A globally distributed mobile genetic element inhibits natural transformation of Vibrio cholerae

Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA⁺ ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence decreased to ∼40% from 2006 to 2011. Thus, IdeA⁺ ICEs may have limited the role of natural transformation in V. cholerae. However, the rise in prevalence of strains lacking IdeA may now increase the role of this conserved mechanism of HGT in the evolution of this pathogen.The causative agent of the diarrheal disease cholera, Vibrio cholerae, is annually responsible for 3.5 million infections worldwide (1). This facultative pathogen naturally resides in temperate aquatic environments and causes disease when ingested in contaminated food or water. A critical nutrient for Vibrio species in the aquatic environment is the chitinous exoskeleton of crustacean zooplankton (2–4). Chitin is an insoluble polysaccharide composed of β-1,4-linked GlcNAc. In addition to serving as a carbon and nitrogen source, chitin also induces a physiological state in V. cholerae known as natural competence (5). In this state, bacteria can take up DNA from the extracellular environment and integrate this DNA into their chromosomes by homologous recombination. This cumulative process of DNA uptake and integration is known as natural transformation and is one mechanism for horizontal gene transfer (HGT) in V. cholerae. HGT by natural transformation is used by pathogenic microbes to evolve in the face of clinical intervention and immune pressure. Indeed, in V. cholerae, this mechanism of HGT is hypothesized to have generated an antigenic variant, the O139 outbreak strain, through homologous recombination and replacement of the locus responsible for O-antigen biosynthesis (6–9).Another mechanism of HGT in V. cholerae is integrating conjugative elements (ICEs) of the SXT/R391 family. These elements can range from ∼80 to 110 Kb in size and contain all of the genes required for conjugative transfer into naive hosts (10, 11); they integrate in a site-specific manner into the 5′ end of the highly conserved prfC (peptide-chain-release factor C) gene (10–12). The first natural transfer of an ICE into V. cholerae likely occurred between 1980 and 1985 (10, 13) and, by the 1990s, virtually all clinical isolates of V. cholerae contained an ICE (13). These elements confer resistance to multiple antibiotics, and it is likely that widespread use of antibiotics has rapidly selected for strains containing ICEs. There are at least 10 genetically distinct ICEs circulating in the V. cholerae population (11). These ICEs share a core set of genes, but have varied gene content at distinct sites. The most common ICE in V. cholerae is VchInd5, which is present in ∼77% of currently sequenced clinical isolates (10, 11). It is hypothesized that the current (seventh) pandemic of cholera originated in the Bay of Bengal, and strains have spread globally from this region in three overlapping waves of transmission (13, 14). Strains containing VchInd5 are globally distributed, indicating that the original transfer of VchInd5 into V. cholerae may have occurred in this region.In 2010, cholera spread to Haiti, a region that previously lacked this disease (15, 16). Phylogenetic and Bayesian analyses indicate that all strains in Haiti share a common ancestor, which was introduced into the region at the outset of the epidemic (16, 17). Consistent with this finding, strains from Haiti ubiquitously harbor a VchInd5 ICE. Throughout the epidemic, strains have acquired mutations that are likely generated intrinsically, and there is no evidence of horizontal gene transfer among these isolates (16). Consistent with this finding, strains from the Haiti outbreak were found to be poorly transformed by chitin-induced natural competence (16).In this study, we identify and characterize an ICE-encoded DNase present on VchInd5 that inhibits HGT by natural transformation in V. cholerae. We also assess the role and prevalence of this DNase in limiting transformation among clinical isolates from Haiti and Bangladesh.     

Identification of divergent type VI secretion effectors using a conserved chaperone domain

The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.Protein secretion systems play a pivotal role in bacterial interspecies interaction and virulence (1, 2). Of the known secretion systems in Gram-negative bacteria, the type VI secretion system (T6SS) enables bacteria to compete with both eukaryotic and prokaryotic species through delivery of toxic effectors (2–4). The T6SS is a multicomponent nanomachine analogous to the contractile bacteriophage tail (5). First characterized in Vibrio cholerae (6) and Pseudomonas aeruginosa (7), the T6SS has now been identified in ∼25% of Gram-negative bacteria, including many important pathogens (2, 8), and has been implicated as a critical factor in niche competition (9–11).The T6SS structure is composed of an Hcp inner tube, a VipAB outer sheath that wraps around the Hcp tube, a tip complex consisting of VgrG and PAAR proteins, and a membrane-bound baseplate (2, 4, 12). Sheath contraction drives the inner Hcp tube and the tip proteins, VgrG and PAAR, outward into the environment and neighboring cells (13, 14). The contracted sheath is then dissembled by an ATPase ClpV and recycled for another T6SS assembly and contraction event (12, 15, 16). Two essential T6SS baseplate components, VasF and VasK, are homologous to the DotU and IcmF proteins of the type IV secretion system (T4SS) in Legionella pneumophila (17).Bacteria often possess multiple copies of VgrG and PAAR genes that form the tip of T6SS, and deletion of VgrG and PAAR genes abolishes T6SS secretion (14). Some VgrG and PAAR proteins carry functional extension domains and thus act as secreted T6SS effectors, as exemplified by the VgrG1 actin cross-linking domain (6), VgrG3 lysozyme domain in V. cholerae (18, 19), and the nuclease domain of the PAAR protein RhsA in Dickeya dadantii (20). Known T6SS effectors can target a number of essential cellular components, including the actin and membrane of eukaryotic cells (18, 21, 22) and the cell wall, membrane, and DNA of bacterial cells (3, 18–20, 23, 24). Each antibacterial effector coexists with an antagonistic immunity protein that confers protection during T6SS-mediated attacks between sister cells (3, 18, 24). Interestingly, T6SS-mediated lethal attacks induce the generation of reactive oxygen species in the prey cells (25), similar to cells treated with antibiotics (26, 27).For non-VgrG/PAAR–related effectors, their translocation requires either binding to the inner tube Hcp proteins as chaperones or binding to the tip VgrG proteins (2, 14, 28). T6SS-dependent effectors can be experimentally identified by comparing the secretomes of WT and T6SS mutants (3, 29–31) and by screening for T6SS-encoded immunity proteins (18). Because known effectors lack a common secretion signal, bioinformatic identification of T6SS effectors is challenging. A heuristic approach based on the physical properties of effectors has been used to identify a superfamily of peptidoglycan-degrading effectors in bacteria (32). A recent study identified a common N-terminal motif in a number of T6SS effectors (31). However, this motif does not exist in the T6SS effector TseL in V. cholerae (18).In this study, we report that VC1417, the gene upstream of tseL, encodes a protein with a highly conserved domain, DUF4123. We show that VC1417 is required for TseL delivery and interacts with VgrG1 (VC1416) and TseL. Because of the genetic linkage of VC1417 and TseL and its importance for TseL secretion, we postulated that genes encoding the conserved DUF4123 domain proteins are generally located upstream of genes encoding putative T6SS effectors. Using the conserved domain sequence, we bioinformatically predicted a large family of effector proteins with diverse functions in Gram-negative bacteria. We validated our prediction by the identification and characterization of a new secreted effector TseC and its antagonistic immunity protein TsiC in Aeromonas hydrophila SSU. Our results demonstrate a new effective approach to identify T6SS effectors with highly divergent sequences.     

