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A new social gene in Dictyostelium discoideum, chtB Santorelli et al. Santorellietal.BMCEvolutionaryBiology2013,13:4 http://www.biomedcentral.com/1471-2148/13/4 Santorellietal.BMCEvolutionaryBiology2013,13:4 http://www.biomedcentral.com/1471-2148/13/4 RESEARCH ARTICLE Open Access A new social gene in Dictyostelium discoideum, chtB Lorenzo A Santorelli1,2,5*, Adam Kuspa1,2,3, Gad Shaulsky1,2, David C Queller4 and Joan E Strassmann4 Abstract Background: Competitive social interactionsare ubiquitous innature, but their geneticbasis is difficult to determine. Much can be learnedfrom single gene knockouts ina eukaryote microbe. The mutants can be competed with theparent to discern the social impact of that specific gene. Dictyostelium discoideum is a social amoeba that exhibits cooperative behavior inthe construction ofa multicellular fruiting body. It is a good model organism to study thegenetic basis of cooperation since it has a sequenced genomeand it is amenable to genetic manipulation. When two strains of D.discoideum are mixed, a cheater strain can exploit its social partner by differentiating more spore than its fair share relative to stalk cells. Cheater strains can be generated in thelab or found inthe wild and genetic analyseshave shown thatcheating behavior can be achieved through many pathways. Results: We have characterizedthe knockout mutant chtB,which was isolated from a screen for cheater mutants that were also able to form normal fruiting bodies ontheirown. When mixed inequal proportionswith parental strain cells, chtBmutants contributed almost 60% ofthetotal numberof spores. To do so, chtB cellsinhibitwild typecells from becoming spores, as indicated by counts and by thewild typecells’reduced expression of the prespore gene, cotB. We found noobvious fitness costs (morphology, doublingtime in liquid medium, spore production, and germination efficiency)associated withthecheating ability ofthechtB knockout. Conclusions: In this study we describe a new gene inD. discoideum,chtB,which when knocked out inhibits the parental strain from producing spores. Moreover, under lab conditions, wedid not detect any fitness costs associated with this behavior. Keywords: Cheating behavior, Social evolution, D.discoideum,Pre-spore marker, chtB Background culture for a number of generations sometime evolve to Microorganisms are able to communicate and cooperate lose the ability to cooperate and instead behave as to perform complex social behaviors once believed to be cheaters. distinctiveofmulticellularorganisms[1-5].Thisincludes Dictyostelium discoideum displays cooperative behavior formation of biofilms, foraging, spore dispersal and pro- that provides a great model for the study of cheating. D. duction of common goods. Cooperative groups are vul- discoideum propagates as free-living unicellular amoebae nerable to exploitation by cheaters, individuals that feeding on bacteria associated with leaf litter, soil, and benefitfromtheproductofcooperationwithoutcontrib- animal dung. In the past few decades, this social amoeba uting their fair share [5-9]. In Pseudomonas aeruginosa has been intensively studied because of its extraordinary [10] and Myxococcus xanthus [11,12], for example, the re- development [13], which is a form of cooperation. When lease of siderophores and digestive enzymes, respectively, starving, cells aggregate and eventually form a multicellu- represent costly activities that could be exploited by non- lar organism capableofmovementtowards lightand heat producers. In both organisms, strains grown in liquid andawayfromchemicalssuchasammonia[14,15].Under the correct conditions, they develop into a fruiting body thatrepresentsthefinalstageofdevelopment[16].About *Correspondence:[email protected] 20%ofthecellsdifferentiateintodeadstalkcellsthatsup- 1DepartmentofEcologyandEvolutionaryBiology,RiceUniversity,Houston, port the other 80%, which differentiate into viable spores. TX77005,USA 