the argument13 that cooperative virulence is a selective trait. Also unknown is whether the outgrowth of fast-growing phe-notypically avirulent cells occurs in response to environmental cues (or even the mutant defectors themselves) or if it is a truly stochas-tic process. If the outgrowth is programmable, then how an optimal balance of virulent and avirulent populations is achieved and maintained becomes an interesting question.

This work provides a rare in vivo analysis of cooperative traits in pathogens and advances the field of social evolution theory for patho-genic microorganisms. The authors’ descrip-tion of S. typhimurium’s elegant mechanism to manipulate phenotypic distribution by

bi stability should allow for deeper investiga-tion of other mixed populations, such as the presence of non-replicating dormant Salmonella in chronic infections. ■

David T. Mulder and Brian K. Coombes are at the Michael G. DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4K1, Canada.e-mail: coombes@mcmaster.ca

1. Diard, M. et al. Nature 494, 353–356 (2013). 2. West, S. A., Griffin, A. S., Gardner, A. & Diggle, S. P.

Nature Rev. Microbiol. 4, 597–607 (2006).3. Dubnau, D. & Losick, R. Mol. Microbiol. 61, 564–572

(2006).4. Veening, J. W., Smits, W. K. & Kuipers, O. P. Annu.

Rev. Microbiol. 62, 193–210 (2008). 5. Stecher, B. et al. PLoS Biol. 5, 2177–2189 (2007).6. Ackermann, M. et al. Nature 454, 987–990 (2008).7. Sturm, A. et al. PLoS Pathog. 7, e1002143 (2011).8. Harrison, F., Browning, L. E., Vos, M. & Buckling, A.

BMC Biol. 4, 21 (2006).9. Rumbaugh, K. P. et al. Curr. Biol. 19, 341–345

(2009).10. Ellermeier, C. D., Ellermeier, J. R. & Slauch J. M.

Mol. Microbiol. 57, 691–705 (2005).11. Baxter, M. A., Fahlen, T. F., Wilson, R. L. & Jones, B. D.

Infect. Immun. 71, 1295–1305 (2003).12. Wickham, M. E., Brown, N. F., Boyle, E. C., Coombes,

B. K. & Finlay, B. B. Curr. Biol. 17, 783–788 (2007).13. Raymond, B., West, S. A., Griffen, A. S. & Bonsall, M. B.

Science 337, 85–88 (2012).

Figure 1 | Bistability prevents the rise of mutant defectors. Diard et al.1 studied mice infected with Salmonella enterica serovar Typhimurium (Salmonella typhimurium). a, The bacteria first enter the lumen of the gut, which also contains numerous non-pathogenic commensal bacterial species. However, expression of virulence proteins by S. typhimurium causes inflammation of gut tissues, killing off many of the commensal bacteria. By the second day of an S. typhimurium infection, hilD-mutant bacteria arise that no longer express some virulence proteins; this allows them to grow faster than the wild-type

bacteria. But the authors show that the infecting population is ‘bistable’ — approximately 60% of the bacteria are phenotypically avirulent through downregulation of virulence-gene expression, and these grow just as fast as the mutants. b, When Diard and colleagues manipulated the bacteria so that the infecting population had a lower proportion of phenotypically avirulent cells, they found that the wild-type bacteria were less effective at keeping the mutant bacteria in check. The relative lack of virulent bacteria in this infection led to the inflammation subsiding faster than in the infection shown in a.

CORRECTIONIn the News & Views article ‘Astrophysics: Going supernova’ by Alexander Heger (Nature 494, 46–47; 2013), supernova SN 1987A was incorrectly stated as having occurred in the Small Magellanic Cloud galaxy. The correct galaxy is the Large Magellanic Cloud.

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