Morphogenesis and cell ordering in confined bacterial biofilms

Biofilms are aggregates of bacterial cells surrounded by an extracellular matrix. Much progress has been made in studying biofilm growth on solid substrates; however, little is known about the biophysical mechanisms underlying biofilm development in three-dimensional confined environments in which the biofilm-dwelling cells must push against and even damage the surrounding environment to proliferate. Here, combining single-cell imaging, mutagenesis, and rheological measurement, we reveal the key morphogenesis steps of Vibrio cholerae biofilms embedded in hydrogels as they grow by four orders of magnitude from their initial size. We show that the morphodynamics and cell ordering in embedded biofilms are fundamentally different from those of biofilms on flat surfaces. Treating embedded biofilms as inclusions growing in an elastic medium, we quantitatively show that the stiffness contrast between the biofilm and its environment determines biofilm morphology and internal architecture, selecting between spherical biofilms with no cell ordering and oblate ellipsoidal biofilms with high cell ordering. When embedded in stiff gels, cells self-organize into a bipolar structure that resembles the molecular ordering in nematic liquid crystal droplets. In vitro biomechanical analysis shows that cell ordering arises from stress transmission across the biofilm–environment interface, mediated by specific matrix components. Our imaging technique and theoretical approach are generalizable to other biofilm-forming species and potentially to biofilms embedded in mucus or host tissues as during infection. Our results open an avenue to understand how confined cell communities grow by means of a compromise between their inherent developmental program and the mechanical constraints imposed by the environment.

The growth of living organisms is critically influenced by the external environment. One form of such environmental influence is the transmission of mechanical stress, which can instruct morphogenesis in systems ranging from stem cells to tissues to the entire organisms (1, 2). In the prokaryotic domain, bacteria commonly live in complex communities encased by an extracellular matrix (3) known as biofilms (4). Biofilm formation is a morphogenetic process whereby a single founder cell develops into a three-dimensional (3D) aggregate in which bacterial cells interact with each other and with the environment (4–8). Recent work has revealed biophysical mechanisms underlying biofilm morphogenesis on solid substrates, which is controlled by cell–substrate adhesion and the resulting shear stress (9–15). In addition to those living on surfaces, bacterial communities are also commonly found inside soft, structured environments, such as hydrogels. Examples include biofilms growing in mucus layers and host tissues during an infection or food contamination (16). Indeed, many common biofilm formers including Pseudomonas aeruginosa and Vibrio cholerae encounter biological hydrogels as their niche during infection (17, 18). Under these conditions, embedded biofilms must grow against 3D confinement and potentially damage the surrounding environment—a process that is fundamentally different from how surface-attached biofilms expand against friction with the surface (10, 13, 15, 19). However, little is known about how biofilms develop under such 3D mechanical constraints, including how cells collectively organize in response to the confinement and how the confining environment, in turn, is modified by cell proliferation. This is in stark contrast to the accumulating knowledge on the growth dynamics of mammalian cell aggregates and tumors under confinement (20, 21).In this study, we integrate single-cell live imaging, mutagenesis, in vitro mechanical testing, and numerical modeling to investigate how the 3D confinement determines the morphodynamics and cell ordering of an embedded biofilm. A model system is established by embedding V. cholerae, the causal agent of the cholera pandemic and a model biofilm former (22, 23), inside agarose gels (24). By using 3D visualization techniques with high spatiotemporal resolution, we reveal that embedded biofilms undergo a shape transition and a series of self-organization events that are distinct from those in surface-attached biofilms. We first show that the stiffness contrast between the biofilm and the confining hydrogels controls a transition between spherical and ellipsoidal biofilms. Furthermore, we discover that embedded biofilms display a core-shell structure with intricate ordering similar to nematic liquid crystal (LC) droplets (25). Finally, we demonstrate that Vibrio polysaccharide (VPS) and cell-to-surface adhesion proteins effectively transmit stress between the environment and the biofilm, giving rise to distinct cell ordering patterns in embedded biofilms.     