2DepartmentofMolecularandHumanGenetics,BaylorCollegeofMedicine, When spores are dispersed to a new source of food, they Houston,TX77030,USA germinate to become amoebae and restart the vegetative Fulllistofauthorinformationisavailableattheendofthearticle ©2013Santorellietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Santorellietal.BMCEvolutionaryBiology2013,13:4 Page2of9 http://www.biomedcentral.com/1471-2148/13/4 stage of eating and dividing by binary fission. Stalk cells laboratory. When mixed with chtB, the parental strain have been described as altruistic, since their death is pre- produces fewer spores and expresses a lower level of the sumedtoaidthedispersalofthesporesthatformthenext pre-sporegenecotB. generation[17].Ifthepopulationconsistsofcellsthatare genetically identical, or related, it is possible to compare Results theseindividuals tosomatic cellsin a multicellularorgan- AmutantcalledchtB(cheaterB)wasrecoveredattheend ism,ortoasocialinsectcolony.Butwhencellsaregenet- of a selection for mutants that preferentially produce ically different, a conflict may arise over which cells sporesratherthanstalksinamixedpopulation[21]. survive and which form the stalk and die. In this case, a In the parental strain AX4, the chtB transcript appears strain that differentiates more than its proportional share early in development and is completely absent in chtB ofsporesiscalledacheaterandtheotheriscalledaloser. mutant cells. This mutant produces a higher number of In several cases, reproductive competition among cells spores than AX4 in chimeras that are made with equal within a chimera has been reported, but the cheater numbers of cells of the two strains. When it is plated strains often carry a fitness cost relative to non-cheaters clonally it shows a normal developmental phenotype, so even to the extent that they are incapable of sporulating it is not dependent on parental cells in a social stage efficiently on their own [18-20]. On the other hand, in chimera. The loss of function of the chtB gene also D. discoideum several strains isolated from the wild are increases expression of cotB, a prespore marker, early in capable of cheating and of producing fruiting bodies in- development,indicatingthemechanismofactionisearly dependent of other clones [17]. A large number of specialization as spore over the ancestral strain. On the knockout or reduced function mutations generated in other hand, parental cells reduce the expression of cotB thelaboratoryalsoshowsuchfacultative cheatingbehav- and differentiate a lower number of spores in chimera. ior [21]. These mutants are capable of producing spores We tested chtB mutants to detect whether fitness costs in pure populations, but preferentially become spores were associated with itscheating ability.Not only didwe andnot stalkcells when mixedwith theancestralstrain. not detect any fitness cost, but we also found that the In D. discoideum, many genes have been reported to mutant presents the same sporulation efficiency and fas- be involved in the disruption of cooperation, but not ter doubling time when grown in liquid, than AX4. much is known about the underlying genetic mechan- Finally, the presence of chtB mutant cells in chimera isms. The analyses performed so far on cheater mutants with parental strain limits the expression of a pre-spore showed that multiple mechanisms and pathways may be marker in the latter. As a consequence the parental involved in cheating behavior, with cheater mutants strain isunabletoproduce itsfairshare ofspores. showing diverse functions including GTPase regulatory activity, polyketide synthesis, nucleotide binding, and IsolationofthechtBmutants phosphoric ester hydrolase activity [21]. Some of these Weisolatedthe chtBmutantduringa selectionforchea- pathways are involved in cell-cell communication, for- ters from a pool of 10,000 mutants [21]. This pool of mation of secondary metabolites, or signal transduction. mutants was subjected to 20 rounds of growth, develop- Theycanaffectthecommunicationpathwaythatregulates