Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria

Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed “farmers”) stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.Symbiotic interactions are ubiquitous in nature and can play a central role in the evolutionary trajectory of organisms. For instance, symbiosis can drive rapid lateral procurement of novel traits as interacting organisms gain access to the genetic capabilities of their partner (1, 2). The evolutionary power of symbiosis is apparent in the many major life forms that owe their very existence to past and present symbiotic partnerships (3, 4). A famous example is the emergence of eukaryotes through their ancestor’s acquisition of bacteria that subsequently evolved into organelles indispensable for energy generation (5). Although many classic examples of symbiosis are conspicuously mutualistic, the characteristics of other symbiotic associations can be complex, dynamic, and less definable. In some cases, symbionts have good or bad effects on their host that vary depending on genotypic and environmental details (6, 7). The length and transmission mode of symbiosis also can have strong effects on the selection and evolution of partner traits. Although older symbiotic associations are often obligate and stable, recent associations can be transient and protean (8). Additionally, vertical transmission may favor mutualistic interactions, whereas horizontal transmission can allow the emergence and spread of more pathogenic characteristics (9–11). However, as a whole, the fates of symbioses are often the result of a delicate balance between mutualism and pathogenesis, requiring pathogenic characteristics at the least to facilitate infection and beneficial properties to promote maintenance (12, 13). Indeed, many cases of mutualistic associations are thought to have evolved from ancient parasitic infections (14, 15). Examining nascent, malleable, or less definable forms of symbiosis may provide insight into the mechanisms that promote or corrode this balance and their subsequent evolutionary consequences.Amoeba–bacteria interactions make a promising system for gaining insight into diverse and dynamic symbiotic relationships. Amoebas interact with bacteria in multiple ways. Most apparently, they are predators of bacteria. However, other amoeba–bacteria interactions are less favorable for the amoebas. Some bacteria can evade amoeba phagocytosis and thereby diminish amoeba predatory prowess and food acquisition (16). Still worse, amoebas can fall victim to bacterial processes or exploitation, with some bacteria producing products detrimental to amoeba fitness or surviving phagocytosis to invade and multiply within amoeba cells (17, 18). There also are stable symbiotic interactions between amoebas and bacteria in which the origins, mechanisms, and impacts on both species are less defined. For instance, several bacterial endosymbionts inhabit amoebas, incurring variable and not always obvious consequences to the amoeba host (19–22). In addition, certain isolates of the soil-dwelling amoeba Dictyostelium discoideum persistently associate with bacteria, an association that has beneficial or detrimental outcomes depending on the environmental conditions (23–25). Amoebas can serve as environmental reservoirs for bacterial pathogens (26, 27) or as training facilities for the adaptation of bacteria to evade eukaryotic phagocytosis or to survive intracellularly after phagocytosis (17, 28). Although, for the eukaryotic host, some of these interactions may have decidedly unfortunate outcomes, such as the emergence of bacterial pathogenesis through intracellular adaptation, others may illuminate important evolutionary advances, such as the transition of bacterial endosymbionts into organelles that provide novel functionality. Thus, the diversity and persistence of amoeba–bacteria interactions may have several ecological and health-related consequences.The stable association between bacteria and some wild isolates of D. discoideum supplies a particularly interesting study system for elucidating symbiotic causes and consequences. D. discoideum is a soil-dwelling amoeba that transitions between solitary and social life stages (29). In the solitary stage, D. discoideum cells consume bacteria through phagocytosis and divide by binary fission. When food is exhausted, amoebas coaggregate, ultimately forming a multicellular structure called a “fruiting body.” As the fruiting body develops, ∼20% of the cells sacrifice themselves to form an erect stalk. The remaining cells ascend the stalk, forming a globular sorus at the top where they differentiate into sturdy spores (29). In the sorus, spores are positioned for dispersal into more favorable environments, presumably through contact and transport upon animals (30). Multiple wild isolates of the amoeba D. discoideum, but not all of them, have been found to be stably associated with several bacteria species (24). These amoebas persistently carry both edible and inedible bacteria with them through the social stage and are termed “farmers” because of their ability to reseed new environments with a food source (23, 24). Interestingly, farmer isolates produce