Saharan dust nutrients promote Vibrio bloom formation in marine surface waters

Vibrio is a ubiquitous genus of marine bacteria, typically comprising a small fraction of the total microbial community in surface waters, but capable of becoming a dominant taxon in response to poorly characterized factors. Iron (Fe), often restricted by limited bioavailability and low external supply, is an essential micronutrient that can limit Vibrio growth. Vibrio species have robust metabolic capabilities and an array of Fe-acquisition mechanisms, and are able to respond rapidly to nutrient influx, yet Vibrio response to environmental pulses of Fe remains uncharacterized. Here we examined the population growth of Vibrio after natural and simulated pulses of atmospherically transported Saharan dust, an important and episodic source of Fe to tropical marine waters. As a model for opportunistic bacterial heterotrophs, we demonstrated that Vibrio proliferate in response to a broad range of dust-Fe additions at rapid timescales. Within 24 h of exposure, strains of Vibrio cholerae and Vibrio alginolyticus were able to directly use Saharan dust–Fe to support rapid growth. These findings were also confirmed with in situ field studies; arrival of Saharan dust in the Caribbean and subtropical Atlantic coincided with high levels of dissolved Fe, followed by up to a 30-fold increase of culturable Vibrio over background levels within 24 h. The relative abundance of Vibrio increased from ∼1 to ∼20% of the total microbial community. This study, to our knowledge, is the first to describe Vibrio response to Saharan dust nutrients, having implications at the intersection of marine ecology, Fe biogeochemistry, and both human and environmental health.Bacteria in the Vibrio genus are globally distributed in marine environments but typically make up a minor portion of the total microbial assemblage (1, 2); however, Vibrio have been shown to bloom in response to often poorly characterized environmental factors (3, 4). Like other opportunistic heterotrophic bacteria, Vibrio can have disproportionately large impacts on carbon and nutrient processing because of their ability to reproduce rapidly and respond to pulses of newly available resources (2, 5–7). Characterized as “opportunitrophs,” Vibrio have a broad genomic and metabolic repertoire (8), allowing them to compete in highly variable nutrient environments ranging from the open ocean to pathogenic associations with animal hosts (3, 9). This genus includes many well-known pathogens of marine organisms and humans, and disease incidence has risen sharply in the last 20 y (10, 11). Common human pathogens include the causative agent of the severe diarrheal disease cholera (Vibrio cholerae), shellfish-associated gastroenteritis (Vibrio parahaemolyticus and Vibrio vulnificus), and seawater-associated wound infections (Vibrio vulnificus and Vibrio alginolyticus) (10). Studies examining the environmental drivers and distribution of Vibrio have largely focused on the role of Vibrio in disease, generally overlooking the importance of Vibrio in the biogeochemical cycling of key nutrients and trace metals (3, 4). Iron (Fe) is an essential micronutrient for Vibrio growth in the environment as well as during host invasion, where it is actively sequestered by the host to prevent bacterial colonization (9). Vibrio have evolved to be adept scavengers of Fe in a variety of conditions (12). Despite the importance of Fe for growth, there has been little characterization of the effects of environmental Fe enrichment on Vibrio population dynamics and the role of these bacteria in Fe cycling in marine systems.Fe can be a limiting micronutrient in marine primary and secondary production (13, 14). As an essential cofactor in many metabolic processes—including aerobic respiration, photosynthesis, and nitrogen fixation—its availability can be a determinant in the cycling of carbon (C) and biologically important macronutrients, like nitrogen (N) and phosphorus (P) (13, 15, 16). Dissolved Fe (dFe) is believed to be the most biologically available fraction of Fe, but is present in vanishingly low amounts in marine systems around the world, especially due to the low solubility of Fe(III) in seawater (17). Heterotrophic bacteria, including Vibrio, play a key role in Fe cycling (14, 15, 18, 19), in part by modulating Fe solubility through secretion of high-affinity Fe-chelating siderophores (20, 21). Fe-siderophore complexes allow active uptake into the bacterial cell (20) and provide a usable exogenous source of Fe for phytoplankton (22, 23). Although most studies of Fe enrichment in marine systems have focused on autotrophs (24, 25), heterotrophic bacteria have been shown to have a higher Fe per biomass quota than many phytoplankton (18), accounting for up to 80% of the total planktonic uptake in some systems (26).Atmospheric dust deposition is a major source of Fe in the global ocean (27) and is estimated to deliver more than triple the amount of dFe as riverine inflow (28). Globally, the Saharan desert is the largest source region of this atmospheric dust–Fe (27). Dust originating in northern Africa is swept across the Atlantic Ocean along easterly trade winds, producing spatiotemporal gradients of dust deposition in the Caribbean and southeastern United States, especially during the summer months (29, 30). Increased atmospheric processing time, associated with long-range transport as well as wet deposition (rain washout), are hypothesized to alter components of atmospheric dust and produce a soluble and highly biologically available form of Fe (17, 27). Although the response of marine autotrophs to dust deposition has been investigated (15, 25), the full biological response to the episodic deposition of dust–Fe to ocean surface water, especially among microbial communities, has yet to be clearly elucidated. Evidence is growing that heterotrophic bacteria, especially among the class γ-proteobacteria, may play a role in processing deposited minerals and nutrients (31, 32), but studies to date, which have largely focused on bulk bacterial response and longer timescales, have shown equivocal results (33, 34). We hypothesized that specific opportunistic bacteria, like Vibrio, can respond quickly to newly available dust-associated Fe and suggest a role of dust–Fe as a driver of Vibrio population dynamics.     