ment, and spore germination in a mixed population so theproportionofsporesandstalkcells[22],ordisruptthe that cheaters that differentiate into spores with a higher ubiquitin-ligasepathwayresponsibleforthebreakdownof efficiency would become enriched in the evolving popu- certain target proteins [19]. Other mechanisms that can lation. At the end of the selection, chtB was one of the lead to cheating [23] might be the expression of spore mutants that were randomly chosen to be tested for genesearlierthanprestalkgenes[24];prematureentrance cheating properties. To confirm that chtB really into development; presence of multinucleated cells that increased its frequency during the selection, we used lead to more cell divisions during development; cannibal- quantitative PCR (Q-PCR) to obtain information about ism, as is present in Dictyostelium caveatum [25]; and the abundance of the chtB allele. We extracted genomic production of a killer factor as reported in Polysphondy- DNA from the entire population after the 1st, 10th, and liumpallidum[26]. 20th generations of the selection and used gene-specific Cheater strains in the Dictyostelidae typically have a primers to quantify the chtB allele. The mutant chtB fitness cost or some deleterious pleiotropic effect that increased 7.4 fold at the 10th generation and 26.4 fold at prevents the mutant from spreading, since otherwise a the 20th (Figure 1), thus supporting the hypothesis that loss of function mutant would easily evolve. This is the thischeaterincreasedinfrequencyduringtheselection. case of mutants chtA, dimA, and csaA [19,27,28]. In this work we characterize a mutant called chtB that can fac- ThechtBgene ultatively cheat when mixed with the parental strain, but The mutant chtB was generated by insertional mutation does not suffer from an obvious fitness cost in the of the pBSR1 plasmid [29] in chromosome 5 at position Santorellietal.BMCEvolutionaryBiology2013,13:4 Page3of9 http://www.biomedcentral.com/1471-2148/13/4 Analysisoffitnesscostassociatedwithcheating chtBhasnoovertmorphologicaldefects To test whether chtB shows any morphological difference relative to its ancestor, we observed cells of both strains at different stages of development. In both parent and knockout, we observed loose aggregates at about 10 hours, tight aggregates at 12 hours, fingers at 16 hours and Mexican hats at 20 hours. Between 20 and 24 hours both strains culminated, leading to the formation of well-proportioned fruiting bodies consisting of stalks cells and spores (Figure 5). We saw no noticeable differences between the phenotypes of AX4 and chtB developed onfilters. Figure1ThechtBmutantincreasesinfrequencyduringthe selection.Q-PCRusingDNAfromtheentirepopulationatthe1st, 10thand20thgeneration(R1,R10andR20)andusingprimers chtBdifferentiatesasimilarnumberofsporesthanAX4ina specifictothechtBallele,showsthatthemutantchtBincreased26.4 purepopulation foldbytheendoftheselection. Afitnesscostforacheatercouldbemanifestedasreduced sporulationwhendevelopinginapurepopulation.Totest 4377789 towards the 3’ end of the ORF of the gene that possibility we developed chtB cells in a pure popula- DDB_G0290617 (Figure 2). The predicted protein has tion or mixed with AX4 cells. The results (see Additional not been studied before and it consists of a FNIP repeat file 1: Figure S1) show that the sporulation efficiency of (named FNIP after the pattern of a conserved motif chtB (83.11±4.69%, n=10) is not significantly different found only in D. discoideum). We named the gene chtB from AX4 (71.53±5.82%, n=10, t-test p<0.14), suggesting because its social behavior resembles the previously that the mutation does not have a sporulation-related described chtA mutant [19]. To verify that the insertion fitness cost. The chimera sporulation efficiency was simi- was responsible for the mutant phenotype, the mutation lar to both pure populations (84.16±7.26%, n=10, t-test was recapitulated by homologous recombination in the p=0.19vs.AX4,p=0.90vs.chtB). AX4 