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In a process termed quorum sensing (QS), the opportunistic bacterial pathogen Pseudomonas aeruginosa uses diffusible signaling molecules to regulate the expression of numerous secreted factors or public goods that are shared within the population. But not all cells respond to QS signals. These social cheaters typically harbor a mutation in the QS r...
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... and culture conditions P. aeruginosa PAO1 was used as the wild-type isogenic parent at the start of all original in vitro evolution experiments (Sandoz et al., 2007;Wilder et al., 2011). All mutants were created via allelic exchange using a suicide vector containing either evolved alleles or in-frame deletions constructed by splicing-overlap-extension PCR (Horton, 1995;Hoang et al., 1998) (see Table 1 for a comprehensive list of strains). For routine culturing, we grew strains at 37 1C on Lennox lysogeny broth (LB) agar or with shaking in Lennox LB broth buffered with 50 mM 3-(N-morpholino)-propanesulfonic acid (MOPS), pH 7.0. ...
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... confirm the identified mutations, and to sequence specific loci of interest, standard dideoxy sequencing of PCR-ampli- fied and purified chromosomal DNA was employed at the Center for Genome Research and Biocomputing at Oregon State University in Corvallis, OR, USA. Primers are listed in Supplementary Table S1. ...
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Quorum sensing describes the ability of microbes to alter gene regulation according to their local population size. Some successful theory suggests that this is a form of cooperation, namely, investment in shared products is only worthwhile if there are sufficient bacteria making the same product. This theory can explain the genetic diversity in th...
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... Verifying that a Collapsing Tragedy of the Commons occurs in microbial populations is both beneficial and necessary because: (i) it provides the opportunity to validate that the existing mathematical theory sufficiently encapsulates the dynamics of the system, (ii) it allows us to identify the predictive limitations of such models, and (iii) it is not uncommon for cooperative populations to evade collapse as a consequence of mechanisms that either increase the cooperator's fitness or decrease the cheater's [19,21,22]. Examples of such mechanisms in microbial populations include non-social adaptations [23,24], punishment of cheaters through the production of toxic substances [25][26][27], reciprocity [28], privatization of public goods [29][30][31], division of labor to reduce individual fitness costs of beneficial behaviors [32][33][34], horizontal gene transfer [35,36], or population structure [17,18,37,38]. By identifying empirical conditions under which the Tragedy of the Commons occurs, we can better understand the conditions necessary to avoid it. ...
... Examples of extracellular public goods include proteases necessary for metabolizing food sources, biosurfactants for swarming, and siderophores for iron scavenging [3]. More specifically, an example of an extracellular protease produced by P. aeruginosa is LasB elastase, which is necessary to digest protein substrates [23,[39][40][41][42]. LasB and many other genes that encode cooperative functions are controlled by a process called quorum sensing (QS) [42,43]. ...
... As a first step, we formulated and tested a new growth medium suitable for our purposes. In several previous studies [21,23,30,41,46,51,54,63], we and others have used the skim milk protein casein as a growth substrate that requires QS dependent proteolysis. Casein, in the form of the soluble salt caseinate, is an inexpensive and efficient protein source for P. ...
Cooperation via shared public goods is ubiquitous in nature, however, noncontributing social cheaters can exploit the public goods provided by cooperating individuals to gain a fitness advantage. Theory predicts that this dynamic can cause a Tragedy of the Commons, and in particular, a ‘Collapsing’ Tragedy defined as the extinction of the entire population if the public good is essential. However, there is little empirical evidence of the Collapsing Tragedy in evolutionary biology. Here, we experimentally demonstrate this outcome in a microbial model system, the public good-producing bacterium Pseudomonas aeruginosa grown in a continuous-culture chemostat. In a growth medium that requires extracellular protein digestion, we find that P . aeruginosa populations maintain a high density when entirely composed of cooperating, protease-producing cells but completely collapse when non-producing cheater cells are introduced. We formulate a mechanistic mathematical model that recapitulates experimental observations and suggests key parameters, such as the dilution rate and the cost of public good production, that define the stability of cooperative behavior. We combine model prediction with experimental validation to explain striking differences in the long-term cheater trajectories of replicate cocultures through mutational events that increase cheater fitness. Taken together, our integrated empirical and theoretical approach validates and parametrizes the Collapsing Tragedy in a microbial population, and provides a quantitative, mechanistic framework for generating testable predictions of social behavior.