Physical evidence of predatory behavior in Tyrannosaurus rex

Feeding strategies of the large theropod, Tyrannosaurus rex, either as a predator or a scavenger, have been a topic of debate previously compromised by lack of definitive physical evidence. Tooth drag and bone puncture marks have been documented on suggested prey items, but are often difficult to attribute to a specific theropod. Further, postmortem damage cannot be distinguished from intravital occurrences, unless evidence of healing is present. Here we report definitive evidence of predation by T. rex: a tooth crown embedded in a hadrosaurid caudal centrum, surrounded by healed bone growth. This indicates that the prey escaped and lived for some time after the injury, providing direct evidence of predatory behavior by T. rex. The two traumatically fused hadrosaur vertebrae partially enclosing a T. rex tooth were discovered in the Hell Creek Formation of South Dakota.One of the most daunting tasks of paleontology is inferring the behavior and feeding habits of extinct organisms. Accurate reconstruction of the lifestyle of extinct animals is dependent on the fossil evidence and its interpretation is most confidently predicated on analogy with modern counterparts (1–6). This challenge to understanding the lifestyle of extinct animals is exemplified by the controversy over the feeding behavior of the Late Cretaceous theropod Tyrannosaurus rex (3, 7–17). Although predation and scavenging have often been suggested as distinct feeding behavior alternatives (3, 7–9, 11–17), these terms merit semantic clarification. In this study, predation is considered a subset of feeding behavior, by which any species kills what it eats. Although the term “predator” is used to distinguish such animals from obligate scavengers, it does not imply that the animal did not also scavenge.Ancient diets can be readily reconstructed on the basis of the available evidence, although their derivation (e.g., predation or scavenging behavior) often remains elusive. Speculation as to dinosaur predation has ranged from inferences based on skeletal morphology, ichnofossils such as bite marks, coprolites, stomach contents, and trackways and, by more rarely, direct predator–prey skeletal associations (3, 4, 18–23).Direct evidence of predation in nonavian dinosaurs other than tyrannosaurids has been observed in rare instances, such as the Deinonychus–Tenontosaurus kill site of the Cloverly Formation where the remains of both were found in close association along with shed teeth (9, 24), and the “fighting dinosaurs” from the Gobi Desert, in which a Velociraptor and Protoceratops were found locked in mortal combat (9, 17). The evidence on tyrannosaurids is more limited. Putative stomach contents, such as partially digested juvenile hadrosaur bones, have been reported in association with tyrannosaurid remains (3, 12, 18). This latter instance only represents physical evidence of the last items consumed before the animal’s death, an indicator of diet but not behavior.Mass death assemblages of ornithischians frequently preserve shed theropod teeth (6, 22, 24). Lockley et al. (23) suggest such shed teeth are evidence of scavenging behavior. It is widely argued that T. rex procured food through obligate scavenging rather than hunting (11, 14, 25–27) despite the fact that there is currently no modern analog for such a large bodied obligate scavenger (26). Horner (25) argued that T. rex was too slow to pursue and capture prey items (14) and that large theropods procured food solely through scavenging, rather than hunting (11, 25). Horner also suggested that the enlarged olfactory lobes in T.rex were characteristic of scavengers (25). More recent studies (28, 29) determined the olfactory lobes of modern birds are “poorly developed,” inferring that enlarged olfactory lobes in T. rex are actually a secondary adaptation for predation navigation “to track mobile, dispersed prey” (30). T. rex has a calculated bite force stronger than that of any other terrestrial predator (7), between 35,000 and 57,000 Newtons (30, 31), and possible ambulatory speeds between 20 and 40 kph (7, 15, 16), documenting that it had the capability to pursue and kill prey items.Healed injuries on potential prey animals provide the most unequivocal evidence of survival of a traumatic event (e.g., predation attempt) (3, 32, 33), and several reports attribute such damage to T. rex (4, 17, 19, 20). These include broken and healed proximal caudal vertebral dorsal spines in Edmontosaurus (17) and healed cranial lesions in Triceratops (4, 19). Although the presence of healed injuries demonstrates that an animal lived long enough after the attack to create new bone at the site of the damage (a rare occurrence in the fossil record) (19), the healing usually obliterates any clear signature linking the injury to a specific predator. Bite traces (e.g., raking tooth marks on bone and puncture wounds in the bones of possible prey animals) attributed to T. rex (2, 4, 19) are ambiguous, because the damage inflicted upon an animal during and after a successful hunt mirrors feeding during scavenging. This makes distinction between the two modes of food acquisition virtually impossible with such evidence (3, 34–38).Tooth marks, reported from dinosaur bone-bearing strata worldwide (e.g., 2–4, 8, 19, 20, 39, 40), are further direct evidence of theropod feeding behavior, attributed by some to specific theropod groups (2, 4, 19, 20). Happ (19) and Carpenter (17) identified theropods to family and genus by matching spaces to parallel marks (traces) with intertooth distance. Happ (19) described opposing conical depressions on a left supraorbital Triceratops horn that was missing its distal third (tip), attributing them to a bite by either a T. rex or a crocodilian. Happ (19) stated that the spacing of the parallel marks present on the left squamosal of the same individual matched the intertooth distance of tyrannosaurids. The presence of periosteal reaction documents healing. This contrasts with the report by Farlow and Holtz (3) and again by Hone and Rauhut (20) of the same Hypacrosaurus fibula containing a superficially embedded theropod tooth. Absence of bone reaction precludes confident attribution to predation.Two coalesced hadrosaur (compare with Edmontosaurus annectens) caudal vertebrae were discovered in the Hell Creek Formation of Harding County, South Dakota (40). Archosaur fauna identified in this site include crocodiles, dinosaurs, and birds (41). Physical evidence of dental penetration and extensive infection (osteomylitis) of the fused vertebral centra and healing (bone overgrowth) document an unsuccessful attack by a large predator. A tooth crown was discovered within the wound, permitting identification of the predator as T. rex. This is unambiguous evidence that T. rex was an active predator, fulfilling the criteria that Farlow and Holtz (3) advanced. As T. rex comprises between 1% and 16% of the Upper Cretaceous dinosaurian fauna in Western North America (41–45), its status as a predator or obligate scavenger is nontrivial and could have significant implications for paleoecological reconstructions of that time period. The present contribution provides unique information demonstrating the ecological role for T. rex as that of an active predator. Despite this documentation of predatory behavior by T. rex, we do not make the argument that T. rex was an obligate predator. Like most modern large predators (27, 45) it almost certainly did also scavenge carcasses (9, 16).     