genetic background using the rescued plasmid as a knockout vector [30]. We confirmed the mutation by chtBsporesareviable Southern blot hybridization using a gene-specific probe We also tested whether a fitness cost may be associated andtested thestrainfor cheating. with the spore germination efficiency. Our results show RT-PCR analysis showed that the parental strain AX4 that chtB was able to germinate a number of spores expressed the chtB mRNA at 0 hours of development (72.3±16.2%) comparable to AX4 (75.5±15.1%, t-test, and mRNA abundance was greatly increased at 4–24 p=0.08, n=6). This result indicates that chtB does not hours (Figure 3). In the mutant cells, chtB mRNA was produce fewer viable spores than its parent (Additional not expressed at anytime. file2:FigureS2). chtBgrowsfasterinliquidmedium ThechtBmutantcheatsontheparentalstrain To compare the growth rates of the chtB mutant and its To be defined as a cheater, the mutant chtB must pro- ancestor, we grew cultures of chtB and AX4 in liquid duce significantly more than 50% of the spores in a pair- media starting at a cell density of 1x105 cells/ml. chtB wise mixing experiment with the parental strain, and reached log phase after about 40 hours, while AX4 took thiswasthe case.Whenmixedatequalproportionswith about 10 hours longer. When in logarithmic phase, chtB AX4, chtB differentiated 59.9±3.3% of the total number cells showed a doubling time of 7.6±0.7 hours (Figure 6) of spores. This result differed significantly from the con- while AX4 cells showed a doubling time of 10.3±1.5 trol chimera between AX4 and AX4 [act15/GFP] hours (t-test, p<0.05, n=3), showing that chtB has a fas- (p<.0001,T-test; Figure4). terdoublingtime than AX4inliquid medium. Figure2ThechtBgeneislocatedonthechromosome5.Thegenecontainsoneshortintron(thinblackline)andtwoexons(thickredlines) asindicatedinthegenemodel.TheredtriangleindicatesthepositionoftheinsertionalmutationgeneratedbyREMI. Santorellietal.BMCEvolutionaryBiology2013,13:4 Page4of9 http://www.biomedcentral.com/1471-2148/13/4 Figure3ThechtBgeneisexpressedduringdevelopmentandiscompletelysilentinourmutant.RT-PCRreactionusingRNAextracted duringatimecourse(timepointsindicatedinhours)showsthatthechtBmRNAisobservedonlyinAX4duringdevelopment.NomRNAis detectedinthemutantchtB. Alterationofcelltypeproportioningascheating alone. When AX4 [cotB/lacZ] cells were mixed at the mechanism sameratiowithchtBcells,theβ-galactosidaseactivitywas We assessed cell-type proportioning by measuring beta- significantly lower than in the mix with AX4 (16, 20 and galactosidase activity in cells that express the marker 24 hour time points, t-test, p<0.05, n=3). These results under the promoters of either cotB [31] (a prespore mar- show that in chimeras chtB is able to reduce the expres- ker) or ecmA [32] (a prestalk marker). We measured the sion of cotB in the wild type cells and that AX4 cells are overallenzymeactivityinthepopulationusinganONPG- forcedtoformfewerprespore-cellsthantheirfairshare. assay and the number of cells of each type using X-gal stainingas described [33].We compared the level of lacZ Prestalkdifferentiation expression in chimerae between the reporter strains and To test whether the presence of chtB cells in chimeras AX4 or chtB to determine the effect of chtB on prespore affects prestalk cell formation in the victim, we per- andprestalkdifferentiationintheAX4victim. formed a similar analysis using the strain AX4[ecmA/ lacZ] alone and in pairwise mixes with AX4 and chtB Presporedifferentiation (Figure 8). In this case, no significant differences were ONPG analysis of AX4 [cotB/lacZ] showed that cotB is seen in the β-galactosidase activity when AX4[ecmA/ not expressed until 12 hours. Then it starts increasing lacZ] cells are mixed with AX4 or with chtB. We con- and reaches saturation at 16 hours (Figure 7). If AX4 clude that the presence of chtB cells in chimeras does [cotB/lacZ]cellsweremixedata1:1ratiowithAX4cells not influence the expression of the prestalk marker that do not