Disentangling mechanisms that mediate the balance between stochastic and deterministic processes in microbial succession

Ecological succession and the balance between stochastic and deterministic processes are two major themes within microbial ecology, but these conceptual domains have mostly developed independent of each other. Here we provide a framework that integrates shifts in community assembly processes with microbial primary succession to better understand mechanisms governing the stochastic/deterministic balance. Synthesizing previous work, we devised a conceptual model that links ecosystem development to alternative hypotheses related to shifts in ecological assembly processes. Conceptual model hypotheses were tested by coupling spatiotemporal data on soil bacterial communities with environmental conditions in a salt marsh chronosequence spanning 105 years of succession. Analyses within successional stages showed community composition to be initially governed by stochasticity, but as succession proceeded, there was a progressive increase in deterministic selection correlated with increasing sodium concentration. Analyses of community turnover among successional stages—which provide a larger spatiotemporal scale relative to within stage analyses—revealed that changes in the concentration of soil organic matter were the main predictor of the type and relative influence of determinism. Taken together, these results suggest scale-dependency in the mechanisms underlying selection. To better understand mechanisms governing these patterns, we developed an ecological simulation model that revealed how changes in selective environments cause shifts in the stochastic/deterministic balance. Finally, we propose an extended—and experimentally testable—conceptual model integrating ecological assembly processes with primary and secondary succession. This framework provides a priori hypotheses for future experiments, thereby facilitating a systematic approach to understand assembly and succession in microbial communities across ecosystems.A major goal in microbial community ecology is to understand the processes that underlie observed patterns in species abundances across space and time (1–3). Two types of processes—deterministic and stochastic—influence the assembly of species into communities. Deterministic processes—in which abiotic and biotic factors determine the presence/absence and relative abundances of species—are associated with ecological selection [sensu Vellend (4)]. Stochastic processes include probabilistic dispersal and random changes in species relative abundances (ecological drift) that are not the consequence of environmentally determined fitness (5, 6).Historically, microbial community assembly has been studied from a deterministic perspective (7, 8), where empirical evidence shows that a variety of environmental factors—such as pH, salinity, and organic carbon—influence community establishment at different scales (9, 10). However, recent studies have provided increasing support for a predominant role of stochasticity in some microbial systems (e.g., ref. 11). As opposed to a dichotomous debate, in which one attempts to reject stochastic processes in favor of deterministic ones (or vice versa), a more comprehensive perspective should integrate both processes and work to understand how and why their relative influences vary across systems, time, and space (3, 6, 12–15).The study of ecological succession provides an ideal setting for understanding mechanisms that govern community assembly processes through time and space. Although ecological succession in microbial communities has been broadly investigated (16–21), little has been done to formally link this theme with the balance in stochastic/deterministic processes. Only two studies have directly related these conceptual domains, and both have focused on secondary succession (i.e., following disturbance) (2, 20). These studies show that disturbance promotes a time-dependent shift in the stochastic/deterministic balance. A full understanding of linkages among community succession, disturbance, and the assembly processes, however, requires a testable conceptual framework that enables systematic evaluation of the stochastic/deterministic interplay during succession in both pristine and disturbed ecosystems.Here we set up a framework that integrates the conceptual domains of microbial succession and the balance in stochastic/deterministic ecological processes. We first devised a conceptual model that links environmental heterogeneity to shifts in these assembly processes during microbial primary succession; for this, we purposefully followed the approach used in Ferrenberg et al. (20) to allow a direct linkage between our model and theirs. Alternative hypotheses within the conceptual model were tested by applying an ecological null modeling approach (3) to data from a soil chronosequence spanning 105 years of primary ecosystem succession (22). The analyses revealed scale dependency with respect to how environmental factors govern the interplay between stochastic and deterministic processes. To better understand the mechanisms underlying the observed patterns, we developed an ecological simulation model that revealed how changes in selective environments cause shifts in the processes underlying community assembly. Finally, to facilitate conceptual synthesis and to generate a priori hypotheses for future experiments, we merged our conceptual model—focused on primary succession—with an extended version of the Ferrenberg et al. (20) secondary succession model.     

Metabolic dependencies drive species co-occurrence in diverse microbial communities