... Notably, mdpA and dppA3 encode a metallo-dipeptidase [17] and a small peptide transport [18], respectively. Through transcriptional repression of mdpA and dppA3, PsdR acts as a dipeptide regulator, governing the transport and processing of dipeptides in P. aeruginosa [16,17,19]. ...
... Mutations in the psdR gene were commonly observed in the laboratory strain PAO1 of P. aeruginosa when it underwent experimental evolution in a minimal medium, in which casein serves as the sole carbon and energy source [19,20]. In this environment, the growth of P. aeruginosa requires QS activation, as QS-controlled extracellular proteases are needed for the degradation of casein. ...
... In this environment, the growth of P. aeruginosa requires QS activation, as QS-controlled extracellular proteases are needed for the degradation of casein. Asfahl et al. described a non-social role of PsdR mutants in this QS-active evolution process, independent of QS circuit regulation [19]. In casein broth, spontaneous psdR mutations quickly emerge and become fixed throughout the entire population, occurring much earlier than LasR mutant cheaters [19,20]. ...
XRE-cupin family proteins containing an DNA-binding domain and a cupin signal-sensing domain are widely distributed in bacteria. In Pseudomonas aeruginosa , XRE-cupin transcription factors have long been recognized as regulators exclusively controlling cellular metabolism pathways. However, their potential functional roles beyond metabolism regulation remain unknown. PsdR, a typical XRE-cupin transcriptional regulator, was previously characterized as a local repressor involved solely in dipeptide metabolism. Here, by measuring quorum-sensing (QS) activities and QS-controlled metabolites, we uncover that PsdR is a new QS regulator in P . aeruginosa . Our RNA-seq analysis showed that rather than a local regulator, PsdR controls a large regulon, including genes associated with both the QS circuit and non-QS pathways. To unveil the underlying mechanism of PsdR in modulating QS, we developed a comparative transcriptome approach named “transcriptome profile similarity analysis” (TPSA). Using this TPSA method, we revealed that PsdR expression causes a QS-null-like transcriptome profile, resulting in QS-inactive phenotypes. Based on the results of TPSA, we further demonstrate that PsdR directly binds to the promoter for the gene encoding the QS master transcription factor LasR, thereby negatively regulating its expression and influencing QS activation. Moreover, our results showed that PsdR functions as a negative virulence regulator, as inactivation of PsdR enhanced bacterial cytotoxicity on host cells. In conclusion, we report on a new QS regulation role for PsdR, providing insights into its role in manipulating QS-controlled virulence. Most importantly, our findings open the door for a further discovery of untapped functions for other XRE-Cupin family proteins.
... PsdR variant mutants are commonly identified within P. aeruginosa QS evolution when bacteria are grown in caseinbased broth ( 59 ,77 ). These PsdR variant cells exhibited a competitive advantage over cells containing a functional PsdR, which was attributed to the non-social role of PsdR variants ( 59 ). In this scenario, the inactive PsdR depressed the expression of its neighboring two genes, mdpA and dppA3 , facilitating dipeptide uptake and processing ( 78 ,79 ). ...
... In this scenario, the inactive PsdR depressed the expression of its neighboring two genes, mdpA and dppA3 , facilitating dipeptide uptake and processing ( 78 ,79 ). This enhanced intracellular dipeptide utilization confers growth fitness to PsdR variants, enabling them to swiftly dominate the entire population ( 59 ). In agreement with previous findings, our work also observed the rapid prevalence of PsdR variants within the evolving population. ...