express lacZ, the level of β-galactosidase ac- ecmAinthewildtype cells. tivity is about half of that produced by AX4 [cotB/lacZ] Developmentalcellfate The results obtained with ONPG analysis show that the presence of chtB cells in a mix with AX4 reduces the promoteractivityofthepre-sporegene cotB.Thisobser- vation could be due to reduced cotB expression in all the wild type prespore cells, or to a reduction in the number of prespore cells in the wild type. We addressed this issue by counting the number of cells that expressed the marker gene. When AX4 [cotB/lacZ] were mixed in equal proportions with chtB (Figure 9A) they produced a significantly lower percentage of stained cells than when mixed with AX4 (for 16 and 20 hour time points, t-test, p<0.05, n=3). Therefore we conclude that, in chimera with wild type, chtB forces the parental cells to reduce the proportion of prespore cells and, as a consequence,toproducefewerspores.Whenthesametestwas Figure4ThechtBmutantcheatsonAX4.Whenmixedwiththe performed using the AX4 [ecmA/lacZ] strain (Figure 9B), parentalstrainAX4[act15/GFP],chtBdifferentiates59.9±3.3%(n=22) ofthetotalspores.ControlAX4cellsmixedwithAX4[act15/GFP] there was no difference between the number of stained cellsdifferentiate50.1±1.0%(n=27)ofthetotalnumberofspores. cells observed in mixes of AX4[ecmA/lacZ] with AX4 or ThechtBstraindifferentiatesasignificantlyhighernumberofspores chtB (for 16, 20 and 24 hours time points, t-test, p>0.26, thantheparentalstrain(Twotailedt-test,p<.0001.Barsrepresent n=3). We conclude that the presence of chtB in chimera standarderrors). did not affect the number of prestalk Acells produced by Santorellietal.BMCEvolutionaryBiology2013,13:4 Page5of9 http://www.biomedcentral.com/1471-2148/13/4 Figure5DevelopmentalmorphologiesofAX4andchtB.Wegrewthecellsinaxenicmediumanddevelopedthemseparately,asindicated, onnitrocellulosefilters.Wephotographedthecellsfromaboveattheindicatedtimes(bar=1mm). the victim strain, butcould havemadethembecome prestalk, but we found no evidence for this; chtB does not stalk B (pstB) cells, which normally produces the basal alter the wild type expression of the prestalk marker disc of the fruiting body, or pstO cells, which occupy the ecmA. It is therefore unclear what happens to the wild rearhalfoftheprestalkregion. type cells thatwouldhaveproduced spores. To testwhetherthechtBmutation wasassociated with Discussion fitness costs, we tested the strain’s growth, developmen- We isolated and characterized a mutant called chtB that tal morphology, sporulation and germination efficiency cheats without overt fitness costs. chtB was isolated after and found no adverse effects compared to the ancestor a genetic selection for cheaters that were able to form AX4.Indeedtherewassomeevidenceof better perform- fruiting bodiesclonally[21]. ance by the mutant. The chtB mutant grows faster than The mechanism by which chtB cheats in chimeras is the wild type in liquid medium. This finding cannot ac- novel. chtB reduced the expression of the prespore mar- count for the increase in frequency of chtB during the ker cotB in the wild type population as a consequence of original selection, because the selection was performed the wild type strain forming fewer prespore cells than it on agar plates, where chtB does not have a growth ad- does in control mixtures. One might expect then that vantage [21]. these missing wild type prespore cells end up making The behavior of the mutant raises both mechanistic and evolutionary questions. On the mechanistic side, we begin by asking whether its higher representation in spores is due to either fixed or facultative allocation strategies [34]. In the fixed allocation strategy, the cheater produces more spores than the loser when each are grown as pure clones. In this case, the cheater has not changed its behavior in chimera; its solitary strategy carriedovertothechimericsettinggivesitanadvantage. In the facultative allocation strategy, the cheater produces more spores in chimeras than expected