Microbial communities populate most environments on earth and play a critical role in ecology and human health. Their composition is thought to be largely shaped by interspecies competition for the available resources, but cooperative interactions, such as metabolite exchanges, have also been implicated in community assembly. The prevalence of metabolic interactions in microbial communities, however, has remained largely unknown. Here, we systematically survey, by using a genome-scale metabolic modeling approach, the extent of resource competition and metabolic exchanges in over 800 communities. We find that, despite marked resource competition at the level of whole assemblies, microbial communities harbor metabolically interdependent groups that recur across diverse habitats. By enumerating flux-balanced metabolic exchanges in these co-occurring subcommunities we also predict the likely exchanged metabolites, such as amino acids and sugars, that can promote group survival under nutritionally challenging conditions. Our results highlight metabolic dependencies as a major driver of species co-occurrence and hint at cooperative groups as recurring modules of microbial community architecture.Microbial communities are ubiquitous in nature and exert a large influence on our environment and health (1–5). These communities exhibit a great compositional variety, ranging from hot-spring assemblies with low species diversity (6) to the human gut microbiota harboring hundreds of species (7, 8). Competition for metabolic resources can affect community composition through competitive exclusion or by facilitating niche differentiation (9–11). Cooperative and syntrophic interactions, such as beneficial metabolic exchanges, are also likely to play an important role because they can substantially alter the nutritional quality of the habitat (8, 9, 11–15). For example, cross-feeding of metabolic by-products such as ethanol and acetate is central to the diversity of cellulose-degrading communities (16). However, such metabolic exchanges are difficult to discover in natural communities, because the metabolites in the environment cannot be easily attributed to a particular donor species or to the abiotic sources. Moreover, species can often use and secrete a large number of metabolites (17, 18), further hampering the elucidation of metabolic exchanges. Here, we tackle these challenges by introducing a modeling approach applicable to large microbial communities. Currently available methods for simulating metabolic exchanges (8, 19–22) are not directly relevant to communities occurring in nature. Whereas some of these methods use only topological information, ignoring mass balance and growth constraints, the others require prior knowledge of metabolic objective functions of the member species (i.e., evolutionarily selected beneficial characteristics such as high growth rate or optimal ATP production)—information that is often not available. In contrast, our modeling approach, termed “species metabolic interaction analysis,” or SMETANA, can be readily applied with as little information as species identity and their genome sequences. Starting with a community metabolic model assembled from the member-species-level models, SMETANA maps all possible interspecies metabolic exchanges. The methodology thus provides an unbiased estimate of the metabolic interaction potential of a community as well as identifies likely exchanged metabolites. We used this approach to interrogate over 800 microbial communities and co-occurring subcommunities therein. To capture interacting species modules beyond pairs, we also considered subcommunities with simultaneous co-occurrence of up to four species. Our results highlight metabolic dependencies as a key biotic force shaping the composition of diverse microbial communities in nature.     