The genetic diversities of subpopulations drive the evolution of pathogens and affect their ability to infect hosts and cause diseases. However, most studies to date have focused on the identification and characterization of adaptive mutations in single colonies, which do not accurately reflect the phenotypes of an entire population. Here, to identify the composition of variant subpopulations within a pathogen population, we developed a streamlined approach that combines high-throughput sequencing of the entire population cells with genotyping of single colonies. Using this method, we reconstructed a detailed quorum-sensing (QS) evolutionary trajectory in Pseudomonas aeruginosa. Our results revealed a new adaptive mutation in the gacS gene, which codes for a histidine kinase sensor of a two-component system (TCS), during QS evolution. This mutation reduced QS activity, allowing the variant to sweep throughout the whole population, while still being vulnerable to invasion by the emerging QS master regulator LasR-null mutants. By tracking the evolutionary trajectory, we found that mutations in gacS facilitated QS-rewiring in the LasR-null mutant. This rapid QS revertant caused by inactive GacS was found to be associated with the promotion of ribosome biogenesis and accompanied by a trade-off of reduced bacterial virulence on host cells. In conclusion, our findings highlight the crucial role of the global regulator GacS in modulating the progression of QS evolution and the virulence of the pathogen population.
... Social cheating can occur among cooperating groups of bacteria under laboratory conditions [58,59], but it seldom leads to the collapse of natural populations [60,61]. Several mechanisms to guard public goods and maintain community stability have been proposed [10,11,15,[18][19][20][21][22][23][24]62]. ...
The type VI secretion system (T6SS) is a bacterial weapon capable of delivering antibacterial effectors to kill competing cells for interference competition, as well as secreting metal ion scavenging effectors to acquire essential micronutrients for exploitation competition. However, no T6SS effectors that can mediate both interference competition and exploitation competition have been reported. In this study, we identified a unique T6SS-1 effector in Yersinia pseudotuberculosis named TepC, which plays versatile roles in microbial communities. First, secreted TepC acts as a proteinaceous siderophore that binds to iron and mediates exploitative competition. Additionally, we discovered that TepC has DNase activity, which gives it both contact-dependent and contact-independent interference competition abilities. In conditions where iron is limited, the iron-loaded TepC is taken up by target cells expressing the outer membrane receptor TdsR. For kin cells encoding the cognate immunity protein TipC, TepC facilitates iron acquisition, and its toxic effects are neutralized. On the other hand, non-kin cells lacking TipC are enticed to uptake TepC and are killed by its DNase activity. Therefore, we have uncovered a T6SS effector, TepC, that functions like a “Trojan horse” by binding to iron ions to provide a valuable resource to kin cells, whereas punishing cheaters that do not produce public goods. This lure-to-kill mechanism, mediated by a bifunctional T6SS effector, may offer new insights into the molecular mechanisms that maintain stability in microbial communities.
... This range is part of the 38.3 kb deletion observed in LasR Q45stop -4. Furthermore, we noted that all four sequenced isolates carried a nonsynonymous mutation in psdR, aligning with prior research that identified frequent psdR mutations in casein evolution experiments (Asfahl et al., 2015;Kostylev et al., 2019). Nevertheless, psdR has been demonstrated to enhance growth on casein independently of QS (Asfahl et al., 2015). ...
... Furthermore, we noted that all four sequenced isolates carried a nonsynonymous mutation in psdR, aligning with prior research that identified frequent psdR mutations in casein evolution experiments (Asfahl et al., 2015;Kostylev et al., 2019). Nevertheless, psdR has been demonstrated to enhance growth on casein independently of QS (Asfahl et al., 2015). We thus infer that MexT and XcpA are instrumental in the observed virulence-associated phenotypic variations in the evolved isolates. ...
... growth. This mutation has been frequently observed in evolution experiments involving casein (Asfahl et al., 2015;Kostylev et al., 2019). Even with psdR deletion in both cooperators and cheaters, the pattern of invasion by the LasR mutant remains largely unaffected (Feng et al., 2019). ...
