Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics

The central questions of bacterial ecology and evolution require a method to consistently demarcate, from the vast and diverse set of bacterial cells within a natural community, the groups playing ecologically distinct roles (ecotypes). Because of a lack of theory-based guidelines, current methods in bacterial systematics fail to divide the bacterial domain of life into meaningful units of ecology and evolution. We introduce a sequence-based approach ("ecotype simulation") to model the evolutionary dynamics of bacterial populations and to identify ecotypes within a natural community, focusing here on two Bacillus clades surveyed from the "Evolution Canyons" of Israel. This approach has identified multiple ecotypes within traditional species, with each predicted to be an ecologically distinct lineage; many such ecotypes were confirmed to be ecologically distinct, with specialization to different canyon slopes with different solar exposures. Ecotype simulation provides a long-needed natural foundation for microbial ecology and systematics.     

Characterization of an electron conduit between bacteria and the extracellular environment

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.     

Essential genes from Arctic bacteria used to construct stable,temperature-sensitive bacterial vaccines

All bacteria share a set of evolutionarily conserved essential genes that encode products that are required for viability. The great diversity of environments that bacteria inhabit, including environments at extreme temperatures, place adaptive pressure on essential genes. We sought to use this evolutionary diversity of essential genes to engineer bacterial pathogens to be stably temperature-sensitive, and thus useful as live vaccines. We isolated essential genes from bacteria found in the Arctic and substituted them for their counterparts into pathogens of mammals. We found that substitution of nine different essential genes from psychrophilic (cold-loving) bacteria into mammalian pathogenic bacteria resulted in strains that died below their normal-temperature growth limits. Substitution of three different psychrophilic gene orthologs of ligA, which encode NAD-dependent DNA ligase, resulted in bacterial strains that died at 33, 35, and 37 °C. One ligA gene was shown to render Francisella tularensis, Salmonella enterica, and Mycobacterium smegmatis temperature-sensitive, demonstrating that this gene functions in both Gram-negative and Gram-positive lineage bacteria. Three temperature-sensitive F. tularensis strains were shown to induce protective immunity after vaccination at a cool body site. About half of the genes that could be tested were unable to mutate to temperature-resistant forms at detectable levels. These results show that psychrophilic essential genes can be used to create a unique class of bacterial temperature-sensitive vaccines for important human pathogens, such as S. enterica and Mycobacterium tuberculosis.     

Subacute bacterial endocarditis caused by Cardiobacterium hominis: A case report

Cardiobacterium hominis, a member of the HACEK group of organisms, is an uncommon but important cause of subacute bacterial endocarditis. First-line therapy is a third-generation cephalosporin due to rare beta-lactamase production. The authors report a case involving endovascular infection due to C hominis that initially tested resistant to third-generation cephalosporins using an antibiotic gradient strip susceptibility method (nitrocephin negative), but later proved to be susceptible using broth microdilution reference methods (a ‘major’ error). There are limited studies to guide susceptibility testing and interpretive breakpoints for C hominis in the medical literature, and the present case illustrates some of the issues that may arise when performing susceptibility testing for fastidious organisms in the clinical microbiology laboratory.     

Interactive effects among ecosystem services and management practices on crop production: Pollination in coffee agroforestry systems

Crop productivity is improved by ecosystem services, including pollination, but this should be set in the context of trade-offs among multiple management practices. We investigated the impact of pollination services on coffee production, considering variation in fertilization, irrigation, shade cover, and environmental variables such as rainfall (which stimulates coffee flowering across all plantations), soil pH, and nitrogen availability. After accounting for management interventions, bee abundance improved coffee production (number of berries harvested). Some management interventions, such as irrigation, used once to trigger asynchronous flowering, dramatically increased bee abundance at coffee trees. Others, such as the extent and type of tree cover, revealed interacting effects on pollination and, ultimately, crop production. The effects of management interventions, notably irrigation and addition of lime, had, however, far more substantial positive effects on coffee production than tree cover. These results suggest that pollination services matter, but managing the asynchrony of flowering was a more effective tool for securing good pollination than maintaining high shade tree densities as pollinator habitat. Complex interactions across farm and landscape scales, including both management practices and environmental conditions, shape pollination outcomes. Effective production systems therefore require the integrated consideration of management practices in the context of the surrounding habitat structure. This paper points toward a more strategic use of ecosystem services in agricultural systems, where ecosystem services are shaped by the coupling of management interventions and environmental variables.     