Stochasticity,succession, and environmental perturbations in a fluidic ecosystem

Unraveling the drivers of community structure and succession in response to environmental change is a central goal in ecology. Although the mechanisms shaping community structure have been intensively examined, those controlling ecological succession remain elusive. To understand the relative importance of stochastic and deterministic processes in mediating microbial community succession, a unique framework composed of four different cases was developed for fluidic and nonfluidic ecosystems. The framework was then tested for one fluidic ecosystem: a groundwater system perturbed by adding emulsified vegetable oil (EVO) for uranium immobilization. Our results revealed that groundwater microbial community diverged substantially away from the initial community after EVO amendment and eventually converged to a new community state, which was closely clustered with its initial state. However, their composition and structure were significantly different from each other. Null model analysis indicated that both deterministic and stochastic processes played important roles in controlling the assembly and succession of the groundwater microbial community, but their relative importance was time dependent. Additionally, consistent with the proposed conceptual framework but contradictory to conventional wisdom, the community succession responding to EVO amendment was primarily controlled by stochastic rather than deterministic processes. During the middle phase of the succession, the roles of stochastic processes in controlling community composition increased substantially, ranging from 81.3% to 92.0%. Finally, there are limited successional studies available to support different cases in the conceptual framework, but further well-replicated explicit time-series experiments are needed to understand the relative importance of deterministic and stochastic processes in controlling community succession.Understanding how local communities assemble from a regional species pool is a central issue in community ecology (1–4). Two types of processes (deterministic vs. stochastic) influence the assembly of species into a local community. However, whether a local community structure is controlled by stochastic or deterministic processes is hotly debated (5–7). Traditional niche-based theory assumes that deterministic factors, including species traits, interspecies interactions (e.g., competition, predation, mutualisms, and tradeoffs), and environmental conditions, control local community compositions (8, 9). Consequently, the resulting local communities generally have little site-to-site variation in species composition (low β-diversity) when the environmental conditions are similar (10). In contrast, neutral theory (6) hypothesizes that all species are ecologically equivalent and ecological drift, i.e., stochastic processes of birth, death, colonization, extinction, and speciation (6, 11), govern the diversity and species composition of local communities independent of their traits and niches. When stochastic processes are coupled with priority effects, where early-arriving species influence the establishment and growth of later-arriving species (12), local communities with greater site-to-site variations (high β-diversity) in species compositions can emerge under similar, even identical, environmental conditions (10, 13, 14). The site-to-site variation in species composition due to stochastic processes (i.e., stochasticity) is unpredictable. To avoid confusion, we refer in this paper to such unpredictable variation in community composition as “compositional stochasticity.”It is now more generally accepted that both deterministic and stochastic processes occur simultaneously in the assembly of local communities (5, 15, 16), but their relative importance remains difficult to resolve (5, 13). It is also known that several factors such as habitat connectivity and size (4), productivity (15), disturbance (13), predation (10), and resource availability (17) influence the relative importance of stochastic vs. deterministic processes in the assembly of local communities. However, it is not clear whether and how their relative importance varies with time. In addition, although the mechanisms shaping the structure of ecological communities have been intensively studied (5, 14–16, 18–21), the drivers controlling ecological succession in response to environmental perturbations are poorly understood.The study of ecological succession remains at the core of ecology because knowledge of the temporal dynamics of ecological communities can help predict changes of biodiversity and ecosystem services in response to environmental change (22, 23). Ecological succession refers to more or less niche-based deterministic development of ecological community structure after perturbations (22, 24). Although, by definition, deterministic succession is expected under identical or rather similar environmental conditions, very few studies have examined the roles of stochastic processes controlling the succession of ecological communities (13, 15, 22). Neutral theory (6) predicts that chance, the stochasticity inherent in various probabilistic biological processes (such as dispersal, colonization, extinction, speciation, biotic interactions, and initial population heterogeneity) could lead to unpredictable variability in community composition (13, 22) (i.e., compositional stochasticity). However, assessing the degree of stochasticity and its role in ecological succession in field studies is challenging (18, 22) because the three components of ecological succession (stages, trajectories, and mechanisms) depend on the individual characteristics of community members, environment, and perturbations (23). Thus, manipulative experiments under similar, if not identical, initial conditions are valuable for disentangling the drivers controlling the succession of natural communities in response to environmental perturbations (18, 22).The physical characteristics of an ecosystem have dramatic impacts on its succession in response to environmental perturbations (23). System fluidity (connectivity) is expected to be a major factor in natural systems ranging from fluidic ecosystems (e.g., groundwater, rivers, ocean, lakes, wetlands, wastewater treatment plants, bioreactors, intestinal tract) to low or nonfluidic ecosystems (e.g., soils, sediments, deep subsurface). One key feature associated with the stochasticity of a fluidic ecosystem, as represented by the planktonic groundwater community examined in this study, is that dispersal is not significantly restricted at local scales due to high hydraulic conductivity. In contrast, microbial dispersal would be much more limited in nonfluidic ecosystems. Thus, it is expected that the stages, trajectories, and mechanisms of succession following a perturbation will vary dramatically between fluidic and nonfluidic ecosystems.In general, environmental perturbations have been classified into two categories (25): (i) increased nutrient input, especially with complex carbon (C) substrates (e.g., nutrient amendment, eutrophication, oil spill) and (ii) disturbances, which increase mortality and decrease biomass (e.g., drought, tillage, toxic chemicals, extreme temperature, salt and pH, predation). The type of environmental perturbation is also thought to influence the relative importance of stochasticity vs. determinism in community assembly (13, 22, 25). Nutrient input is believed to increase compositional stochasticity by enhancing ecological drift (e.g., stochastic processes of birth, death, colonization, and possibly extinction, and random change in species relative abundance) (5), and weakening niche selection by providing more resources (C, energy, nutrients). In contrast, it is generally believed that extreme disturbances such as drought often decrease compositional stochasticity by acting as selection factors (13), eliminating a large portion of the regional species pool. In addition, perturbations can also be classified as pulses or presses, depending on their duration (26). Whereas pulse perturbations are relatively discrete and short term, press perturbations are continuous and of long duration.Microorganisms play integral and unique roles in various ecosystem processes and functions and are of enormous importance in global biogeochemistry, human health (27), energy (28), environmental remediation (29), engineering (30), and agriculture (31). However, in contrast to their known ecological primacy, the drivers controlling the succession of microbial communities in response to environmental perturbations are poorly understood. Thus, in this study, we developed a novel theoretical framework to conceptualize the relationships of succession to stochasticity. This framework is composed of four different models to predict the relative importance of stochastic processes in mediating microbial community succession with respect to ecosystem characteristics (fluidic vs. nonfluidic) and environmental perturbations (nutrient input vs. disturbance). We then tested one of these models for a specific fluidic system: a groundwater system perturbed by addition of emulsified vegetable oil (EVO) for uranium immobilization as a part of a long-term field-scale bioremediation experiment (32). The advantages of this system are that it is open and well connected with high hydraulic conductivity so that dispersal appeared to not be a major limiting factor in community assembly and that the study had sufficient replicate time-series data on many community variables (i.e., genes/functional populations) to allow null model analysis.Our main objectives of this study were to answer the following questions: (i) How does a groundwater microbial community’s structure respond to the environmental perturbation of C amendment? (ii) What are the relative roles of deterministic and stochastic factors in determining community composition and succession? (iii) Does the relative importance of deterministic and stochastic factors change over time? Our results suggest that stochastic processes play predominant roles in controlling the succession of the groundwater microbial communities in response to the nutrient amendment but that the relative importance of stochastic and deterministic processes is time dependent. To the best of our knowledge, this is the first study to conceptualize the relationships between stochasticity and ecological succession and to demonstrate the importance and dynamic behavior of stochastic processes in controlling the succession of microbial communities.     