The Gram-negative opportunistic pathogen Pseudomonas aeruginosa possesses hierarchical quorum sensing (QS) systems. The intricate QS network of P. aeruginosa synchronizes a suite of virulence factors, contributing to the mortality and morbidity linked to the pathogenicity of this bacterium. Previous studies have revealed that variations in the lasR gene are frequently observed in chronic isolates of cystic fibrosis (CF). Specifically, LasR Q45stop was identified as a common variant among CF, lasR mutants during statistical analysis of the clinical lasR mutants in the database. In this study, we introduced LasR Q45stop into the chromosome of P. aeruginosa PAO1 through allelic replacement. The social traits of PAO1 LasR Q45stop were found to be equivalent to those of PAO1 LasR-null isolates. By co-evolving with the wild-type in caseinate broth, elastase-phenotypic-variability variants were derived from the LasR Q45stop subpopulation. Upon further examination of four LasR Q45stop sublines, we determined that the variation of T2SS-peptidase xcpA and mexT genes plays a pivotal role in the divergence of various phenotypes, including public goods elastase secretion and other pathogenicity traits. Furthermore, XcpA mutants demonstrated a fitness advantage compared to parent strains during co-evolution. Numerous phenotypic variations were associated with subline-specific genetic alterations. Collectively, these findings suggest that even within the same parental subline, there is ongoing microevolution of individual mutational trajectory diversity during adaptation.
... Our study focused more on the genes that directly affect QS in P. aeruginosa, and the interaction between lasR and mexT still needs further investigation. Also mutations in genes such as psdR which influences the fitness of the bacteria and non-cooperative cheating in the presence of lasR mutants (Asfahl et al., 2015;Kostylev et al., 2019) are currently being studied in our lab. Mutations in the intergenic region of transcriptional regulator, PsdR, has been shown to arise early in the evolution of P. aeruginosa strains growing in the presence of casein, and enhances fitness in the presence of lasR cheaters (Dandekar et al., 2012). ...
... DppA3 is a dipeptide binding protein with specificity for the transport of L-amino acids (Pletzer et al., 2014;Fernández et al., 2019). Derepression of this function of DppA3 by PsdR has been shown to enhance non-cooperative cheating in the P. aeruginosa population under QS-inducing conditions (Asfahl et al., 2015). As most of the regulatory systems in P. aeruginosa are highly coordinated and exhibit crosstalk, it may be a bit daunting to directly link phenotypes to specific microevolution events. ...
The Pseudomonas aeruginosa strain PAO1 has routinely been used as a laboratory model for quorum sensing (QS). However, the microevolution of P. aeruginosa laboratory strains resulting in genetic and phenotypic variations have caused inconsistencies in QS research. To investigate the underlying causes of these variations, we analyzed 5 Pseudomonas aeruginosa PAO1 sublines from our laboratory using a combination of phenotypic characterization, high throughput genome sequencing, and bioinformatic analysis. The major phenotypic variations among the sublines spanned across the levels of QS signals and virulence factors such as pyocyanin and elastase. Furthermore, the sublines exhibited distinct variations in motility and biofilm formation. Most of the phenotypic variations were mapped to mutations in the lasR and mexT, which are key components of the QS circuit. By introducing these mutations in the subline PAO1-E, which is devoid of such mutations, we confirmed their influence on QS, virulence, motility, and biofilm formation. The findings further highlight a possible divergent regulatory mechanism between the LasR and MexT in the P. aeruginosa. The results of our study reveal the effects of microevolution on the reproducibility of most research data from QS studies and further highlight mexT as a key component of the QS circuit of P. aeruginosa.
... A previous publication has shown that after the first several days of growth on casein, a PsdR mutant sweeps through the population. Mutations in psdR enhance growth on casein (20). The psdR gene codes for a repressor of a peptide transporter, and enhanced growth can be explained, at least in part, by improved uptake of peptide products of LasR-induced extracellular proteases. ...
... There is some information about adaptation to environments where P. aeruginosa QS is required for growth (2). In a relatively short time during QS-dependent growth on casein, a mutation in psdR, which codes for a repressor of a small-peptide uptake system, sweeps through the population and results in a substantial fitness gain reflected by more rapid growth in this environment (20). Inactivating mutations in the transcription factor mexT or the mexEF-oprN efflux pump genes also improve growth on casein, at least in part, by increasing activity of the Rhl QS system (34,35). ...