From the Cover: A single macrolichen constitutes hundreds of unrecognized species

The number of Fungi is estimated at between 1.5 and 3 million. Lichenized species are thought to make up a comparatively small portion of this figure, with unrecognized species richness hidden among little-studied, tropical microlichens. Recent findings, however, suggest that some macrolichens contain a large number of unrecognized taxa, increasing known species richness by an order of magnitude or more. Here we report the existence of at least 126 species in what until recently was believed to be a single taxon: the basidiolichen fungus Dictyonema glabratum, also known as Cora pavonia. Notably, these species are not cryptic but morphologically distinct. A predictive model suggests an even larger number, with more than 400 species. These results call into question species concepts in presumably well-known macrolichens and demonstrate the need for accurately documenting such species richness, given the importance of these lichens in endangered ecosystems such as paramos and the alarming potential for species losses throughout the tropics.Fungi make up the second largest kingdom, with an estimated number of 1.5–3 million species (1–3). Lichenization plays an important role in fungal evolution, particularly in the Ascomycota, where lichens make up 30% of the currently recognized species (4–6). Transition toward a lichenized lifestyle appears to have taken place at least 10 times in the Ascomycota and 5 times in the Basidiomycota (7–9), but the distribution of lichen formers favors the Ascomycota, with the Basidiomycota accounting for less than 0.3% of all lichenized Fungi (7, 10). Altogether, ∼18,000 lichenized species are currently accepted, but estimates suggest that this represents only 50–65% of the true species richness (4, 6).Global species richness of lichenized Basidiomycota appears to be especially underestimated. The Dictyonema clade, which includes some of the best-known basidiolichens, until recently was considered to represent five species in a single genus, Dictyonema (11, 12). Subsequent taxonomic and molecular phylogenetic studies suggested that this concept did not reflect the true diversity in this clade (7, 12, 13). Currently, a total of 43 species are recognized in five genera (14, 15). Two genera, Cora and Corella, are foliose macrolichens, with a total of 16 species, corresponding to what was considered a single species, Dictyonema glabratum (11, 12, 16). This name is well known in the scientific community and even among nonspecialists and is included in the Listing of Interesting Plants of the World (17). The 16-fold increase in the number of species now recognized is a striking figure that even surpasses recent findings reported from the large macrolichens Lobariella and Sticta in the Ascomycota (18, 19). The dramatic change in the taxonomic concept of these basidiolichens has important implications for recognizing their role in ecosystem function and as model organisms. Species of Cora abound in tropical montane regions and, with their cyanobacterial photobionts capable of fixing atmospheric nitrogen, serve as biological fertilizers (20). Cora is also one of the best studied lichens in terms of ecomorphology, ecophysiology, and biochemistry (10, 21–28).     

LRP1 is a receptor for Clostridium perfringens TpeL toxin indicating a two-receptor model of clostridial glycosylating toxins