Soil biodiversity and soil community composition determine ecosystem multifunctionality

Biodiversity loss has become a global concern as evidence accumulates that it will negatively affect ecosystem services on which society depends. So far, most studies have focused on the ecological consequences of above-ground biodiversity loss; yet a large part of Earth’s biodiversity is literally hidden below ground. Whether reductions of biodiversity in soil communities below ground have consequences for the overall performance of an ecosystem remains unresolved. It is important to investigate this in view of recent observations that soil biodiversity is declining and that soil communities are changing upon land use intensification. We established soil communities differing in composition and diversity and tested their impact on eight ecosystem functions in model grassland communities. We show that soil biodiversity loss and simplification of soil community composition impair multiple ecosystem functions, including plant diversity, decomposition, nutrient retention, and nutrient cycling. The average response of all measured ecosystem functions (ecosystem multifunctionality) exhibited a strong positive linear relationship to indicators of soil biodiversity, suggesting that soil community composition is a key factor in regulating ecosystem functioning. Our results indicate that changes in soil communities and the loss of soil biodiversity threaten ecosystem multifunctionality and sustainability.It has long been recognized that biodiversity can be the mechanism behind the performance of an ecosystem, particularly in communities of above-ground organisms (1–5). In soils below ground, however, the functioning of biodiversity is not well understood (6). Soils are highly diverse. It has been estimated that 1 g of soil contains up to 1 billion bacteria cells consisting of tens of thousands of taxa, up to 200 m fungal hyphae, and a wide range of mites, nematodes, earthworms, and arthropods (7, 8). This vast and hidden diversity contributes to the total terrestrial biomass and is intimately linked to above-ground biodiversity (9, 10).In recent years several studies have shown that anthropogenic activities, such as agricultural intensification and land use change, reduce microbial and faunal abundance and the overall diversity of soil organisms (11–13). This has triggered increasing concern that reduced biodiversity in soils may impair numerous ecosystem functions, such as nutrient acquisition by plants and the cycling of resources between above- and below-ground communities (6, 11, 13, 14). However, to date research has largely focused on the effects of specific groups of organisms, such as soil microbes (15, 16), mycorrhizal fungi (17, 18), and soil fauna (19, 20), or on large-scale correlative analysis in the field (13). However, soil organisms interact within complex food webs, and therefore changes in diversity within one trophic group or functional guild may alter the abundance, diversity, and functioning of another (21, 22). Hence, it is important to know how changes in soil biodiversity and the simplification of the soil community composition influences ecosystem functioning. However, whether reductions of biodiversity in soil communities have consequences for the overall performance of an ecosystem remains unresolved. Moreover, recent studies show that above-ground plant diversity influences multiple ecosystem functions, defined as ecosystem multifunctionality (23). However, it is still unclear whether ecosystem multifunctionality is likewise influenced by soil biodiversity.Here we manipulated soil biodiversity and soil community composition in model grassland microcosms simulating European grassland. We tested whether changes in soil biodiversity and soil community composition influenced multiple ecosystem functions. To manipulate soil biodiversity and soil community composition, we inoculated the grassland microcosms with different soil communities. The soil inoculum was prepared by fractionating soil communities according to size, using filters of decreasing mesh size (19). This method reduces the abundance of different groups of soil organisms at different mesh sizes, thus altering the community composition and the overall diversity of soil organisms simultaneously (19). To maintain the different soil community treatments and to prevent microbial contamination, we maintained the communities in self-contained microcosms in which we could restrict external contamination (24). Additionally, the experiment was repeated and performed for a longer period to confirm initial results and include additional measures on ecosystem characteristics. We hypothesized that soil biodiversity loss reduces ecosystem functioning and multifunctionality. Specifically, we hypothesized that plant diversity, decomposition, and the recycling of nutrients is impaired when the diversity and abundance of various groups of soil biota (e.g., fungi, mycorrhizal fungi, bacteria, and nematodes) are reduced.     

From the Cover: PNAS Plus: Cell-cycle regulation of formin-mediated actin cable assembly

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.Eukaryotic cells contain populations of actin structures with distinct architectures and protein compositions, which mediate varied cellular processes (1). Understanding how F-actin polymerization is regulated in time and space is critical to understanding how actin structures provide mechanical forces for corresponding biological processes. Branched actin filament arrays, which concentrate at sites of clathrin-mediated endocytosis (2, 3) and at the leading edge of motile cells (4), are nucleated by the actin-related protein-2/3 (Arp2/3) complex. In contrast, bundles of unbranched actin filaments, which sometimes mediate vesicle trafficking or form myosin-containing contractile bundles, often are nucleated by formin proteins (5–14).Much has been learned about how branched actin filaments are polymerized by the Arp2/3 complex and how these filaments function in processes such as endocytosis (2, 15). In contrast, relatively little is known about how actin cables are assembled under physiological conditions. In previous studies, branched actin filaments derived from the Arp2/3 complex have been reconstituted using purified proteins (16–19) or cellular extracts (20–25). When microbeads were coated with nucleation-promoting factors for the Arp2/3 complex and then were incubated in cell extracts, actin comet tails were formed by sequential actin nucleation, symmetry breaking, and tail elongation. Importantly, the motility behavior of F-actin assembled by the Arp2/3 complex using defined, purified proteins differs from that of F-actin assembled by the Arp2/3 complex in the full complexity of cytoplasmic extracts (19, 26–28).Formin-based actin filament assembly using purified proteins also has been reported (29, 30). However, reconstitution of formin-derived actin cables under the more physiological conditions represented by cell extracts has not yet been reported.The actin nucleation activity of formin proteins is regulated by an inhibitory interaction between the N- and C-terminal domains, which can be released when GTP-bound Rho protein binds to the formin N-terminal domain, allowing access of the C terminus (FH1-COOH) to actin filament barbed ends (31–40). In yeast, the formin Bni1 N terminus also has an inhibitory effect on actin nucleation through binding to the C terminus (41).Interestingly, several recent reports provided evidence for cell-cycle regulation of F-actin dynamics in oocytes and early embryos (42–45). However, which specific types of actin structures are regulated by the cell cycle and what kind of nucleation factors and actin interacting-proteins are involved remain to be determined.Here, we report a reconstitution of actin cables in yeast extracts from microbeads derivatized with Bni1 FH1-COOH, identifying the proteins involved, increasing the inventory of the proteins that regulate actin cable dynamics and establishing that the actin cable reconstitution in cytoplasmic extracts is cell-cycle regulated.     