Pseudomonas aeruginosa uses quorum sensing (QS) to activate expression of dozens of genes (the QS regulon). Because there is strain-to-strain variation in the size and content of the QS regulon, we asked how the regulon might evolve during long-term P. aeruginosa growth when cells require some but not all the functions activated by QS.
... Hence, cooperation can be maintained when the benefits of any mutations outweigh the cost of the cooperative trait. Examples of adaptive mutations include the de-repression of growth-limiting private metabolism (Asfahl et al., 2015), enhanced nutrient uptake (Waite & Shou, 2012) and phage resistance (Morgan et al., 2012). ...
... Competitive interactions can lead to the loss of strains that produce public goods or strains that have unique metabolic pathways. In the case of public goods, this competition can involve the exploitation of secreted products by non-producers to outcompete and exclude producers, resulting in reduced productivity (Asfahl et al., 2015;Lindsay et al., 2019;Özkaya et al., 2018;Piccardi et al., 2019;Sanchez & Gore, 2013;Wang et al., 2015). However, interactions with secondary traits can prevent productivity loss, and in certain situations can even enhance productivity. ...
... SelecƟon on cooperaƟon -(exploitaƟon by nonproducers) Griffin et al. 2004Ӧzkaya et al. 2018Sanchez & Gore 2013 ProducƟon of biofilm + Drescher et al. 2014 ProducƟon of QS molecules + Allen et al. 2016Bruger et al. 2020 Non-producƟon of secondary public goods - Granato et al. 2018Ross-Gillespie et al. 2015 ProducƟon of alternaƟve inefficient public good + Inglis et al. 2016Inglis et al. Ӧzkaya et al. 2018 ProducƟon of external toxins + Yan et al. 2019Castañeda-Tamez et al. 2018Wang et al. 2015 Public good producƟon (providing addiƟonal funcƟon) + Jin et al. 2018Jousset et al. 2009 - Popat et al. 2017 Secondary private trait + Dandekar et al. 2012Asfahl et al. 2015Waite & Shou 2012Morgan et al. 2012Foster et al. 2004 Self-restraint metabolism - Lindsay et al. 2018 Public good producƟon (and its exploitaƟon due to compeƟƟon) Diversity, producƟvity and stability population of public good producers can inadvertently increase virulence because public good production interacts with another trait, resource-use efficiency (Lindsay et al., 2016). This discovery has put into question the current understanding regarding aspects of the evolution of cooperation and virulence (Box 1). ...
Understanding how microbial traits affect the evolution and functioning of microbial communities is fundamental for improving the management of harmful microorganisms, while promoting those that are beneficial. Decades of evolutionary ecology research has focused on examining microbial cooperation, diversity, productivity and virulence but with one crucial limitation. The traits under consideration, such as public good production and resistance to antibiotics or predation, are often assumed to act in isolation. Yet, in reality, multiple traits frequently interact, which can lead to unexpected and undesired outcomes for the health of macroorganisms and ecosystem functioning. This is because many predictions generated in a single‐trait context aimed at promoting diversity, reducing virulence or controlling antibiotic resistance can fail for systems where multiple traits interact. Here, we provide a much needed discussion and synthesis of the most recent research to reveal the widespread and diverse nature of multi‐trait interactions and their consequences for predicting and controlling microbial community dynamics. Importantly, we argue that synthetic microbial communities and multi‐trait mathematical models are powerful tools for managing the beneficial and detrimental impacts of microbial communities, such that past mistakes, like those made regarding the stewardship of antimicrobials, are not repeated. Decades of evolutionary ecology research has focused on examining microbial cooperation, diversity, productivity and virulence but with one crucial limitation. The traits under consideration, such as public good production and resistance to antibiotics or predation, are often assumed to act in isolation. Yet, in reality, multiple traits frequently interact, which can lead to unexpected and undesired outcomes for the health of macroorganisms and ecosystem functioning.
... Mutations in ptsP were genetic adaptations to the antibiotic tobramycin. Adaptive mutations have been shown to influence cooperation and cheating in other studies (43)(44)(45). For example, adaptive mutations can improve the fitness of cooperators so they can outcompete cheaters (19,44) or cause rewiring of cooperative traits and allow the cheaters to cooperate again (43,52). ...
