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.     

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.     

Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis

The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical–basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.The phytohormone auxin plays critical roles in almost every aspect of plant development including embryogenesis, architecture formation, and tropic growth. One of the most fascinating topics in plant biology is how auxin can have so many diverse and context-specific functions (1). Dynamic auxin gradients, which are regulated mainly by local auxin synthesis, catabolism, conjugation, and polar auxin transport, are essential for integration of various environmental and endogenous signals (2, 3). Auxin perception and signaling systems are responsible for a read-out of the auxin gradients (1).Local auxin biosynthesis is important for leaf development, shade avoidance, root-specific ethylene sensitivity, vascular patterning, flower patterning, and embryogenesis (4). Indole-3-acetic acid (IAA), the naturally occurring principal auxin in plants, is biosynthesized from tryptophan (Trp) through four proposed routes according to their key intermediates, namely indole-3-acetaldoxime (IAOx), indole-3-pyruvic acid (IPyA), indole-3-acetamide (IAM), and tryptamine (TAM) (5). The TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA)/YUCCA (YUC) linear pathway has been considered as a predominant Trp-dependent auxin biosynthetic pathway (4, 6–8). However, labeling studies and analyses of Trp auxotrophic mutants have long predicted the existence of a Trp-independent IAA biosynthetic pathway (9–13). When the Arabidopsis and maize seedlings were fed with isotope-labeled precursor, IAA was more enriched than Trp, and the incorporation of label into IAA from Trp is low, suggesting that IAA can be produced de novo without Trp as an intermediate (11, 12). The Trp biosynthetic mutant trp1, defective in phosphoribosylanthranilate transferase (PAT), and indole-3-glycerol phosphate synthase (IGS) antisense plants are deficient in steps earlier than indole-3-glycerol phosphate (IGP) formation and display decreased levels of both IAA and Trp (10, 14). However, the trp3 and trp2 mutants, defective in tryptophan synthase α (TSA) and tryptophan synthase β (TSB) subunits, respectively, accumulate higher levels of IAA than the wild type despite containing lower Trp levels (10, 11, 15, 16). These data strongly suggested the existence of the Trp-independent IAA biosynthetic pathway that might branch from IGP and/or indole (10). Further studies suggested that a cytosol-localized indole synthase (INS) may be involved in the Trp-independent biosynthesis of indole-containing metabolite (17). However, the molecular basis, biological functions, and spatiotemporal regulation of the Trp-independent IAA biosynthetic pathway have remained a mystery.In higher eukaryotes, embryogenesis initiates the generation of the species-specific body plan. During embryogenesis, a single-celled zygote develops into a functional multicellular organism with cells adopting specific fates according to their relative positions. In higher plants, essential architecture features, such as body axes and major tissue layers, are established in embryogenesis, and auxin plays a vital role in apical-basal patterning and embryo axis formation (18, 19). Local auxin biosynthesis, polar auxin transport facilitated by PIN-FORMED1/3/4/7 (PIN1/3/4/7), and auxin response coordinately regulate apical–basal pattern formation during embryogenesis (7, 20–22). The TAA and YUC families in Trp-dependent IAA biosynthesis predominantly regulate embryogenesis at or after the globular stage (7, 22). However, the auxin source for embryogenesis before the globular stage remains elusive. In this study, we provide, to our knowledge, the first genetic and biochemical evidence that the cytosol-localized INS is a key component in the long predicted Trp-independent auxin biosynthetic pathway and is critical for apical–basal pattern formation during early embryogenesis in Arabidopsis.     

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

Efficient plant male fertility depends on vegetative nuclear movement mediated by two families of plant outer nuclear membrane proteins