Functional ecology of an Antarctic Dry Valley

The McMurdo Dry Valleys are the largest ice-free region in Antarctica and are critically at risk from climate change. The terrestrial landscape is dominated by oligotrophic mineral soils and extensive exposed rocky surfaces where biota are largely restricted to microbial communities, although their ability to perform the majority of geobiological processes has remained largely uncharacterized. Here, we identified functional traits that drive microbial survival and community assembly, using a metagenomic approach with GeoChip-based functional gene arrays to establish metabolic capabilities in communities inhabiting soil and rock surface niches in McKelvey Valley. Major pathways in primary metabolism were identified, indicating significant plasticity in autotrophic, heterotrophic, and diazotrophic strategies supporting microbial communities. This represents a major advance beyond biodiversity surveys in that we have now identified how putative functional ecology drives microbial community assembly. Significant differences were apparent between open soil, hypolithic, chasmoendolithic, and cryptoendolithic communities. A suite of previously unappreciated Antarctic microbial stress response pathways, thermal, osmotic, and nutrient limitation responses were identified and related to environmental stressors, offering tangible clues to the mechanisms behind the enduring success of microorganisms in this seemingly inhospitable terrain. Rocky substrates exposed to larger fluctuations in environmental stress supported greater functional diversity in stress-response pathways than soils. Soils comprised a unique reservoir of genes involved in transformation of organic hydrocarbons and lignin-like degradative pathways. This has major implications for the evolutionary origin of the organisms, turnover of recalcitrant substrates in Antarctic soils, and predicting future responses to anthropogenic pollution.The largest ice-free regions on the Antarctic continent are the McMurdo Dry Valleys, designated by international treaty as an Antarctic Special Managed Area (1) to reflect their environmental significance. The Dry Valleys are among the most threatened environments from climate change due to their polar location and unique ecology (2, 3). The terrestrial landscape is dominated by oligotrophic mineral soils (4) and extensive rocky outcrops with life restricted mainly to microbial communities due to the extreme environmental stress (5).Extensive recent research has focused on elucidating the biodiversity of Dry Valleys landscapes (5–16). Classical microbiological studies identified edaphic Antarctic taxa by morphology and revealed a general recalcitrance to cultivation (17, 18). Molecular interrogations greatly expanded understanding of taxonomic community structure (5–16, 19) and speculated on the origin of inocula such as from streams that freeze dry and subsequently are dispersed by wind in winter (5).These and other studies have provided major insight into the biodiversity of soil and rock niches largely from the rRNA gene perspective, although a caveat to any solely molecular study concerning biodiversity and functionality is that DNA can be isolated from nonviable propagules (20) or from inactive material blown in from elsewhere such as dessicated microbial mats on streams, dry lake beds, glaciers, coastal ice, and wetlands or brought in by snowfall (21). A limited number of in situ respirometry studies also indicated microbial contributions to carbon and nitrogen transformations may be significant (22, 23). A picture of the extent of bacterial colonization has emerged, but also soil and rock niches support algae (24), fungi (20), lichen (25), mosses (26), and invertebrates (27). A high degree of niche specialization in terms of rRNA gene-defined community assembly has been recorded between open soil, hypolith (colonized ventral surface of quartz), chasmoendolith (colonized cracks and fissures in sandstone and granite), and cryptoendolith (colonized pore spaces in weathered sandstone) (5).Despite this, almost nothing is known about the contribution of Dry Valleys microbial communities in soils and rocks toward metabolic processes essential to biological mineral transformation and the stress tolerance mechanisms that allow them to flourish in such harsh extremes. The less extreme sub-Antarctic and maritime peninsula Antarctic locations received relatively greater attention, and the GeoChip-based functional gene arrays were successfully applied to indicate pathways and taxonomic identity of soil microbial carbon and nitrogen utilization (28–31) but they represent a fundamentally different biome compared with the extreme polar desert of the Dry Valleys ecosystem (32).Here, we present findings from a comprehensive study using GeoChip to address key issues related to microbial contributions in geobiological processes, including carbon cycling genes, i.e., involved in autotrophy, acetogenesis, and methanogenesis; nitrogen cycling genes, i.e., nitrification and assimilatory and dissimilatory nitrogen reductions; and also, importantly, the stress tolerance strategies available to microbes to survive in these harsh environmental conditions. The data were compared with PCR-based diversity of 16S rRNA genes and biomass estimates and demonstrated autotrophic, heterotrophic, and diazotrophic pathways in Antarctic Dry Valleys microorganisms. Strikingly, we identified unique stress response pathways that can be directly related to environmental stressors in this Antarctic environment. Finally, we highlighted soil microbial pathways that indicate a potential ability to transform lignin-like molecules and anthropogenic pollutants and discussed this in view of the evolutionary origin of the microorganisms, increasing human exposure, and potential future contamination in the Dry Valleys system.     

Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest

The phyllosphere—the aerial surfaces of plants, including leaves—is a ubiquitous global habitat that harbors diverse bacterial communities. Phyllosphere bacterial communities have the potential to influence plant biogeography and ecosystem function through their influence on the fitness and function of their hosts, but the host attributes that drive community assembly in the phyllosphere are poorly understood. In this study we used high-throughput sequencing to quantify bacterial community structure on the leaves of 57 tree species in a neotropical forest in Panama. We tested for relationships between bacterial communities on tree leaves and the functional traits, taxonomy, and phylogeny of their plant hosts. Bacterial communities on tropical tree leaves were diverse; leaves from individual trees were host to more than 400 bacterial taxa. Bacterial communities in the phyllosphere were dominated by a core microbiome of taxa including Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria, and Sphingobacteria. Host attributes including plant taxonomic identity, phylogeny, growth and mortality rates, wood density, leaf mass per area, and leaf nitrogen and phosphorous concentrations were correlated with bacterial community structure on leaves. The relative abundances of several bacterial taxa were correlated with suites of host plant traits related to major axes of plant trait variation, including the leaf economics spectrum and the wood density–growth/mortality tradeoff. These correlations between phyllosphere bacterial diversity and host growth, mortality, and function suggest that incorporating information on plant–microbe associations will improve our ability to understand plant functional biogeography and the drivers of variation in plant and ecosystem function.The phyllosphere—the aerial surfaces of plants—is an important and ubiquitous habitat for bacteria (1). It is estimated that on a global scale, the phyllosphere spans more than 10⁸ km² and is home to up to 10²⁶ bacterial cells (2). Bacteria are also important to their plant hosts. Leaf-associated bacteria represent a widespread and ancient symbiosis (3, 4) that can influence host growth and function in many ways, including the production of growth-promoting nutrients and hormones (5, 6) and protection of hosts against pathogen infection (7, 8). Phyllosphere bacteria have the potential to influence plant biogeography and ecosystem function through their influence on plant performance under different environmental conditions (9–11), but the drivers of variation in leaf-associated bacterial biodiversity among host plants are not well understood.The ability to quantify microbial community structure in depth with environmental sequencing technologies has led to an increasing focus not only on the ecology of individual microbial taxa but on the entire genomic content of communities of microbes in different habitats, or “microbiomes” (12). Numerous studies of host-associated microbiomes have shown that microbial biodiversity is a trait (13) that forms part of the extended phenotype of the host organism (4, 14, 15) with important effects on the health and fitness (16–18) and evolution (19–21) of the host. Because of the importance of the microbiome for host fitness and function, there is a growing desire to model and manage host–microbiome interactions (22, 23), and understanding the drivers of host-associated microbial community assembly has thus become a cornerstone of microbiome research (24).In animals, the assembly of host-associated microbiomes is known to be driven by ecologically important host attributes, such as diet, that covary with host evolutionary history (20, 25, 26). A similar understanding of the drivers of plant microbiome assembly is lacking. Most of our knowledge of plant–bacterial associations on leaves has been based on studies of individual bacterial strains and individual host species. Different plant species possess characteristic bacterial phyllosphere communities (27, 28), and there are several examples of variation in bacterial biodiversity on leaves among plant genotypes (29–31) as well as among species and higher taxonomic ranks (32). Although these patterns are presumably due to phylogenetic variation in ecologically important plant functional traits (33) among host populations and species, the influence of host functional traits on variation in phyllosphere community structure across host species has not been directly quantified. As a result, we have very little understanding of the potential of plant–microbe interaction networks to influence the distribution and functional biogeography of their hosts at large scales in the face of global change (34).A first step toward integrating phyllosphere microbial communities into the study of plant biogeography will require establishing correlations between microbial community structure on leaves and the functional traits of plant hosts. To address this goal, we used high-throughput sequencing to characterize the structure of the bacterial phyllosphere microbiome on the leaves of multiple host tree species in a diverse neotropical forest in Panama. We combined phyllosphere microbiome data with a rich dataset on the attributes of plant hosts, including functional traits and evolutionary relationships, to (i) quantify the magnitude of leaf-associated bacterial biodiversity in a diverse natural forest community, (ii) identify the host plant attributes that influence microbiome community assembly on leaves, and (iii) understand relationships between bacterial biodiversity and suites of host plant traits and functions and discuss their implications for our understanding of plant functional biogeography.     