from its solitary production. The spore production of chtB is not significantly higher than that of AX4, suggesting it is a facultative cheater. However the point estimate is higher, so it is worth considering whether, if that non-significant difference were real, it could explain cheating as a fixed Figure6GrowthcurvesofAX4andchtBcellsinliquidHL5 strategy. In fact it cannot, as the 59.9% representation medium.Celldensitiesofthetwostrains,asindicated,areplotted observed in chimeras is significantly more than the asafunctionoftime(hrs). expected fixed allocation of 54.0% (calculated from chtB Santorellietal.BMCEvolutionaryBiology2013,13:4 Page6of9 http://www.biomedcentral.com/1471-2148/13/4 the entire population [35]. High relatedness is an important control of cheaters, because it means that like groups with like, so cheaters have reduced opportunity to exploit cooperators [36,37]. This explanation does not seem to apply to chtB because it does at least as well as wild type even when alone, so it would appear to be favored even at high relatedness, with an added boost from cheating inany chimeric mixtures. With or without high relatedness, disadvantageous pleiotropic effects can also keep a mutant from spreading [27]. We detected no pleiotropic disadvantages of chtB. However, there are two important caveats. First, all the cheating experiments reported so far were per- Figure7ONPGanalysisofAX4cellsexpressingthelacZgene formed in the laboratory under controlled environments. underthepresporepromotercotB.Cellsweredevelopedaloneor In the wild, however, factors like temperature, pH, and mixedata1:1ratiowithAX4andchtBcells.Developmentaltime moisture could influence both cheating efficiency and points(hours)arerepresentedonthex-axisandbeta-galactosidase activityonthey-axis.WhenAX4[cotB/lacZ]aremixedwithchtB pleiotropic effects. The chtB knockout behaves as a cells,theβ-galactosidaseactivityislowerthanwhenmixedwith cheater under the conditions that we have used, but it AX4(16,20and24hourtimepoints,t-test,p<0.05,n=3). maynotbeabletocheatinnature.Likewise,inactivation of chtB had no apparent adverse consequences in the la- on its own divided by the sum of chtB on its own and boratory, but it must have a cost that would limit its wildtype on its own, or 83.1/(71.5 + 83.1)). Thus chtB propagation in nature, or the gene would have been lost. appears to be a facultative cheater, indicating that an Second, selective forces too small to effectively be additional cheating mechanism is at play besides fixed detected in the laboratory could still be important in na- higher spore production. Several wild clones of Dictyos- ture. One hypothesis that combines these two caveats is telium discoideum [17] and many laboratory mutants that chtB might suffer from reduced dispersal from [21,22]arealsocheatersthatdonotexhibitovertpheno- shorter stalks. If chtB produces more spores, it may typic abnormalities, but the previous studies did not ex- produce shorter stalks. We did not attempt stalk mea- plore possible costs that might be associated with the surements because they are far less accurate and more cheating behavior. Cheater strains in a population can variable than spore measurements. Shorter stalks might be viewed as parasites since they use common resources reduce dispersal, presumably by animal vectors. Another withoutpaying the cost, which could leadto the collapse possibilityisthatthemechanismbywhichchtBknockouts of the entire social machinery. Several mechanisms have achievetheirsuccessinchimerainvolvesearlierfruiting.If been suggested to explain why cheaters do not take over this is the case, it could be that this is normally a disad- vantage,keepingthemutantfromspreading. Conclusion In this study we describe a novel mechanism by which a D. discoideum cheater exploits parental strain cells by inhibiting them from producing spores. One way to understand how cooperative behaviours have evolved and are maintained is to identify the mechanisms cheaters use to exploit such