A second wave of Sonic hedgehog expression during the development of the bat limb

Sonic hedgehog (Shh) plays an integral role in both the anterior-posterior (A-P) patterning and expansion of developing vertebrate limbs through a feedback loop involving Fgfs, Bmps, and Gremlin. In bat limbs A-P patterning and the size of the digital field are unique. The posterior digits of the forelimb are elongated and joined by tissue, whereas the thumb is short. The hindlimb digits often are uniform in length. Here, we reveal novel expression patterns for Shh and its target, Patched 1 (Ptc1), during limb development in two bat species. Early Shh expression in the zone of polarizing activity is wider in the bat forelimb than in the mouse forelimb, correlating with the reported expansion of Fgf8 expression in the apical ectodermal ridge and the early loss of symmetry in the bat forelimb. Later in limb development, Shh and Ptc1 expression is reinitiated in the interdigital tissue. Shh is graded along the A-P axis in forelimb and is expressed uniformly at a lower level across the hindlimb interdigital tissue. We also show that the reported Fgf8 expression in the interdigital tissue precedes the expression of Shh. We propose that the reinitiation of Shh and Fgf8 expression in bat limbs reactivates the Shh-Fgf feedback loop in the interdigital tissue of stage 16 bat embryos. The cell survival and proliferation signals provided by the Shh-Fgf signaling loop probably contribute to the lengthening of the posterior forelimb digits, the survival of the forelimb interdigital webbing, and the extension of the hindlimb digits to a uniform length.     

From the Cover: A Trojan horse mechanism of bacterial pathogenesis against nematodes

Understanding the mechanisms of host–pathogen interaction can provide crucial information for successfully manipulating their relationships. Because of its genetic background and practical advantages over vertebrate model systems, the nematode Caenorhabditis elegans model has become an attractive host for studying microbial pathogenesis. Here we report a “Trojan horse” mechanism of bacterial pathogenesis against nematodes. We show that the bacterium Bacillus nematocida B16 lures nematodes by emitting potent volatile organic compounds that are much more attractive to worms than those from ordinary dietary bacteria. Seventeen B. nematocida-attractant volatile organic compounds are identified, and seven are individually confirmed to lure nematodes. Once the bacteria enter the intestine of nematodes, they secrete two proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. This Trojan horse pattern of bacterium–nematode interaction enriches our understanding of microbial pathogenesis.     

Epigenetic reprogramming during vegetative phase change in maize

An important step during plant development is the transition from juvenile to adult growth. It is only after this transition that plants are reproductively competent. Given the great danger that transposon activity represents to the germ line, this may also be an important period during development with respect to transposon regulation and silencing. We demonstrate that a change in expression of a key component of the RNA silencing pathway is associated with both vegetative phase change and shifts in epigenetic regulation of a maize transposon.     

Complete Nucleotide Sequence and Molecular Characterization of Bacillus Phage TP21 and its Relatedness to Other Phages with the Same Name

Three different Bacillus bacteriophages designated TP21 are known from the literature. We have determined the sequence and structure of the TP21-L genome, and compared it to the other phages. The genome is 37.5 kb in size, possesses fixed invariable genome ends and features the typical modular organization of a temperate siphovirus. TP21-L is neither identical to TP21 isolated by Thorne (TP21-T), as shown by a PCR-based approach nor to TP21 isolated by He et al. (TP21-H), as estimated from phage dimensions. For reasons of clarity, we suggest renaming the different TP21 isolates.     

Vacuolar ATPase depletion affects mitochondrial ATPase function,kinetoplast dependency,and drug sensitivity in trypanosomes

Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F₁-γ-subunit of the two-sector F₁F_o-ATP synthase allow for F_o-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal F_o-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.Kinetoplastid parasites, including African trypanosomes, South American trypanosomes, and Leishmania spp., cause a range of lethal and debilitating diseases in humans and animals, and there is an urgent need for new therapies (1). The kinetoplastids transmitted by tsetse flies, and the diseases they cause, are restricted to the African tsetse belt. These parasites include the major causes of nagana in livestock, Trypanosoma vivax and Trypanosoma congolense, and Trypanosoma brucei brucei, the animal parasite most often manipulated in the laboratory. Although Trypanosoma brucei gambiense, the most important cause of human infection, is not thought to circulate in cattle, Trypanosoma brucei rhodesiense is zoonotic, passing among humans and animal reservoirs. Trypanosoma equiperdum and Trypanosoma evansi are similar trypanosomes (2), but with defects in the kinetoplast, the large DNA network comprising the mitochondrial genome (3). These parasites cause dourine or surra disease in horses, camels, or water buffalo. These dyskinetoplastic parasites are not transmitted by tsetse flies because passage through the insect midgut requires kinetoplast-dependent mitochondrial elaboration (4). Instead, they are mechanically transmitted by other biting insects or sexually transmitted in Africa and outside Africa.Several anti-trypanosomal drugs target the kinetoplast (5). Although this mitochondrial genome is essential in T. brucei, it is dispensable in T. equiperdum and T. evansi (6). As outlined below, studies on the mitochondrial F₁F_o ATP synthase, also known as complex V, led to an explanation for this difference. ATP synthase is a large, multisubunit complex of the electron transport chain that couples the mitochondrial membrane potential, ΔΨ^mito, to proton transport and ATP synthesis in insect-stage T. brucei. In bloodstream-form T. brucei, which rely on glycolysis for energy production, the more minimal mitochondrion lacks cytochromes, Krebs cycle enzymes, and oxidative phosphorylation (4), and ATP synthase works in the other direction, hydrolyzing ATP to generate ΔΨ^mito (7–9); ΔΨ^mito is still required for mitochondrial protein import, redox homeostasis, and Fe-S cluster assembly. In this life cycle stage, the kinetoplast encodes a single essential protein, the F_o-A6-subunit of the membrane-sector of ATP synthase. A mutation in the nuclear genome-encoded γ-subunit in T. evansi or T. equiperdum can compensae for this A-subunit requirement and allows for kinetoplast loss (6). Indeed, these dyskinetoplastic trypanosomes have been referred to as petite mutants of T. brucei (10), sharing similarities with petite mutants of yeast (11). Thus, dyskinetoplastic trypanosomes use an alternative, F_o-independent mode of ΔΨ^mito generation involving the uncoupled F₁ sector of ATP synthase and an ADP^3-/ATP^4- carrier (6, 12).Isometamidium chloride (ISM; samorin, veridium, trypamidium) and diminazene aceturate (berenil) are the most important veterinary trypanocidal drugs. ISM, a phenanthridine related to ethidium bromide, is mainly used as a prophylactic against nagana disease in cattle, with millions of doses administered each year. This drug accumulates in the kinetoplast, and cells with mutations in the γ-subunit of ATP synthase are ISM resistant (5). Ethidium bromide has itself also been used against nagana. The diamidine, pentamidine, is another kinetoplast-targeting drug used against human trypanosomal diseases (13). Although kinetoplast biology is intimately linked to parasite biology and antiparasite therapy, our understanding of kinetoplast dependency, drug mode of action, and potential resistance mechanisms remains incomplete.To further explore these areas, we screened a genome-scale RNA interference library in bloodstream-form T. brucei for ISM resistance mechanisms. Among 30 genes identified were those encoding possibly all 14 V-type H⁺ ATPase (V-ATPase) subunits, all 4 adaptin-3 subunits, and 5 endoplasmic-reticulum (ER) membrane complex subunits. Depletion of V-ATPase subunits, or the other complex subunits, was associated with probable ATP synthase uncoupling, kinetoplast-independent growth, and multidrug resistance. Our results reveal an unexpected link between V-ATPase function, mitochondrial ATP synthase function, and kinetoplast dependency and also provide candidate mechanisms to explain dyskinetoplasty and/or drug resistance in the field.     