fewer spores under optimal conditions than their non–bacteria-carrying counterparts; however, this cost is countered by farmers’ advantage in being able to replenish their food supply when dispersing to food-poor environments (24). Additionally, some of the bacterial isolates that farmers carry produce compounds detrimental to nonfarmer clones but not to their host farmer, giving the host farmer a competitive advantage in a mixed population (23, 25).The ease of D. discoideum manipulation coupled with the variability inherent in the farming symbiosis provide a promising platform for addressing questions concerning symbiosis between microbes and eukaryotes. For instance, are mutualistic associations and their resulting phenotypes driven by the host, by the bacteria, or by a very specific interaction between the two? How much coevolution is required to reach a beneficial outcome? In aphids and other insects, defensive symbionts can sweep through populations and be horizontally transferred to new hosts (31, 32). Even Buchnera, a vertically transferred obligate symbiont of aphids, still confers its benefits to new hosts after experimental infection (33). Taken together, this horizontal transfer suggests that little coevolution may be needed for the formation of novel symbiosis. However, in some plant–rhizobia or –mycorrhizal associations, the symbiont phenotype can vary substantially among hosts, and the outcome of the association is determined by the interaction of host and symbiont-derived factors (34–38). Similarly, a synergistic interplay between host and symbiont components mediates the initiation and persistence of the bobtail squid–Vibrio fischeri symbiosis (39–41). For the D. discoideum farming symbiosis, it previously had been assumed that farmers were genetically distinct from nonfarmers, suggesting the role of a host-specific factor in establishing the symbiosis (24). This study aims to characterize more thoroughly the partner dynamics within the D. discoideum farming symbiosis by specifically analyzing the role of bacterial associates in farming phenotypes.To determine the role bacterial partners play in farming, we first examined the diversity of bacterial passengers associated with our present collection of stable farmer clones. We confirmed that several different bacterial species can be isolated from farmer D. discoideum; however isolates belonging to the Burkholderia genus were ubiquitous among our tested farmers. These Burkholderia isolates fail to support amoeba growth when provided as the only food source and therefore are considered inedible. Because of their prevalence in our farmer clones and because Burkholderia species form symbiotic relationships with diverse organisms, we hypothesized that these Burkholderia isolates could be crucial for the symbiotic relationship with D. discoideum that results in the farming phenomenon (42). Therefore we asked whether nonfarmer D. discoideum could be colonized by farmer-associated Burkholderia isolates and whether colonization could induce secondary bacterial carriage. We established that the Burkholderia associated with D. discoideum fall into two distinct phylogenetic clades. We found that the tested Burkholderia isolates from each clade robustly colonized nonfarmer D. discoideum sori, with this colonization persisting through multiple rounds of D. discoideum spore dispersal, germination, and vegetative growth. Like their farmer counterparts, nonfarmers colonized with Burkholderia can carry bacterial food, allowing them to reseed new territories with food bacteria following spore dispersal. Inversely, removal of Burkholderia from wild farmers by antibiotic treatment results in the loss of detectable bacterial food carrying. These findings suggest that Burkholderia colonization drives secondary bacterial carriage (farming) in D. discoideum. In total, these results suggest that specific Burkholderia isolates stably colonize D. discoideum and induce a novel adaptive trait of ecological relevance, the carriage of bacterial food. Our initial evidence suggests that the consequences of Burkholderia carriage may differ according to symbiont and host genotypes. For instance, some Burkholderia isolates impose a higher cost to their hosts, and the extent of this cost appears to be more severe for newer hosts than for the original host. We also observe Burkholderia (and occasionally, our laboratory food bacterium, Klebsiella pneumoniae) inside colonized D. discoideum spores after fruiting body formation. This observation, together with Burkholderia’s ability to be horizontally transmitted to new hosts and to associate stably with old hosts, suggests a mixed mode of Burkholderia transmission. The less severe fitness costs exerted by Burkholderia colonization in the original hosts suggests that long-term vertical transmission may lead to compensatory host adaptation.     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

From the Cover: Form of an evolutionary tradeoff affects eco-evolutionary dynamics in a predator–prey system