Large glycosylating toxins are major virulence factors of various species of pathogenic Clostridia. Prototypes are Clostridium difficile toxins A and B, which cause antibiotics-associated diarrhea and pseudomembranous colitis. The current model of the toxins’ action suggests that receptor binding is mediated by a C-terminal domain of combined repetitive oligopeptides (CROP). This model is challenged by the glycosylating Clostridium perfringens large cytotoxin (TpeL toxin) that is devoid of the CROP domain but still intoxicates cells. Using a haploid genetic screen, we identified LDL receptor-related protein 1 (LRP1) as a host cell receptor for the TpeL toxin. LRP1-deficient cells are not able to take up TpeL and are not intoxicated. Expression of cluster IV of LRP1 is sufficient to rescue toxin uptake in these cells. By plasmon resonance spectroscopy, a K_D value of 23 nM was determined for binding of TpeL to LRP1 cluster IV. The C terminus of TpeL (residues 1335–1779) represents the receptor-binding domain (RBD) of the toxin. RBD-like regions are conserved in all other clostridial glycosylating toxins preceding their CROP domain. CROP-deficient C. difficile toxin B is toxic to cells, depending on the RBD-like region (residues 1349–1811) but does not interact with LRP1. Our data indicate the presence of a second, CROP-independent receptor-binding domain in clostridial glycosylating toxins and suggest a two-receptor model for the cellular uptake of clostridial glycosylating toxins.Clostridial glycosylating toxins are major pathogenicity factors that are responsible for numerous severe diseases in humans and animals. Prototypes of these toxins are Clostridium difficile toxins A and B, the causative agents of antibiotics-associated diarrhea and pseudomembranous colitis (1, 2). During recent years, morbidity and mortality of C. difficile-induced infections (CDI) largely increased (3, 4). In fact, CDI advanced to one of the most important nosocomial infections in developed countries. Other members of the family of clostridial glycosylating toxins are Clostridium sordellii lethal and hemorrhagic toxin, and the α-toxin from Clostridium novyi, which cause gas gangrene syndromes (5). All these toxins have a very similar primary structure comprising an amino acid sequence identity of 40–90% (1, 5). Recently, an ABCD model has been proposed for these toxins with an N-terminally located glycosyltransferase domain (domain A), a subsequent cysteine protease domain for autoproteolytic cleavage (domain C), a putative pore-forming and delivery domain (domain D), and a C-terminal binding domain (domain B) (6). After binding to cell surface receptors, the toxins are endocytosed in a clathrin-dependent and dynamin-dependent manner (7). At a low pH of endosomes, the toxins insert into endosomal membranes and form pores, which probably allow translocation of the glycosyltransferase (GT) and cysteine protease domains into the cytosol where inositol hexakisphosphate activates the protease for autoproteolytic cleavage and release of the GT into the cytosol (8–10). In the cytosol, Rho and/or Ras proteins are glucosylated or modified by GlcNAcylation, resulting in inhibition of these switch proteins and, eventually, in inflammation and cell death (11–13). Although autoproteolytic processing and toxin-induced glycosylation of Rho/Ras proteins are well characterized, the interaction of the toxins with cell surface receptors is still enigmatic. Cell surface-binding is suggested to be mediated by C-terminal polypeptide repeats (B domain) termed combined repetitive oligopeptides (CROP) that might recognize cell surface carbohydrate structures (14–17). Recombinant fragments of this C-terminal toxin part blocks toxin binding and cytotoxicity (18). Moreover, monoclonal antibodies raised against this toxin portion prevent cell intoxication (19). Putative receptors have been described for C. difficile toxin A, including carbohydrates, glycophospholipids, and proteins (16, 17, 20, 21). The hypothesis that the C-terminal part of clostridial glycosylating toxins is solely responsible for receptor interaction has been challenged. For example, after removal of the CROP domain of toxin A or toxin B, the toxins were still cytotoxic (10, 22). Thus, we and others hypothesized that a receptor binding site different from the CROP domain might be involved in toxin binding.Recently, TpeL, the most recent member of the family of clostridial glycosylating toxins that is produced by Clostridium perfringens type A, B, and C strains, was described (23). TpeL exhibits 30–40% amino acid sequence identity with other clostridial glycosylating toxins and shares with them the glycosyltransferase domain, the cysteine protease domain, and the delivery domain. However, it does not possess a CROP domain. Nevertheless, TpeL intoxicates cells and kills mice (23). Toxic effects of TpeL are probably due to GlcNAcylation of Ras proteins at threonine-35 (24). In addition, TpeL-induced modification of Rac has been reported (25).Thus, TpeL is a model toxin to unravel the interaction of clostridial glycosylating toxins with target cells. Here, we identify the low-density lipoprotein receptor-related protein 1 (LRP1) as a target molecule for binding and cell entry of TpeL. We report that the C-terminal part of TpeL binds to LRP1 and represents the receptor-binding domain. Furthermore, we show that the respective part within C. difficile toxin B, which resembles to the receptor-binding domain of TpeL, binds to cells. Our study strongly supports a two-receptor model of clostridial glycosylating toxins and offers an additional perspective in the understanding of the pathogenicity of this group of clinically important toxins.     

Infection of phytoplankton by aerosolized marine viruses

Marine viruses constitute a major ecological and evolutionary driving force in the marine ecosystems. However, their dispersal mechanisms remain underexplored. Here we follow the dynamics of Emiliania huxleyi viruses (EhV) that infect the ubiquitous, bloom-forming phytoplankton E. huxleyi and show that EhV are emitted to the atmosphere as primary marine aerosols. Using a laboratory-based setup, we showed that the dynamic of EhV aerial emission is strongly coupled to the host–virus dynamic in the culture media. In addition, we recovered EhV DNA from atmospheric samples collected over an E. huxleyi bloom in the North Atlantic, providing evidence for aerosolization of marine viruses in their natural environment. Decay rate analysis in the laboratory revealed that aerosolized viruses can remain infective under meteorological conditions prevailing during E. huxleyi blooms in the ocean, allowing potential dispersal and infectivity over hundreds of kilometers. Based on the combined laboratory and in situ findings, we propose that atmospheric transport of EhV is an effective transmission mechanism for spreading viral infection over large areas in the ocean. This transmission mechanism may also have an important ecological impact on the large-scale host–virus “arms race” during bloom succession and consequently the turnover of carbon in the ocean.Oceanic phytoplankton blooms are the major primary producers that constitute the base of marine food webs, and are key components of large biogeochemical cycles in the ocean (1). Emiliania huxleyi (Prymnesiophyceae, Haptophyta) is a dominant, bloom-forming phytoplankton that plays a pivotal role in carbon and sulfur cycles owing to its high productivity, calcification rates, and DMS production and emission to the atmosphere (2–4). In recent years it has become evident that E. huxleyi blooms are largely influenced by the activity of EhV, a lytic large double-stranded DNA coccolithovirus (Phycodnaviridae) that specifically infects E. huxleyi cells, accelerating the turnover and determining the fate of phytoplankton biomass (5–7). Bloom dynamics in the ocean is often characterized by a rapid demise of E. huxleyi cells owing to viral infection (5–10) that occurs over thousands of kilometers. Until recently, virus dispersal was thought to be solely mediated by physical processes within the water body, such as diffusion, advection, and mixing (11, 12). Recently, it has been shown that zooplankton can further enhance viral dispersal (13). These viral-dispersal mechanisms are restricted to processes within the water body. Recent evidence suggests that marine primary aerosols produced by wind-induced bubble bursting in the ocean (14) can be highly enriched with microorganisms (15–19). Nevertheless, there is very limited information on the presence of aerosolized marine viruses and their possible role as a transmission mechanism affecting large-scale host–virus interactions during algal bloom succession.     