The Pseudomonas aeruginosa LasR-I quorum-sensing system regulates secreted proteases that can be exploited by cheaters, such as quorum sensing receptor-defective ( lasR ) mutants. lasR mutants emerge in populations growing on casein as a sole source of carbon and energy. These mutants are exploitative cheaters because they avoid the substantial cost of engaging in quorum sensing. Previous studies showed that quorum sensing increases resistance to some antibiotics, such as tobramycin. Here, we show that tobramycin suppressed the emergence of lasR mutants in casein-passaged populations. Several mutations accumulated in those populations indicating evidence of antibiotic adaptation. We found that mutations in one gene, ptsP, increased antibiotic resistance and also pleiotropically increased production of a quorum sensing-controlled phenazine, pyocyanin. When passaged on casein, ptsP mutants suppressed cheaters in a manner that was tobramycin independent. We found the mechanism of cheater suppression in ptsP mutants relied on pyocyanin, which acts as a policing toxin by selectively blocking growth of cheaters. Thus, tobramycin suppresses lasR mutants through two mechanisms: first, through direct effects on cheaters and second, by selecting mutations in ptsP that suppressed cheating in a tobramycin-independent manner. This work demonstrates how adaptive mutations can alter the dynamics of cooperator-cheater relationships, which might be important for populations adapting to antibiotics during interspecies competition or infections.
IMPORTANCE
The opportunistic pathogen Pseudomonas aeruginosa is a model for understanding quorum sensing, a type of cell-cell signaling important for cooperation. Quorum sensing controls production of cooperative goods, such as exoenzymes, which are vulnerable to cheating by quorum sensing-defective mutants. Because uncontrolled cheating can ultimately cause a population to collapse, much focus has been on understanding how P. aeruginosa can control cheaters. We show that an antibiotic, tobramycin, can suppress cheaters in cooperating P. aeruginosa populations. Tobramycin suppresses cheaters directly because the cheaters are more susceptible to tobramycin than cooperators. Tobramycin also selects for mutations in a gene, ptsP, that suppresses cheaters independent of tobramycin through pleiotropic regulation of a policing toxin, pyocyanin. This work supports the idea that adaptation to antibiotics can have unexpected effects on the evolution of quorum sensing and has implications for understanding how cooperation evolves in dynamic bacterial communities.
... In a well-mixed environment, since all individuals share equal access to the cooperative benefit regardless of their contributions, cheaters are favored over cooperators (Harcombe, 2010). An exception is that cooperators can stochastically purge cheaters if cooperators happen to be better adapted to an environmental stress than cheaters (Asfahl et al., 2015;Morgan et al., 2012;Waite and Shou, 2012). Finally, pleiotropy -a single mutation affecting multiple phenotypes -can stabilize cooperation if reducing benefit supply to partner also elicits a crippling effect on self (Foster et al., 2004;Dandekar et al., 2012;Oslizlo et al., 2014;Harrison and Buckling, 2009;Sathe et al., 2019). ...
Cooperation, paying a cost to benefit others, is widespread. Cooperation can be promoted by pleiotropic 'win-win' mutations which directly benefit self ('self-serving') and partner ('partner-serving'). Previously, we showed that partner-serving should be defined as increased benefit supply rate per intake benefit (Hart & Pineda et al., 2019). Here, we report that win-win mutations can rapidly evolve even under conditions unfavorable for cooperation. Specifically, in a well-mixed environment we evolved engineered yeast cooperative communities where two strains exchanged costly metabolites lysine and hypoxanthine. Among cells that consumed lysine and released hypoxanthine, ecm21 mutations repeatedly arose. ecm21 is self-serving, improving self's growth rate in limiting lysine. ecm21 is also partner-serving, increasing hypoxanthine release rate per lysine consumption and the steady state growth rate of partner. ecm21 also arose in monocultures evolving in lysine-limited chemostats. Thus, even without any history of cooperation or pressure to maintain cooperation, pleiotropic win-win mutations may readily evolve.