Evolution on a time scale similar to ecological dynamics has been increasingly recognized for the last three decades. Selection mediated by ecological interactions can change heritable phenotypic variation (i.e., evolution), and evolution of traits, in turn, can affect ecological interactions. Hence, ecological and evolutionary dynamics can be tightly linked and important to predict future dynamics, but our understanding of eco-evolutionary dynamics is still in its infancy and there is a significant gap between theoretical predictions and empirical tests. Empirical studies have demonstrated that the presence of genetic variation can dramatically change ecological dynamics, whereas theoretical studies predict that eco-evolutionary dynamics depend on the details of the genetic variation, such as the form of a tradeoff among genotypes, which can be more important than the presence or absence of the genetic variation. Using a predator–prey (rotifer–algal) experimental system in laboratory microcosms, we studied how different forms of a tradeoff between prey defense and growth affect eco-evolutionary dynamics. Our experimental results show for the first time to our knowledge that different forms of the tradeoff produce remarkably divergent eco-evolutionary dynamics, including near fixation, near extinction, and coexistence of algal genotypes, with quantitatively different population dynamics. A mathematical model, parameterized from completely independent experiments, explains the observed dynamics. The results suggest that knowing the details of heritable trait variation and covariation within a population is essential for understanding how evolution and ecology will interact and what form of eco-evolutionary dynamics will result.Evolutionary dynamics, changes in intraspecific genotype frequency over generations, can have a time scale similar to that of ecological dynamics (1–3). Selection mediated by ecological interactions causes evolutionary dynamics, and evolution of traits, in turn, changes ecological interactions. Thus, understanding population dynamics needs to take account of the feedbacks between trait evolution and ecological interactions (i.e., eco-evolutionary feedbacks). These feedbacks have increasingly attracted ecologists’ attention since Pimentel (4) proposed genetic feedback as a mechanism regulating animal populations (e.g., refs. 5–11). This integration of evolutionary biology and ecology has important implications in both basic and applied problems in biology (12–17).Empirical studies have shown that rapid evolution can affect many ecological interactions, including predator–prey (18–20), host–parasite (21), herbivore–plant (22), competitive interactions (23), and interactions with abiotic environments (24–27). Previous empirical studies on eco-evolutionary feedbacks have usually compared the dynamics of populations with and without genetic variation, but recent theoretical models predicted that not only the presence or absence of genetic variation (28–30) but also the form of the evolutionary tradeoff among genotypes is important in generating qualitatively different dynamics (31–35). Indeed, the forms of evolutionary tradeoffs within populations are known to be remarkably variable in plants and microbes (36–38). Thus, there should be various eco-evolutionary dynamics depending on the form of evolutionary tradeoffs existing in wild populations. Nevertheless, to our knowledge, no empirical study has directly demonstrated the theoretically predicted effects of the evolutionary tradeoff on eco-evolutionary dynamics, and it is still unclear how different forms of an evolutionary tradeoff in real organisms can result in different eco-evolutionary dynamics.Here, using a predator–prey (rotifer–algal) system cultured in continuous flow-through microcosms (chemostats), we examined how different forms of an evolutionary tradeoff between defense and growth in algal prey (Chlorella vulgaris) affect the population dynamics of the predator–prey system and the evolutionary changes in the clonal frequency of the algal prey. Experimental studies using laboratory microcosms have been a powerful approach in exploring eco-evolutionary dynamics and testing theoretical predictions because of the constant environment and simple community structure (39–41). We used two different pairs of algal clones originally obtained from the University of Texas (UTEX) algal collection that showed different forms of a fitness tradeoff between antipredator defense and competitive ability to obtain the resource limiting population growth in the experimental system (inorganic nitrogen). Each pair of algal clones was cultured with an obligately asexual lineage of rotifer predators (Brachionus calyciflorus). Population dynamics of the predators and prey and clonal frequency changes in the algal pair were observed in long-term chemostat runs. We recorded evolutionary dynamics (genotype frequency change) by using an allele-specific quantitative PCR (AsQ-PCR) technique based on microsatellite DNA that allowed us to measure the relative abundance of algal clones (42). We also developed a mathematical model for the experimental system, based on a model of Jones and Ellner (43), parameterized the model using data from separate experiments, and compared the model’s predictions to the observed population and genotype dynamics.     