cooperation. Moreover, for the first time we concluded that under our experimental conditions there are no fitness costs associated with the cheater trait investigated, unlike the disadvantageous pleiotropic effects that are suggested mechanisms pre- ventingcheatersfrom spreadinginapopulation. Figure8ONPGanalysisofAX4cellsexpressingthelacZgene Methods undertheprestalkpromoterecmA.Cellsweredevelopedalone ormixedata1:1ratiowithAX4andchtBcells.Developmentaltime Strainsandgrowthconditions points(hours)arerepresentedonthex-axisandbeta-galactosidase The Dictyostelium discoideum strains used were the activityonthey-axis.β-galactosidaseactivitydidnodifferwhenAX4 axenic strain AX4 [38], AX4 [actin15/GFP], and the [ecmA/lacZ]cellsaremixedwithAX4orwithchtB. REMI mutant chtB [21]. Cells were grown in suspension Santorellietal.BMCEvolutionaryBiology2013,13:4 Page7of9 http://www.biomedcentral.com/1471-2148/13/4 Figure9Changesintheproportionofpresporecellsbutnotinprestalkcells.(A)CellsexpressinglacZunderthecotBpromoteror(B) undertheecmApromoterweredevelopedaloneormixedwithAX4orchtBina1:1ratio.Developmentaltimepoints(hours)arerepresentedon thex-axisandpercentageofstainedcellsonthey-axis.AX4[cotB/lacZ]mixedwithchtBatthe16and20hourstimepoint,showsasignificantly lowerpercentageofstainedcellsthanwhenmixedwithAX4(forthe16and20hourstimepoints,t-test,p<0.05,n=3).AX4[ecmA/lacZ]mixed withchtBdoesnotshowasignificantdifferenceinthepercentageofstainedcellsthanwhenmixedwithAX4.(For16,20and24hourtime points,t-test,p>0.26,n=3). in HL-5 medium or on SM agar plates in association Developmentalmorphology with Klebsiella aerogenes [39]. HL-5 was supplemented Wild type and mutant cells were grown in HL5 liquid withtheantibioticsG418orBlasticidinS(bothat5μg/ml), medium. Once in log phase (between 1×106 and 5×106 asrequired. cells/ml), cells were washed twice with KK2 buffer (16.3mM K HPO , 3.7mM KH PO , pH 6.2) and resus- 2 4 2 4 pended in PDF (20mM KCl, 5mM MgCl , 9mM IncreaseinfrequencyofthechtBgeneduringthescreen 2 QuantitativePCRwasperformedusingtheDNAOpticonW K2HPO4, 13mM KH2PO4, 0.3mM streptomycin sulfate, pH 6.4) at a density of 5×107 cells/ml. 1 ml of this cell EnginesystemBIO-RAD[40].Astemplateforthereaction suspension was deposited on Black nitrocellulose filters we used genomic DNA extracted from the 1st, 10th and [38] and incubated at 22°C. Pictures were taken every 20throundofselection.SpecificchtBprimerswereusedto two hours. obtain amplification. IG7 (a constitutively expressed gene) was used as the loading control. SYBR Green was used as Sporulationefficiency thedsDNAfluorescentdye. We plated 5x107 cells on a KK2 agar plate both clonally and in 1:1 ratio. After 30 hours the entire contents of RNAextraction the plates were collected and resuspended in 1 ml of AX4 and chtB cells were plated on two black filters each KK2 with 0.1% NP40 so that only spores could survive. (5x107 cells/filter) and the entire populations were col- To assess spore number, a measured aliquot from each lected at different developmental time points. We sample was counted with a haematocytometer. The extracted RNA using the TRIzol reagent (Invitrogen) sporulation efficiency was calculated by dividing the according to the manufacturer’s recommended proce- number of spores collected at the end of development dures anddissolveditin100μlof1XMOPSbuffer[41]. bythetotalnumberofcells plated initially. Germinationefficiency chtBgeneexpression ™ Spores were counted and plated out at low density Total RNA was reverse transcribed using SuperScript (100–500 per plate) on SM plates with 300 μl of Klebsi- II Reverse Transcriptase kit (Invitrogen) and oligo d(T) ella aerogenes. After a few days, single plaques were primers. Q-PCR was performed on the resulting cDNA observedinthebacteriallawn,eachrepresentingaviable using chtB gene-specific primers. The products were spore. The proportion between the number of plaques resolved by electrophoresis on a 0.8% agarose gel, stained observed and the number of the spores plated indicated