Ancestral polymorphisms shape the adaptive radiation of Metrosideros across the Hawaiian Islands

Some of the most spectacular adaptive radiations begin with founder populations on remote islands. How genetically limited founder populations give rise to the striking phenotypic and ecological diversity characteristic of adaptive radiations is a paradox of evolutionary biology. We conducted an evolutionary genomics analysis of genus Metrosideros, a landscape-dominant, incipient adaptive radiation of woody plants that spans a striking range of phenotypes and environments across the Hawaiian Islands. Using nanopore-sequencing, we created a chromosome-level genome assembly for Metrosideros polymorpha var. incana and analyzed whole-genome sequences of 131 individuals from 11 taxa sampled across the islands. Demographic modeling and population genomics analyses suggested that Hawaiian Metrosideros originated from a single colonization event and subsequently spread across the archipelago following the formation of new islands. The evolutionary history of Hawaiian Metrosideros shows evidence of extensive reticulation associated with significant sharing of ancestral variation between taxa and secondarily with admixture. Taking advantage of the highly contiguous genome assembly, we investigated the genomic architecture underlying the adaptive radiation and discovered that divergent selection drove the formation of differentiation outliers in paired taxa representing early stages of speciation/divergence. Analysis of the evolutionary origins of the outlier single nucleotide polymorphisms (SNPs) showed enrichment for ancestral variations under divergent selection. Our findings suggest that Hawaiian Metrosideros possesses an unexpectedly rich pool of ancestral genetic variation, and the reassortment of these variations has fueled the island adaptive radiation.

Adaptive radiations exhibit extraordinary levels of morphological and ecological diversity (1). Although definitions of adaptive radiation vary (2–7), all center on ecological opportunity as a driver of adaptation and, ultimately, diversification (2, 8–10). Divergent selection, the primary mechanism underlying adaptive radiations, favors extreme phenotypes (11) and selects alleles that confer adaptation to unoccupied or under-utilized ecological niches. Differential adaptation results in divergence and, ultimately, reproductive isolation between populations (12). Adaptive radiations demonstrate the remarkable power of natural selection as a driver of biological diversity and provide excellent systems for studying evolutionary processes involved in diversification and speciation (13).Adaptive radiations on remote oceanic islands are especially interesting, as colonization of remote islands is expected to involve population bottlenecks that restrict genetic variation (14). Adaptive radiations in such settings are especially impressive and even paradoxical, given the generation of high species richness from an initially limited gene pool (15). Several classic examples of adaptive radiation occur on oceanic islands, such as Darwin’s finches from the Galapagos islands (16), anole lizards from the Caribbean islands (9), Hawaiian Drosophilids (17), and Hawaiian silverswords (18), to name a few.Recent advances in genome sequencing and analyses have greatly improved our ability to examine the genetics of speciation and adaptive radiation. By examining sequences of multiple individuals from their natural environment, it has become possible to “catch in the act” the speciation processes between incipient lineages (19). Genomic studies of early stage speciation show that differentiation accumulates in genomic regions that restrict the homogenizing effects of gene flow between incipient species (20). The number, size, and distribution of these genomic regions can shed light on evolutionary factors involved in speciation (19). Regions of high genomic differentiation can also form from evolutionary factors unrelated to speciation, such as linkage associated with recurrent background selection or selective sweeps on shared genomic features (21, 22).Genomic studies of lineages undergoing rapid ecological diversification have begun to reveal the evolutionary mechanisms underlying adaptive radiations. Importantly, these studies highlight the pivotal role of hybridization between populations and the consequent exchange of adaptive alleles that facilitates rapid speciation and the colonization of diverse niches (23–25). Most genomic studies of adaptive radiation involve animal systems, however, in particular, birds and fishes. In plants, genomic studies of adaptive radiation are sparse (26–28), and all examine continent-wide radiations. There are no genomics studies of plant adaptive radiations in geographically restricted systems such as remote islands. Because the eco-evolutionary scenarios associated with adaptive radiations are diverse (5, 29), whether commonalities identified in adaptive radiations in animals (23, 30) are applicable to plants is an open question. For example, the genetic architecture of animal adaptive radiations typically involves differentiation at a small number of genomic regions (31–33). In contrast, the limited insights available for plants suggest a more complex genetic architecture (26).We investigated the evolutionary genomics of adaptive radiation in Metrosideros Banks ex Gaertn. (Myrtaceae) across the Hawaiian Islands. Hawaiian Metrosideros is a landscape-dominant, hypervariable, and highly dispersible group of long-lived (possibly >650 y) (34) woody taxa that are nonrandomly distributed across Hawaii’s heterogeneous landscape, including cooled lava flows, wet forests and bogs, subalpine zones, and riparian zones (35, 36). About 25 taxa or morphotypes are distinguished by vegetative characters ranging from prostate plants that flower a few centimeters above ground to 30-m-tall trees, and leaves range dramatically in size, shape, pubescence, color, and rugosity (35, 37, 38); a majority of these forms are intraspecific varieties or races (provisional varieties) of the abundant species, Metrosideros polymorpha (35, 36, 38). Variation in leaf mass per area within the four Metrosideros taxa on Hawaii Island alone matches that observed for woody species globally (39). Common garden experiments (38, 40–44) and parent–offspring analysis (45) demonstrate heritability of taxon-diagnostic vegetative traits, indicating that taxa are distinct genetic groups and not the result of phenotypic plasticity. Metrosideros taxa display evidence of local adaptation to contrasting environments (46, 47), suggesting ecological divergent selection is responsible for diversification within the group (48). This diversification, which spans the past ∼3.1 to 3.9 million years (49, 50), has occurred despite the group’s high capacity for gene flow by way of showy bird-pollinated flowers and tiny wind-dispersed seeds (36, 51). Lastly, the presence of partial reproductive isolating barriers between taxa is consistent with the early stages of speciation (52). Here, we generated several genomic resources for Hawaiian Metrosideros and used these in population genomics analyses to gain deeper insights into the genomic architecture and evolutionary processes underlying this island adaptive radiation.     