Increasing evidence suggests that nuclear migration is important for eukaryotic development. Although nuclear migration is conserved in plants, its importance for plant development has not yet been established. The most extraordinary plant nuclear migration events involve plant fertilization, which is starkly different from that of animals. Instead of evolving self-propelled sperm cells (SCs), plants use pollen tubes to deliver SCs, in which the pollen vegetative nucleus (VN) and the SCs migrate as a unit toward the ovules, a fundamental but barely understood process. Here, we report that WPP domain-interacting proteins (WIPs) and their binding partners the WPP domain-interacting tail-anchored proteins (WITs) are essential for pollen nuclear migration. Loss-of-function mutations in WIT and/or WIP gene families resulted in impaired VN movement, inefficient SC delivery, and defects in pollen tube reception. WIPs are Klarsicht/ANC-1/Syne-1 Homology (KASH) analogs in plants. KASH proteins are key players in animal nuclear migration. Thus, this study not only reveals an important nuclear migration mechanism in plant fertilization but also, suggests that similar nuclear migration machinery is conserved between plants and animals.Nuclear migration is essential for cell differentiation, polarization, and migration, which influence organism development (1–3). Examples range from Caenorhabditis elegans P-cell development to mammalian neural development (1–3). The key players in opisthokont nuclear migration are the inner nuclear membrane Sad1/UNC-84 (SUN) proteins and outer nuclear membrane Klarsicht/ANC-1/Syne-1 Homology (KASH) proteins. SUN and KASH proteins form the linkers of the nucleoskeleton and the cytoskeleton complexes at the nuclear envelope (NE) and transfer cytoplasmic forces to the nucleus (1–3). In plants, nuclear migration is associated with a number of developmental events and environmental responses, including fertilization, root and leaf hair formation, and plant–microbe interactions (4, 5). So far, little is known about the mechanism of plant nuclear migration. Although SUN proteins are conserved in plants (6, 7), absence of animal KASH homologs in plants suggests that plants may have evolved different molecular solutions to achieve nuclear migration. Recently, WPP domain-interacting proteins (WIPs) were identified as KASH proteins in plants (8), and their outer nuclear membrane binding partners WPP domain-interacting tail anchored proteins (WITs) were shown to interact with myosin XI-I (9). The WIT–myosin XI-I complexes regulate nuclear movement in root and mesophyll cells, but no developmental events have been linked to these nuclear movements (9).Essential for plant fertility, pollen tube growth harbors the most dramatic nuclear movement in plants. Unlike animals, which have sperm cells (SCs) that travel through self-propelled flagellum, flowering plants use pollen tubes to deliver SCs to ovules (10–13). In Arabidopsis, pollen tube growth is guided by chemical cues in carpel tissues and attracted by small peptides secreted by synergid cells in the vicinity of ovules (14–18). Pollen tube reception is completed by pollen tube burst, SC release, and degeneration of synergid cells (12). If this process fails, a second pollen tube can be attracted to the same ovule for a second attempt, resulting in polytubey (19). The SCs [or their progenitor the generative cell (GC)] are enclosed by an endocytic membrane tethered to the pollen vegetative nucleus (VN) (20). During pollen tube elongation, the VN and the SCs/GC are usually closely associated and move as a male germ unit (MGU) (13, 21). For decades, the movement of the MGU has been analyzed using cytoskeleton-depolymerizing reagents or heterogeneous antimyosin antibodies (22–27). However, no genes have been implicated in MGU movement, and the function of the joint migration of VN and GC/SC remains hypothetical.Here, we have identified the Arabidopsis WIT and WIP protein families as key players in VN movement. WIP1 and WIT1 are localized at the vegetative nuclear envelope (VNE). Loss of either WIT or WIP family proteins impaired VN movement, resulting in defective pollen tube reception and inefficient SC-to-ovule migration. This study has not only identified a molecular mechanism regulating the VN movement but also, revealed an important function of the VN in plant fertilization.     

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.The emergence of multidrug-resistant bacteria is a mounting global health crisis. In particular, multidrug-resistant Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and is resistant to most antibiotics commonly used for patient treatment (1). Hospital intensive care units in many countries, including the United States, now report methicillin-resistant S. aureus infection rates exceeding 50% (2, 3). Antibiotic resistance in contemporary infectious S. aureus strains, such as in hospitals, is often encoded by plasmids that can be transmitted between strains via horizontal DNA transfer mechanisms. These plasmids are typically classified as small (<5 kb) multicopy plasmids, which usually encode only a single resistance gene; medium-sized (8–40 kb) multirestance plasmids that confer resistance to multiple antibiotics, disinfectants, and/or heavy metals; and large (>40 kb) conjugative multiresistance plasmids that additionally encode a conjugative DNA transfer mechanism (4–6). Importantly, sequence analyses have shown that most staphylococcal conjugative and nonconjugative multiresistance plasmids encode a highly conserved replication initiation protein, denoted RepA_N (5–15). RepA_N proteins are also encoded by plasmids from other Gram-positive bacteria as well as by some phage, underscoring their ubiquitous nature (10). These RepA proteins are essential for replication of multiresistance plasmids, and hence plasmid carriage and dissemination, yet the mechanisms by which these proteins function in replication are currently unknown.The DNA replication cycle can be divided into three stages: initiation, elongation, and termination. Replication initiation proteins (RepA) mediate the crucial first step of initiation. Bacterial chromosome replication is initiated by the chromosomal replication initiator protein, DnaA, which binds the origin and recruits the replication components known as the primosome (16). In Gram-negative bacteria the primosome includes DnaG primase, the replicative helicase (DnaB), and DnaC (17). Replication initiation in Gram-positive bacteria involves DnaG primase and helicase (DnaC) and the proteins DnaD, DnaI, and DnaB (18–22). DnaD binds first to DnaA at the origin. This is followed by binding of DnaI/DnaB and DnaG, which together recruit the replicative helicase (23, 24). Instead of DnaA, plasmids encode and use their own specific replication initiator binding protein. Structures are only available for RepA proteins (F, R6K, and pPS10 Rep) harbored in Gram-negative bacteria. These proteins contain winged helix-turn-helix (winged HTH) domains and bind iteron DNA as monomers to, in some still unclear manner, drive replicon assembly (25–27).Replication mechanisms used by plasmids harbored in Gram-positive bacteria are less well understood and are distinct from their Gram-negative counterparts. Indeed, most plasmid RepA proteins in Gram-negative and Gram-positive bacteria show no sequence homology and seem to be unrelated. The multiresistance RepA proteins are arguably among the most abundant of plasmid Rep proteins, yet how they function is not known. Data suggest that these proteins are composed of three main regions: an N-terminal domain (NTD) consisting of ∼120 aa, a long and variable linker region (∼30–50 residues), and a C-terminal domain (CTD) of ∼120 residues (28–31). The NTD and CTD are both essential for replication. The NTD exhibits the highest level of sequence conservation, which has resulted in the designation of plasmids that encode these proteins as the RepA_N replicon family (10). Although not as well conserved as the NTD, RepA CTD regions show homology between plasmids found in genus-specific clusters, suggesting that this domain may perform a host-specific role (28–32). Although the function of the RepA CTD remains enigmatic, recent studies have indicated that the NTD mediates DNA binding and interacts with iterons that reside within the plasmid origin (30). The essential roles played by RepA proteins in multiresistance plasmid retention marks them as attractive targets for the development of specific chemotherapeutics. However, the successful design of such compounds necessitates structural and mechanistic insight. Here, we describe a detailed dissection of the RepA proteins from the multiresistance plasmids pSK41 and pTZ2126. The combined data reveal the molecular underpinnings of a minimalist replication assembly mediated by multiresistance RepA proteins.     