The pathogen Batrachochytrium dendrobatidis disturbs the frog skin microbiome during a natural epidemic and experimental infection

Symbiotic microbial communities may interact with infectious pathogens sharing a common host. The microbiome may limit pathogen infection or, conversely, an invading pathogen can disturb the microbiome. Documentation of such relationships during naturally occurring disease outbreaks is rare, and identifying causal links from field observations is difficult. This study documented the effects of an amphibian skin pathogen of global conservation concern [the chytrid fungus Batrachochytrium dendrobatidis (Bd)] on the skin-associated bacterial microbiome of the endangered frog, Rana sierrae, using a combination of population surveys and laboratory experiments. We examined covariation of pathogen infection and bacterial microbiome composition in wild frogs, demonstrating a strong and consistent correlation between Bd infection load and bacterial community composition in multiple R. sierrae populations. Despite the correlation between Bd infection load and bacterial community composition, we observed 100% mortality of postmetamorphic frogs during a Bd epizootic, suggesting that the relationship between Bd and bacterial communities was not linked to variation in resistance to mortal disease and that Bd infection altered bacterial communities. In a controlled experiment, Bd infection significantly altered the R. sierrae microbiome, demonstrating a causal relationship. The response of microbial communities to Bd infection was remarkably consistent: Several bacterial taxa showed the same response to Bd infection across multiple field populations and the laboratory experiment, indicating a somewhat predictable interaction between Bd and the microbiome. The laboratory experiment demonstrates that Bd infection causes changes to amphibian skin bacterial communities, whereas the laboratory and field results together strongly support Bd disturbance as a driver of bacterial community change during natural disease dynamics.Symbiotic interactions between microbes and multicellular organisms are ubiquitous. In recent years, research to understand the complex microbial communities living in or on multicellular organisms (termed the microbiome) has sparked fundamental changes in our understanding of the biology of metazoans (1–5). The microbiome can affect host health directly by influencing metabolism (6), development (7), inflammation (8), or behavior (9), but it may also influence host health indirectly through interactions with infectious pathogens. The microbiome may interact with pathogens through competition for resources, release of antimicrobial compounds, contact-dependent antagonism, or modulation of the host immune response (10), and an “imbalanced” microbiome may leave the host more susceptible to pathogen infection (11, 12). At the same time, an invading pathogen may disrupt the microbiome (10, 13–15). Thus, the microbiome may play a role in disease resistance, or may itself be disturbed or altered by invading pathogens. Although a wealth of recent research has described associations between microbiome composition and a variety of syndromes in both humans and animals (16–25), documentation of microbiome responses to natural epidemics of known infectious pathogens is rare.Chytridiomycosis is an emerging infectious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Bd is an aquatic fungus that infects the skin of amphibians and disrupts osmoregulation, a critical function of amphibian skin (26). Chytridiomycosis can be fatal, and the severity of disease symptoms has been linked to Bd load, which is a measure of the density of Bd cells infecting the host (27, 28). Bd has a broad host range spanning hundreds of amphibian species, and has been implicated in population extinctions and species declines worldwide (29–34). Efforts to understand and mitigate the effects of Bd have led to research examining the potential for symbiotic bacteria to increase resistance to infection by the pathogen (35, 36). Bacterial species isolated from the skin of amphibians have been shown to inhibit the growth of Bd and other fungal pathogens in culture (37–39), possibly by producing antifungal metabolites (40, 41). In a controlled laboratory experiment, inundation of Rana muscosa with the bacterium Janthinobacterium lividium protected frogs from subsequent Bd infection (42). These and other studies highlight the possible role of bacteria in resistance to chytridiomycosis, but critical questions remain. First, most research has focused on the ability of cultured bacteria to prevent Bd infection, whereas very little is known about whether Bd infection alters the diverse skin microbiome. Examining this latter concept is critical both to a basic understanding of how the microbiome interacts with pathogens and to conservation efforts because Bd-induced perturbations of the microbiome could undermine attempts to mitigate effects of Bd infection through augmentation with particular bacteria. A second knowledge gap is the paucity of comprehensive culture-independent assessments of the amphibian microbiome, which are important because the vast majority of environmental and symbiotic microbes are not readily cultured, and culture-based methods can lead to severe underestimates of diversity and biased assessment of community composition (43). Few studies have applied next-generation sequencing methods to characterize the microbial communities on amphibian skin (44–47), and, to our knowledge, none have done so in the context of Bd infection. A final challenge to understanding interactions between Bd and bacteria stems from the difficulties of drawing direct connections between laboratory and field studies. Laboratory studies are essential for definitive identification of cause and effect. However, complex natural microbiomes can be impossible to recreate in the laboratory, and field studies are needed to show whether processes identified in the laboratory are relevant in nature.We present paired laboratory and field studies using high-throughput 16S amplicon pyrosequencing both to document associations between Bd infection and the amphibian skin bacterial microbiome in nature and to deduce causal relationships in an experiment. Our work centers on the Sierra Nevada yellow-legged frog, Rana sierrae, which is severely threatened by, and has already suffered drastic declines due to, Bd (28, 48). We surveyed frogs from four distinct R. sierrae populations to test if differences in skin bacterial communities are associated with the intensity of pathogen infection. We then conducted a laboratory experiment to establish causal relationships underlying Bd-bacterial community associations. The data establish a strong effect of Bd infection on the composition of the amphibian skin bacterial microbiome that is consistent between the laboratory experiment and naturally occurring Bd dynamics in wild frog populations.     