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon—Shigella IpaC, Salmonella SipC, or Chromobacterium CipC—determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.Intracellular bacterial pathogens use a variety of elaborate means to survive within host cells. Postinvasion, some such as Legionella, Salmonella, and Chlamydia species modify bacteria-containing vacuoles to avoid death via phagosomal acidification or lysosomal fusion. Others, including Shigella, Listeria, Rickettsia, and Burkholderia species, rapidly escape from phagosomes into the cytosol of infected cells. Although escape from phagosomes by the classic intracytoplasmic Gram-positive bacterium Listeria monocytogenes is well understood (1), much less is known regarding how Gram-negative pathogens, including the model professional intracytoplasmic Shigella species, enter the cytosol.During the course of an infection, many Gram-negative pathogens, including Shigella, Salmonella, enteropathogenic Escherichia coli, and Yersinia species, use type III secretion systems (T3SSs) as injection devices to deliver multiple virulence proteins, referred to as effectors, directly into the cytosol of infected cells (2). T3SSs are composed of ∼20 proteins and sense host cell contact via a tip complex at the distal end of a needle filament, which then acts as a scaffold for the formation of a translocon pore in the host cell membrane. Although components of their type III secretion apparatus (T3SA) are relatively well conserved, each pathogen delivers a unique repertoire of effectors into host cells, likely accounting for the establishment of a variety of replicative niches. For example, Salmonella and Shigella secreted effectors promote the uptake of these bacteria into nonphagocytic cells, whereas those from Yersinia inhibit phagocytosis by macrophages.All four pathogenic Shigella species—Shigella flexneri, Shigella sonnei, Shigella boydii, and Shigella dysenteriae—deliver ∼30 effectors into host cells, the majority of which are encoded on a large virulence plasmid (VP) alongside the genes for all of the proteins needed to form a T3SA (3). These secreted proteins play major roles in Shigella pathogenesis, including host cell invasion and modulation of innate immune response. One effector, IpgD, promotes the efficiency of Shigella phagosomal escape, although it is not absolutely required for this process (4). Interestingly, IpaB and IpaC, components of the Shigella translocon, the portion of the T3SA that inserts into the host cell membrane, have been implicated to mediate phagosomal escape based on the behavior of recombinant proteins (5–7). The physiologic relevance of these findings has not yet been directly addressed, as strains that lack either of these two proteins are completely impaired in the delivery of Shigella effectors into host cells (8).Here, using a reductionist approach, we directly tested a role for the Shigella translocon apparatus in phagosomal escape. Using an innovative reengineering approach, we introduced a functional effectorless Shigella T3SA into a nonpathogenic laboratory strain of DH10B E. coli. Remarkably, upon entry into host epithelial cells, these bacteria efficiently escape from phagosomes. This demonstrates for the first time, to our knowledge, in the context of an infection, a direct role for the Shigella T3SA in mediating vacuole lysis. Despite structural conservation across T3SS families, we further observed that, in the absence of any type III effectors, the Ysc T3SA mediates little to no Yersinia phagosomal escape, suggesting that not all injectisomes have equivalent functions. Lastly, by exploring the functional interchangeability of translocon components of the Shigella, Salmonella, and Chromobacterium T3SA, we demonstrate that one translocon protein controls the extent to which these intracellular pathogens escape into the cytosol of infected cells, thus demonstrating a major role for the T3SA in determining the site of the replicative niche of intracellular bacteria.     

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

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

Native root-associated bacteria rescue a plant from a sudden-wilt disease that emerged during continuous cropping