Patterned progression of bacterial populations in the premature infant gut

In the weeks after birth, the gut acquires a nascent microbiome, and starts its transition to bacterial population equilibrium. This early-in-life microbial population quite likely influences later-in-life host biology. However, we know little about the governance of community development: does the gut serve as a passive incubator where the first organisms randomly encountered gain entry and predominate, or is there an orderly progression of members joining the community of bacteria? We used fine interval enumeration of microbes in stools from multiple subjects to answer this question. We demonstrate via 16S rRNA gene pyrosequencing of 922 specimens from 58 subjects that the gut microbiota of premature infants residing in a tightly controlled microbial environment progresses through a choreographed succession of bacterial classes from Bacilli to Gammaproteobacteria to Clostridia, interrupted by abrupt population changes. As infants approach 33–36 wk postconceptional age (corresponding to the third to the twelfth weeks of life depending on gestational age at birth), the gut is well colonized by anaerobes. Antibiotics, vaginal vs. Caesarian birth, diet, and age of the infants when sampled influence the pace, but not the sequence, of progression. Our results suggest that in infants in a microbiologically constrained ecosphere of a neonatal intensive care unit, gut bacterial communities have an overall nonrandom assembly that is punctuated by microbial population abruptions. The possibility that the pace of this assembly depends more on host biology (chiefly gestational age at birth) than identifiable exogenous factors warrants further consideration.The vertebrate digestive system hosts a profound transition from a state of complete or near-sterility in utero to dense bacterial colonization within weeks of birth. This event has lasting effects on the host (1), influencing health and development (2–4), infection resistance (5, 6), predisposition to inflammatory (7) and metabolic disorders (8), and immune function (9), but remarkably little is known about this process. Gut colonization has been partly characterized in term infants (10–12) who reside in open venues, and who will, therefore, experience many exposures (e.g., contact with older children, adults, and pets, varying diets, oral antibiotics) that could drive microbial population assembly (1, 11–13).A delineation of the dynamics of the natural de novo assembly of this microbial community would form a basis for better understanding how the gut acquires its founding microbiome, and how the bacteria in the gut start their transition to population equilibrium (1, 14, 15). In view of the importance of bacterial gut colonization, we sought to determine if the initial assembly of host intestinal microbial populations follows discernible patterns, and if interventions such as antibiotics or nutrition alter this progression. A discernibly patterned progression would suggest that host biology influences bacterial community assembly more than do random encounters of individuals with microbes, whereas stochastic assembly would suggest that random encounters sculpt population structure. In this latter scenario, the gut serves as a passive culture chamber. Fine interval enumeration of gut contents from multiple subjects in as controlled an environment as possible is needed to answer this question.Here, we demonstrate that the gut microbiota of premature infants residing in a tightly controlled environment of a neonatal intensive care unit (NICU) progresses through a choreographed succession of bacterial classes from Bacilli to Gammaproteobacteria to Clostridia interrupted by abrupt population changes. The rate of assembly is slowest for the most premature of these infants.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Tetratricopeptide repeat protein protects photosystem I from oxidative disruption during assembly

A Chlamydomonas reinhardtii mutant lacking CGL71, a thylakoid membrane protein previously shown to be involved in photosystem I (PSI) accumulation, exhibited photosensitivity and highly reduced abundance of PSI under photoheterotrophic conditions. Remarkably, the PSI content of this mutant declined to nearly undetectable levels under dark, oxic conditions, demonstrating that reduced PSI accumulation in the mutant is not strictly the result of photodamage. Furthermore, PSI returns to nearly wild-type levels when the O₂ concentration in the medium is lowered. Overall, our results suggest that the accumulation of PSI in the mutant correlates with the redox state of the stroma rather than photodamage and that CGL71 functions under atmospheric O₂ conditions to allow stable assembly of PSI. These findings may reflect the history of the Earth’s atmosphere as it transitioned from anoxic to highly oxic (1–2 billion years ago), a change that required organisms to evolve mechanisms to assist in the assembly and stability of proteins or complexes with O₂-sensitive cofactors.Although the structure and function of photosystem I (PSI) in plants, algae, and cyanobacteria have been elucidated at high spatial and temporal resolution (1–7), PSI assembly is poorly understood but is a topic of growing interest (7). Unlike PSII, there are essentially no inhibitors of PSI, and PSI assembly intermediates are difficult to separate from mature complexes (7–9). Furthermore, PSI abundance is not highly controlled by environmental conditions (8), and mutants with much lower levels of PSI than WT cells can still grow under photoautotrophic conditions (10, 11), although they often are light sensitive (12, 13) and the level of PSI in a mutant may not show a linear correlation with its rate of photoautotrophic growth.Progress in understanding PSI assembly has come largely from studies of mutants in putative assembly factors (7, 10, 11, 14, 15), including hypothetical chloroplast open reading frame 3 (Ycf3), Ycf3-interacting protein 1 (Y3IP1), Ycf4, plant-specific putative DNA-binding protein 1 (PPD1), and Ycf37/pale yellow green7-1 (Pyg7-1). Ycf3 is a plastid-encoded protein with tetratricopeptide repeat (TPR) domains believed to interact transiently with PsaA and PSAD (16), whereas Y3IP1 interacts with Ycf3 (10). Ycf4 has two transmembrane domains and is necessary for PSI assembly in Chlamydomonas, but tobacco mutants lacking Ycf4 accumulate sufficient PSI to grow photoautotrophically (11). ALB3 (ALBINO3) mediates the insertion of the chloroplast-encoded core PSI proteins, PsaA and PsaB, into thylakoid membranes (17) but also is involved in the biogenesis of other photosynthetic complexes (7, 18, 19). PPD1 is required for establishing proper structure/function relationships for the luminal portion of PSI (15).One of the least understood of the proteins associated with PSI assembly is the Chlamydomonas protein CGL71. This protein is part of the GreenCut, a bioinformatically assembled set of proteins present in all green lineage organisms examined; many of these proteins are associated with photosynthetic function (20–25). CGL71 is orthologous to Ycf37 of Synechocystis (26) and PYG7 of Arabidopsis (27). In this study, we present evidence that supports a role for CGL71 in PSI assembly and, more specifically, in protecting the complex from oxidative disruption during assembly. The requirement of CGL71 for proper assembly of PSI may reflect an evolutionary adaptation that is linked to oxygenation of the Earth’s atmosphere.     