Neural substrate for higher-order learning in an insect: Mushroom bodies are necessary for configural discriminations

Learning theories distinguish elemental from configural learning based on their different complexity. Although the former relies on simple and unambiguous links between the learned events, the latter deals with ambiguous discriminations in which conjunctive representations of events are learned as being different from their elements. In mammals, configural learning is mediated by brain areas that are either dispensable or partially involved in elemental learning. We studied whether the insect brain follows the same principles and addressed this question in the honey bee, the only insect in which configural learning has been demonstrated. We used a combination of conditioning protocols, disruption of neural activity, and optophysiological recording of olfactory circuits in the bee brain to determine whether mushroom bodies (MBs), brain structures that are essential for memory storage and retrieval, are equally necessary for configural and elemental olfactory learning. We show that bees with anesthetized MBs distinguish odors and learn elemental olfactory discriminations but not configural ones, such as positive and negative patterning. Inhibition of GABAergic signaling in the MB calyces, but not in the lobes, impairs patterning discrimination, thus suggesting a requirement of GABAergic feedback neurons from the lobes to the calyces for nonelemental learning. These results uncover a previously unidentified role for MBs besides memory storage and retrieval: namely, their implication in the acquisition of ambiguous discrimination problems. Thus, in insects as in mammals, specific brain regions are recruited when the ambiguity of learning tasks increases, a fact that reveals similarities in the neural processes underlying the elucidation of ambiguous tasks across species.Learning can be categorized into two levels of complexity termed elemental and configural (nonelemental) (1–3). Simple and unambiguous links between events characterize elemental learning (4). By contrast, ambiguity and nonlinearity characterize configural learning, where associations involve conjunctions of elemental stimuli, which may have different, contradictory outcomes. As a consequence, solving configural tasks typically requires treating stimulus conjunctions as being different from the simple sum of their elemental components (5–8). For example, in a negative patterning task (9–11), subjects have to discriminate a nonreinforced conjunction of two elements A and B from its reinforced elements (i.e., AB– vs. A+ and B+), which requires treating AB as being different from the simple sum of A and B (12, 13). The ambiguity of the task lies in the fact that each element (A and B) is as often reinforced (when presented alone) as nonreinforced (when presented as a compound). In mammals, different brain structures have been associated with these two learning forms: Whereas the hippocampus seems to be dispensable for learning elemental associations (6, 8), it is required for fast formation of conjunctive representations during learning tasks, such as spatial learning or contextual fear conditioning (6, 8, 10, 14–19). Moreover, the cortical system is necessary to form configural representations over extended training, thus supporting the learning of nonlinear discriminations,Here, we ask whether the specialization of different brain centers for learning tasks of different complexity is a property that can be extended to an insect brain. Insects offer the possibility of studying sophisticated behaviors and simultaneously accessing the neural bases of these behaviors (20). Several studies have shown that insects, in particular the honey bee Apis mellifera, possess higher-order cognitive abilities (5, 21), which raises the question of which neural mechanisms support these capacities in a brain whose size is only 1 mm³ (22).The mushroom bodies (MBs) are paired structures in the insect brain that have been historically associated with olfactory learning and memory. Their function has been extensively studied in a variety of elemental learning protocols, mainly in the honey bee and the fruit fly Drosophila melanogaster (23–29). In both species, MBs play a fundamental role for the encoding, storing, and retrieval of appetitive and aversive elemental memories, but no study has clearly established their role for nonelemental learning and memory (30). In fruit flies, this missing information may be due to the incapacity of these insects to solve nonelemental problems, such as negative patterning (31). By contrast, honey bees exhibit elaborated nonelemental learning abilities (32–36), which have been suggested to require intact MB function (5).Here, we used a combination of nonelemental conditioning protocols, disruption of MB function, and optophysiological recordings of neural activity to determine whether MBs are necessary for nonelemental forms of learning. Our results show that acquisition of olfactory patterning discriminations is impaired in bees in which neural activity in the MBs was blocked by procaine injection (37, 38), but not in control animals injected with saline solution. By contrast, MB blockade by procaine affected neither olfactory processing upstream of the MBs nor elemental olfactory discriminations. To uncover the neural mechanisms underlying the necessity of MBs for patterning discriminations, we focused on GABAergic feedback neurons (39), which provide inhibitory feedback to the MBs of the bee (40–43). We blocked GABAergic signaling by locally injecting picrotoxin (PTX), a GABA antagonist, into the MB calyces or into the MB lobes. We show that GABAergic feedback to the calyces—but not to the lobes—is required for patterning discriminations. These results uncover a previously unidentified role for MBs: namely, the disambiguation between elemental and conjunctive odor representations, thus supporting the learning of nonlinear discriminations.     

Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.The bacterium Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSIs), including cellulitis, furunculosis, and folliculitis (1–4), and a common etiologic agent of impetigo (5), erysipelas (6), and superinfection in atopic dermatitis (7). This bacterium is a significant cause of surgical or traumatic wound infections (8, 9), as well as decuibitus and diabetic skin lesions (10). Moreover, SSSI is an important risk factor for systemic infection. The skin is a key portal of entry for hematogenous dissemination, particularly in association with i.v. catheters. S. aureus is now the second most common bloodstream isolate in healthcare settings (11), and SSSI is a frequent source of invasive infections such as pneumonia or endocarditis (12, 13). Despite a recent modest decline in rates of methicillin-resistant S. aureus (MRSA) infection in some cohorts (13), infections due to S. aureus remain a significant problem (14, 15). Even with appropriate therapy, up to one-third of patients diagnosed with S. aureus bacteremia succumb—accounting for more attributable annual deaths than HIV, tuberculosis, and viral hepatitis combined (16).The empiric use of antibiotics in healthcare-associated and community-acquired settings has increased S. aureus exposure to these agents, accelerating selection of resistant strains. As a result, resistance to even the most recently developed agents is emerging at an alarming pace (17, 18). The impact of this trend is of special concern in light of high rates of mortality associated with invasive MRSA infection (e.g., 15–40% in bacteremia or endocarditis), even with the most recently developed antistaphylococcal therapeutics (19, 20). Moreover, patients who experience SSSI due to MRSA exhibit high 1-y recurrence rates, often prompting surgical debridement (21) and protracted antibiotic treatment.Infections due to MRSA are a special concern in immune-vulnerable populations, including hemodialysis (22), neutropenic (23, 24), transplantation (25), and otherwise immunosuppressed patients (26, 27), and in patients with inherited immune dysfunctions (28–31) or cystic fibrosis (32). Patients having deficient interleukin 17 (IL-17) or IL-22 responses (e.g., signal transduction mediators STAT3, DOCK8, or CARD9 deficiencies) exhibit chronic or “cold” abscesses, despite high densities of pathogens such as S. aureus (33, 34). For example, patients with Chronic Granulomatous Disease (CGD; deficient Th1 and oxidative burst response) have increased risk of disseminated S. aureus infection. In contrast, patients with Job’s Syndrome (deficient Th17 response) typically have increased risk to SSSI and lung infections, but less so for systemic S. aureus bacteremia (35, 36). This pattern contrasts that observed in neutropenic or CGD patients (37). These themes suggest efficacious host defenses against MRSA skin and invasive infections involve complementary but distinct molecular and cellular immune responses.From these perspectives, vaccines or immunotherapeutics that prevent or lessen severity of MRSA infections, or that enhance antibiotic efficacy, would be significant advances in patient care and public health. However, to date, there are no licensed prophylactic or therapeutic vaccine immunotherapies for S. aureus or MRSA infection. Unfortunately, efforts to develop vaccines targeting S. aureus capsular polysaccharide type 5 or 8 conjugates, or the iron-regulated surface determinant B protein, have not been successful thus far (38, 39). Likewise, passive immunization using monoclonal antibodies targeting the S. aureus adhesin clumping factor A (ClfA, tefibazumab) (40) or lipoteichoic acid (pagibaximab) (41) have not shown efficacy against invasive infections in human clinical studies to date. Moreover, the striking recurrence rates of SSSI due to MRSA imply that natural exposure does not induce optimal preventive immunity or durable anamnestic response to infection or reinfection. Thus, significant challenges exist in the development of an efficacious vaccine targeting diseases caused by S. aureus (42) that are perhaps not optimally addressed by conventional approaches.The NDV-3 vaccine reflects a new strategy to induce durable immunity targeting S. aureus. Its immunogen is engineered from the agglutinin-like sequence 3 (Als3) adhesin/invasin of Candida albicans, which we discovered to be a structural homolog of S. aureus adhesins (43). NDV-3 is believed to cross-protect against S. aureus and C. albicans due to sequence (T-cell) and conformational (B-cell) epitopes paralleled in both organisms (44). Our prior data have shown that NDV-3 is efficacious in murine models of hematogenous and mucosal candidiasis (45), as well as S. aureus bacteremia (46–48). Recently completed phase I clinical trials demonstrate the safety, tolerability, and immunogenicity of NDV-3 in humans (49).     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Novel serologic biomarkers provide accurate estimates of recent Plasmodium falciparum exposure for individuals and communities