From the Cover: Canine sexual dimorphism in Ardipithecus ramidus was nearly human-like

Body and canine size dimorphism in fossils inform sociobehavioral hypotheses on human evolution and have been of interest since Darwin’s famous reflections on the subject. Here, we assemble a large dataset of fossil canines of the human clade, including all available Ardipithecus ramidus fossils recovered from the Middle Awash and Gona research areas in Ethiopia, and systematically examine canine dimorphism through evolutionary time. In particular, we apply a Bayesian probabilistic method that reduces bias when estimating weak and moderate levels of dimorphism. Our results show that Ar. ramidus canine dimorphism was significantly weaker than in the bonobo, the least dimorphic and behaviorally least aggressive among extant great apes. Average male-to-female size ratios of the canine in Ar. ramidus are estimated as 1.06 and 1.13 in the upper and lower canines, respectively, within modern human population ranges of variation. The slightly greater magnitude of canine size dimorphism in the lower than in the upper canines of Ar. ramidus appears to be shared with early Australopithecus, suggesting that male canine reduction was initially more advanced in the behaviorally important upper canine. The available fossil evidence suggests a drastic size reduction of the male canine prior to Ar. ramidus and the earliest known members of the human clade, with little change in canine dimorphism levels thereafter. This evolutionary pattern indicates a profound behavioral shift associated with comparatively weak levels of male aggression early in human evolution, a pattern that was subsequently shared by Australopithecus and Homo.

A small canine tooth with little sexual dimorphism is a well-known hallmark of the human condition. The small and relatively nonprojecting deciduous canine of the first known fossil of Australopithecus, the Taung child skull, was a key feature used by Raymond Dart for his inference that the fossil represented an early stage of human evolution (1). However, recovery of additional Australopithecus fossils led to the canine of Australopithecus africanus to be characterized as large (compared to that of humans or “robust australopithecines”) and its morphology primitive, based on a projecting main cusp and crown structures lacking or hardly expressed in Homo (2). Later, the perception of a large and primitive canine was enhanced by the discovery and recognition of Australopithecus afarensis and Australopithecus anamensis (3–8), the latter species extending back in time to 4.2 million years ago (Ma). Although assessments of canine size variation and sexual dimorphism in Au. afarensis were hampered by limited sample sizes (9, 10), some suggested that the species had a more dimorphic canine than do humans, equivalent in degree to the bonobo (11) or to chimpanzees and orangutans (12). Initially, Au. anamensis was suggested to express greater canine dimorphism than did Au. afarensis (13, 14). However, based on a somewhat larger sample size, this is now considered to be the case with the tooth root but not necessarily its crown (15–17).Throughout the 1990s and 2000s, a pre-Australopithecus record of fossils spanning >6.0 to 4.4 Ma revealed that the canines of these earlier forms did not necessarily exceed those of Au. afarensis or Au. anamensis in general size (18–28). However, all these taxa apparently possessed canine crowns on average about 30% larger than in modern humans, which makes moderately high levels of sexual dimorphism potentially possible. Canine sexual dimorphism, combined with features such as body size dimorphism, inform sociobehavioral and ecological adaptations of past and present primates, and therefore have been of considerable interest since Darwin’s 1871 considerations (29–57). In particular, the relationship of canine size dimorphism (and/or male and female relative canine sizes) with reproductive strategies and aggression/competition levels in primate species have been a continued focus of interest (14, 33, 35–45, 49–56). Conspecific-directed agonistic behavior in primates related to mate and/or resource competition can be particularly intense among males both within and between groups (14, 44, 57). It is widely recognized that a large canine functions as a weapon in intra- and intergroup incidences of occasional lethal aggression (45, 58–61), and a large, tall canine has been shown or inferred to significantly enhance male fitness (50, 56). Hence, canine size and dimorphism levels in fossil species provide otherwise unavailable insights into their adaptive strategies.Here, we apply a recently developed method of estimating sexual size dimorphism from fossil assemblages of unknown sex compositions, the posterior density peak (pdPeak) method (62), and reexamine canine sexual dimorphism in Ardipithecus ramidus at ∼4.5 Ma. We include newly available fossils recovered from the Middle Awash and Gona paleoanthropological research areas in the Afar Rift, Ethiopia (26, 63, 64) in order to obtain the most reliable dimorphism estimates currently possible. We apply the same method to Australopithecus, Homo, and selected fossil apes, and evaluate canine sexual dimorphism through evolutionary time.We operationally define canine sexual dimorphism as the ratio between male and female means of basal canine crown diameters (the m/f ratio). Because the canines of Ar. ramidus, Au. anamensis, and extant and fossil apes are variably asymmetric in crown shape, we examine the maximum basal dimension of the crown. This can be either the mesiodistal crown diameter or a maximum diameter taken from the distolingual to mesiobuccal crown base (7, 27, 65). In the chronologically later Au. afarensis and all other species of Australopithecus sensu lato and Homo, we examine the more widely available conventional metric of buccolingual breadth, which corresponds to or approximates the maximum basal crown diameter. In anthropoid primates, canine height is more informative than basal canine diameter as a functional indicator of aggression and/or related display (14, 41–44). We therefore also examine available unworn and minimally worn fossil canines with reliable crown heights.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.