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.     

From the Cover: Socially mediated induction and suppression of antibiosis during bacterial coexistence

Despite their importance for humans, there is little consensus on the function of antibiotics in nature for the bacteria that produce them. Classical explanations suggest that bacteria use antibiotics as weapons to kill or inhibit competitors, whereas a recent alternative hypothesis states that antibiotics are signals that coordinate cooperative social interactions between coexisting bacteria. Here we distinguish these hypotheses in the prolific antibiotic-producing genus Streptomyces and provide strong evidence that antibiotics are weapons whose expression is significantly influenced by social and competitive interactions between competing strains. We show that cells induce facultative responses to cues produced by competitors by (i) increasing their own antibiotic production, thereby decreasing costs associated with constitutive synthesis of these expensive products, and (ii) by suppressing antibiotic production in competitors, thereby reducing direct threats to themselves. These results thus show that although antibiotic production is profoundly social, it is emphatically not cooperative. Using computer simulations, we next show that these facultative strategies can facilitate the maintenance of biodiversity in a community context by converting lethal interactions between neighboring colonies to neutral interactions where neither strain excludes the other. Thus, just as bacteriocins can lead to increased diversity via rock–paper–scissors dynamics, so too can antibiotics via elicitation and suppression. Our results reveal that social interactions are crucial for understanding antibiosis and bacterial community dynamics, and highlight the potential of interbacterial interactions for novel drug discovery by eliciting pathways that mediate interference competition.The discovery and development of antibiotics to fight bacterial diseases is one of the great triumphs in modern medicine (1). However, increasing rates of antimicrobial resistance require innovative strategies to replenish antimicrobial drug pipelines (2, 3). Several novel antibiotics have been discovered in previously unexplored habitats (4) or uncultured microbes (5). By contrast, a second potential source of novel agents, silent antibiotic gene clusters in well-characterized organisms, remains unexploited because the factors that elicit their production are unknown (1). Identifying these factors requires understanding the ecological and evolutionary roles of antibiotics in the competitive and social context in which they are used in nature (6, 7). Here we test the role of social and competitive dynamics on antibiosis in the prolific antibiotic-producing bacterial genus Streptomyces. Simultaneously, we distinguish competing hypotheses for the role of antibiotics in nature.Streptomycetes are a diverse group of filamentous bacteria that produce some two-thirds of all known antibiotics (8). Although the antibiotics they produce have classically been viewed as intermicrobial weapons (6, 9), this perspective is increasingly questioned on two grounds (10–13). First, antibiotic concentrations in soil are believed to be too low to kill or inhibit competing bacteria (9). Second, subinhibitory (sub-MIC) concentrations of antibiotics induce responses in exposed organisms, such as increased biofilm formation (14) or expression of virulence genes (11, 15) that may benefit these target cells (10). Thus, rather than weapons, these arguments have led to the idea that antibiotics are cooperative signals (16) used for intercellular communication, that they are “collective regulators of the homeostasis of microbial communities” (12).However, evidence of response to sub-MIC antibiotic concentrations does not imply that antibiotics are signals or a form of communication. Communication can be partitioned according to the costs and benefits associated with production and response (17). A signal is a form of mutually beneficial communication between the sender of a signal and its recipient. A cue, by contrast, elicits a response that benefits only the recipient, sometimes to the detriment of the sender. Finally, suppression or attenuation (18) elicits a response that harms the recipient and benefits the producer (19, 20). Whereas signals are a form of cooperation, the unidirectional benefits associated with cues and suppression imply that these are forms of competition.Distinguishing whether antibiotics are cooperative signals or competitive weapons requires partitioning communication into these contrasting modes (6, 19, 20) and examining the role of antibiotics in the competitive and social context in which they are used.