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual’s recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86–0.93), whereas responses to six antigens accurately estimated an individual’s malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.Many countries have extensive programs to reduce the burden of Plasmodium falciparum (Pf), the parasite responsible for most malaria morbidity and mortality (1). Effectively using limited resources for malaria control or elimination and evaluating interventions require accurate measurements of the risk of being infected with Pf (2–15). To reflect the rate at which individuals are infected with Pf in a useful way, metrics used to estimate exposure in a community need to account for dynamic changes over space and time, especially in response to control interventions (16–18).A variety of metrics can be used to estimate Pf exposure, but tools that are more precise and low cost are needed for population surveillance. Existing metrics have varying intrinsic levels of precision and accuracy and are subject to a variety of extrinsic factors, such as cost, time, and availability of trained personnel (19). For example, entomological measurements provide information on mosquito to human transmission for a community but are expensive, require specially trained staff, and lack standardized procedures, all of which reduce precision and/or make interpretation difficult (19–22). Parasite prevalence can be measured by detecting parasites in the blood of individuals from a cross-sectional sample of a community and is, therefore, relatively simple and inexpensive to perform, but results may be imprecise, especially in areas of low transmission (19, 23), and biased by a number of factors, including immunity and access to antimalarial treatment (5, 6, 19, 23–25). The burden of symptomatic disease in a community can be estimated from routine health systems data; however, such data are frequently unreliable (5, 26–28) and generally underestimate the prevalence of Pf infection in areas of intense transmission. Precise and quantitative information about exposure at an individual level can be reliably obtained from cohort studies by measuring the incidence of asymptomatic and/or symptomatic Pf infection (i.e., by measuring the molecular force of infection) (29–35). Unfortunately, the expense of cohort studies limits their use to research settings. The end result is that most malaria-endemic regions lack reliable, timely data on Pf exposure, limiting the capabilities of malaria control programs to guide and evaluate interventions.Serologic assays offer the potential to provide incidence estimates for symptomatic and asymptomatic Pf infection, which are currently obtained from cohort studies, at the cost of cross-sectional studies (36–38). Although Pf infections are transient, a record of infection remains detectable in an individual’s antibody profile. Thus, appropriately chosen antibody measurements integrated with age can provide information about an individual’s exposure history. Antibodies can be measured by simple ELISAs and obtained from dried blood spots, which are easy to collect, transport, and store (39–41). Serologic responses to Pf antigens have been explored as potential epidemiological tools (42–45), and estimated rates of seroconversion to well-characterized Pf antigens accurately reflect stable rates of exposure in a community, whereas distinct changes in these rates are obtained from successful interventions (22, 39, 41, 46–53). However, current serologic assays are not designed to detect short-term or gradual changes in Pf exposure or measure exposure to infection at an individual level. The ability to calibrate antibody responses to estimates of exposure in individuals could allow for more flexible sampling of a population (e.g., not requiring age stratification), improve accuracy of exposure estimates from small sample sizes, and better characterize heterogeneity in exposure within a community.Different Pf antigens elicit antibody responses with different magnitudes and kinetics, providing a large and diverse set of potential biomarkers for exposure (38, 54–58). We hypothesized that new and more highly informative serologic biomarkers better able to characterize an individual’s recent exposure history could be identified by analyzing antibody responses to a large number of candidate Pf antigens in participants with well-characterized exposure histories. To test this hypothesis, we probed plasma from participants in two cohort studies in Uganda against a protein microarray containing 856 Pf antigens. The primary aim of this analysis was to identify responses to select antigens that were most informative of recent exposure using robust, data-adaptive statistical methods. Each participant’s responses to these selected antigens were used as predictors for two primary outcomes of their recent exposure to Pf: (i) days since last Pf infection and (ii) the incidence of symptomatic malaria in the last year. These individual-level estimates were then aggregated across a population to assess community-level malaria exposure. The selection strategy presented here identified accurate biomarkers of exposure for children living in areas of moderate to high Pf exposure and illustrates the utility of this flexible and broadly applicable approach.     