withethidiumbromide,andobservedunderUVlight. thegerminationefficiency. Cheatingassays Doublingtime The strain chtB was tested for cheating behavior in pair- Cells were inoculated at a density of 1×105 cells/ml in wise mixing experiments using the GFP-labeled strain 250ml Pyrex flasks containing 50 ml of HL5. The flasks methodasdescribed [21]. wereshakenat200rpmat22°Cuntiltheculturesreached Santorellietal.BMCEvolutionaryBiology2013,13:4 Page8of9 http://www.biomedcentral.com/1471-2148/13/4 stationary phase at a density of about 2.5 × 107cells/ml. collected,treatedwithdetergentandthesporescounted.The Samples were collected at 12 hour intervals and the cells sporulationefficiencyofchtBisnotsignificantlydifferentfromAX4. werecounted.Eachexperimentwasrepeatedthreetimes. Additionalfile2:FigureS2.Germinationefficiencyassay.Sporesof chtBandAX4strainswereplatedonSMplatesinassociationwith ONPG(ortho-Nitrophenyl-β-galactoside)analysisandX-gal bacteriaandthenumberofviablesporeswasinferredfromthenumber ofplaquesformed.chtBproduceapercentageofviablespores staining comparabletoAX4. The strains used for this analysis were TL1 (AX4 [cotB/ lacZ]),TL6 (AX4 [ecmA/lacZ]), the chtB mutant and the Competinginterests parentalstrainAX4.StrainsexpressingthelacZgenewere Theauthorsdeclarethattheyhavenocompetinginterests. grown in the presence of 5μg/ml G418. Cells were grown Authors’contributions in HL5 liquid medium and then plated on black filters as Conceivedanddesignedtheexperimentandadvisedonexperimental describedpreviously.EachlacZstrainwasplatedaloneor procedure,analysisandwriting:LAS,AK,GS,DCQandJES.Performedthe experimentsLAS;analyzedthedataandwrotethepaper:LAS,AK,GS,DCQ, mixed in 1:1 ratio with AX4 and chtB. AX4 [cotB/lacZ] andJES.Allauthorsreadandapprovedthefinalmanuscript. cells were plated in a pure population or mixed at a 1:1 ratiowithAX4andchtBonwhitenitrocellulosefilters. Acknowledgements WethankthemembersoftheKuspa,Queller,ShaulskyandStrassmannlabs forfriendlysupportandmuchadviceduringtheproject. ONPGanalysis The contents of each filter was washed in KK2 and resus- Funding pended in 1ml of Z buffer with 1% Triton in order to ex- ThismaterialispartlybaseduponworksupportedbytheNationalScience tract the protein content. For each sample 5 μl were used FoundationunderGrantNos.EF0626963,andDEB0918931. to determine the protein concentration with a Bradford Authordetails assay [42]. After normalizing the protein concentration of 1DepartmentofEcologyandEvolutionaryBiology,RiceUniversity,Houston, all the samples, 200 μl ofthe protein extract was added to TCXol7le7g0e05o,fUMSeAd.i2cDineep,aHrtomuestnotno,fTXMo7l7e0c3u0la,rUaSnAd.3HDuempaarntmGeennteotifcsB,ioBcahyleomristry 200 μl of Z buffer and 200 μl of ONPG solution (4 mg andMolecularBiology,BaylorCollegeofMedicine,Houston,TX77030,USA. ortho-nitrophenyl-b-D-galactopyranosidein1mlof0.1MZ 4BiologyDepartment,WashingtonUniversityinSt.Louis,St.Louis,MO63130, USA.5Presentaddress:DepartmentofZoology,UniversityofOxford,Oxford buffer). This mixture was incubated at room temperature OX13PS,UK. and gently shaken until the yellow color developed to the desired intensity. The reaction was stopped by adding 400 Received:7June2012Accepted:29November2012 μl of 1M Na CO and the time recorded. The absorbance Published:9January2013 2 3 ofthesolutionwasmeasuredwithaspectrophotometerat References 420nmwavelength.β-galactosidaseactivitywascalculated 1. CrespiBJ:Theevolutionofsocialbehaviorinmicroorganisms.TrendsEcol Evol2001,16:178–183. usingthefollowingformula: 2. VelicerGJ:Socialstrifeinthemicrobialworld.TrendsMicrobiol2003, 11:330–337. Specif(cid:1)icβ(cid:1)gal:activity(cid:3) 3. WmeicsrtoSoArg,GanriifsfimnsA.SN,aGtaRrnevdeMricAr,oDbiioglg2le00S6P,:4S:o5c9i7a–l6e0v7o.lutiontheoryfor ¼ A (cid:3)2(cid:3)106 =ð4700ðA =mol ONPGÞ 4. WestSA,DiggleSP,BucklingA,GardnerA,GriffinAS:Thesociallivesof 420 420 Microbes.AnnuRevEcolEvolSyst2007,38:53–77. (cid:3)proteinmg= (cid:3)volðmlÞ(cid:3)timeðminÞÞ 5. 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