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

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

Stoichiometry and specific assembly of Best ion channels

Human Bestrophin 1 (hBest1) is a calcium-activated chloride channel that regulates neuronal excitability, synaptic activity, and retinal homeostasis. Mutations in hBest1 cause the autosomal-dominant Best macular dystrophy (BMD). Because hBest1 mutations cause BMD, but a knockout does not, we wondered if hBest1 mutants exert a dominant negative effect through interaction with other calcium-activated chloride channels, such as hBest2, 3, or 4, or transmembrane member 16A (TMEM16A), a member of another channel family. The subunit architecture of Best channels is debated, and their ability to form heteromeric channel assemblies is unclear. Using single-molecule subunit analysis, we find that each of hBest1, 2, 3, and 4 forms a homotetrameric channel. Despite considerable conservation among hBests, hBest1 has little or no interaction with other hBests or mTMEM16A. We identify the domain responsible for assembly specificity. This domain also plays a role in channel function. Our results indicate that Best channels preferentially self-assemble into homotetramers.Bestrophin 1 is calcium-activated chloride channel (CACC) and has been shown to express in a variety of tissues (1). In the brain, Best1 plays a crucial role in the regulation of neuronal excitability and synaptic activity by releasing gliotransmitters such as glutamate and GABA from astrocytes upon G-protein–coupled receptor (GPCR) activation (2–5). In retinal pigment epithelium (RPE) cells, Best1 plays an important role in retinal homeostasis (1), and mutations in human Best1 have been implicated in several retinal degenerative diseases including Best Macular Dystrophy (BMD) (6–12) and Retinitis Pigmentosa (13).The human bestrophin family includes three additional members; hBest2, 3, and 4 (14, 15). All four members function in heterologous cells (15–18) as anion-selective channels, whose main physiological charge carrier is chloride (15, 17, 19–22), but which also permeate glutamate and GABA (3, 4).hBest1 contains six hydrophobic segments (S1–S6), with both N and C termini residing inside the cell. Two topology models have been proposed for hBest1 (15, 23). In the first model, S1, S2, S4, and S6 traverse the membrane, whereas S3 is intracellular and S5 forms a reentrant loop from outside (15). A more recent model has S1, S2, S5, and S6 traversing the membrane and S3 and S4, although hydrophobic, remaining on the intracellular side (23).Best1 is activated by Ca²⁺ with a K_d of ∼150 nM (24). Several pieces of evidence suggest that this activation is due to direct binding of Ca²⁺ (25, 26) to an EF hand located immediately after S6 (24). It is unclear how Ca²⁺-binding gates the channel and whether the EF hand is part of the gate or communicates with it.Although much progress has been made on Best channels (15, 17, 19–22, 27), several fundamental aspects of the structure and function of this channel family are not understood. First, previous biochemical analysis has indicated that Best1 is multimeric (22, 27) but led to conflicting assessments of the number of subunits in the channel, with experiments on human Best1 suggesting a tetramer or pentamer (22) but experiments on porcine Best1 suggesting a dimer (27). Second, although coimmunoprecipitation suggests that hBest1 interacts with hBest2 (22), it is unclear if this is direct interaction. Moreover, virtually nothing is known about the determinants of assembly.In this study, we used single-molecule subunit counting and colocalization to address four major questions about the subunit assembly and function of hBest channels: (i) What is the subunit stoichiometry of hBest channels? (ii) Does hBest1 coassemble with any other member of the hBest family or with a member of different CACC family, transmembrane member 16A (TMEM16A)? (iii) How is subunit assembly specified? (iv) Does the assembly determinant play any role in channel function?     

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.