Recombinant transfer in the basic genome of Escherichia coli

An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.The increasing availability of complete genome sequences of many different bacterial and archaeal species, as well as metagenomic sequencing of mixed populations from natural environments, has stimulated theoretical and computational approaches to understand mechanisms of speciation and how prokaryotic species should be defined (1–8). Much genome analysis and comparison has been at the level of gene content, identifying core genomes (the set of genes found in most or all genomes in a group) and the continually expanding pan-genome. Population genomics of Escherichia coli has been particularly well studied because of its long history in laboratory research and because many pathogenic strains have been isolated and completely sequenced (9–14). Proposed models of how related groups or species form and evolve include isolation by ecological niche (7–9, 11, 15), decreased homologous recombination as divergence between isolated populations increases (2–4, 8, 14, 16), and coevolving phage and bacterial populations (6).E. coli genomes are highly variable, containing an array of phage-related mobile elements integrated at many different sites (17), random insertions of multiple transposable elements (18), and idiosyncratic genome rearrangements that include inversions, translocations, duplications, and deletions. Although E. coli grows by binary cell division, genetic exchange by homologous recombination has come to be recognized as a significant factor in adaptation and genome evolution (9, 10, 19). Of particular interest has been the relative contribution to genome variability of random mutations (single base-pair differences referred to as SNPs) and replacement of genome regions by homologous recombination with fragments imported from other genomes (here referred to as recombinant transfers or transferred regions). Estimates of the rate, extent, and average lengths of recombinant transfers in the core genome vary widely, as do methods for detecting transferred regions and assessing their impact on phylogenetic relationships (12–14, 20, 21).In a previous comparison of complete genome sequences of the K-12 reference strain MG1655 and the reconstructed genome of the B strain of Delbrück and Luria referred to here as B-DL, we observed that SNPs are not randomly distributed among 3,620 perfectly matched pairs of coding sequences but rather have two distinct regimes: sharply decreasing numbers of genes having 0, 1, 2, or 3 SNPs, and an abrupt transition to a much broader exponential distribution in which decreasing numbers of genes contain increasing numbers of SNPs from 4 to 102 SNPs per gene (22). Genes in the two regimes of the distribution are interspersed in clusters of variable lengths throughout what we referred to as the basic genome, namely, the ∼4 Mbp shared by the two genomes after eliminating mobile elements. We speculated that genes having 0 to 3 SNPs may primarily have been inherited clonally from the last common ancestor, whereas genes comprising the exponential tail may primarily have been acquired by horizontal transfer from diverged members of the population.The current study was undertaken to extend these observations to a diverse set of 32 completely sequenced E. coli genomes and to analyze how SNP distributions in the basic genome change as a function of evolutionary divergence between the 496 pairs of strains in this set. We have taken a simpler approach than those of Touchon et al. (13), Didelot et al. (14), and McNally et al. (21), who previously analyzed multiple alignments of complete genomes of E. coli strains. The appreciably larger basic genome derived here is not restricted to protein-coding sequences and retains positional information.