Plants maintain microbial associations whose functions remain largely unknown. For the past 15 y, we have planted the annual postfire tobacco Nicotiana attenuata into an experimental field plot in the plant’s native habitat, and for the last 8 y the number of plants dying from a sudden wilt disease has increased, leading to crop failure. Inadvertently we had recapitulated the common agricultural dilemma of pathogen buildup associated with continuous cropping for this native plant. Plants suffered sudden tissue collapse and black roots, symptoms similar to a Fusarium–Alternaria disease complex, recently characterized in a nearby native population and developed into an in vitro pathosystem for N. attenuata. With this in vitro disease system, different protection strategies (fungicide and inoculations with native root-associated bacterial and fungal isolates), together with a biochar soil amendment, were tested further in the field. A field trial with more than 900 plants in two field plots revealed that inoculation with a mixture of native bacterial isolates significantly reduced disease incidence and mortality in the infected field plot without influencing growth, herbivore resistance, or 32 defense and signaling metabolites known to mediate resistance against native herbivores. Tests in a subsequent year revealed that a core consortium of five bacteria was essential for disease reduction. This consortium, but not individual members of the root-associated bacteria community which this plant normally recruits during germination from native seed banks, provides enduring resistance against fungal diseases, demonstrating that native plants develop opportunistic mutualisms with prokaryotes that solve context-dependent ecological problems.Eukaryotes maintain many complex relationships with the microbes they host, which can be so abundant and diverse that they frequently are considered a eukaryote’s second genome. The complex relationships mediated by microbial associates are being revealed rapidly, thanks to the advances in sequencing, microbial culturing techniques, and the reconstitution of associated microbial communities in gnotobiotic systems (1, 2), even if some of these putative functional roles may need to be evaluated more critically (3).When plants germinate from their seed banks, they typically acquire a selection of the diverse fungi and bacteria that exist in native soils, and a subset of this community becomes root-associated. The best characterized are the bacterial microbiomes of Arabidopsis thaliana. Approximately half of the bacterial community in the plant root is representative of the soil flora; the remainder is a conserved core consisting of a smaller number of bacterial lineages from three phyla: Actinobacteria, Proteobacteria, and Bacteroidetes (2, 4). Because these bacterial communities occur in nondiseased plants, they are thought to represent commensalistic or possibly mutualistic associations.Root-associated microbes could benefit plants in many ways, and a recent review (5) highlighted the parallel functional roles of the microbiomes of the human gut and those of plant roots. The best-characterized beneficial functions for plants are (i) the plant growth-promoting rhizobacteria (PGPR), which promote growth by a variety of direct and indirect means that include increasing nutrient availability, interfering with ethylene (ET) signaling, and preventing diseases (6), and (ii) the bacteria that elicit induced systemic resistance (ISR) (7) by activating jasmonic acid (JA) and ET signaling (8). PGPR and ISR have been studied in a variety of cultivated and model plants, usually with model microbes (5), but little is known about their ecological context or whether they increase the growth and fitness of native plants. Whether PGPR and ISR functions occur among the well-characterized root-associated bacterial communities of Arabidopsis, either collectively or individually, also remains unknown.The well-described agricultural phenomenon of disease-suppressive soils that harbor microbiomes that suppress particular soil-borne pathogens (9) illustrates the complexity of the dynamics involved. Native soils have a certain degree of pathogen-suppressive ability, frequently seen when a crop is grown continuously in a soil, suffers an outbreak of a disease, and subsequently becomes resistant to the disease (5). Perhaps the mechanisms involved are best understood in a root disease of wheat caused by Gaeumannomyces graminis var Tritici infections, known as “take-all” disease. After many years of continuous wheat cropping with several disease outbreaks, the disease suddenly wanes, apparently because of the build-up of antagonistic Pseudomonas spp. (9). Whether any of these interactions also occur in native plants remains unknown.Nicotiana attenuata, a native annual tobacco of North America, germinates from long-lived seed banks to grow in the immediate postfire environment (10). When N. attenuata seeds germinate from their seed banks, they acquire a root-associated microbiome from their native soils which has been characterized by pyrosequencing and culture-dependent approaches (11–14). The composition of the root-associated microbiome is not influenced by a plant’s ability to elicit JA signaling (14), but ET signaling, as mediated by the ability both to produce and to perceive ET, plays a decisive role in shaping the “immigration policy” for the root-associated microbiome (12). A certain Bacillus strain, B55, was isolated from the roots of an ET-insensitive N. attenuata plant (35S etr-1) and was able to rescue the impaired-growth and high-mortality phenotype of ET-insensitive plants under field conditions (15). Beneficial effects were attributed to B55’s ability to reduce sulfur and produce dimethyl disulfide, which N. attenuata uses to alleviate sulfur deficiencies. This rescue provided one of the first demonstrations that the soil bacteria recruited by plants during germination can form opportunistic mutualistic relationships with their host based on the host plant’s ecological context. Here we provide a second example that involves protection against a sudden wilt disease, which accumulated in a field plot after consecutive planting of N. attenuata seedlings.     

Rapid changes in the gut microbiome during human evolution

Humans are ecosystems containing trillions of microorganisms, but the evolutionary history of this microbiome is obscured by a lack of knowledge about microbiomes of African apes. We sequenced the gut communities of hundreds of chimpanzees, bonobos, and gorillas and developed a phylogenetic approach to reconstruct how present-day human microbiomes have diverged from those of ancestral populations. Compositional change in the microbiome was slow and clock-like during African ape diversification, but human microbiomes have deviated from the ancestral state at an accelerated rate. Relative to the microbiomes of wild apes, human microbiomes have lost ancestral microbial diversity while becoming specialized for animal-based diets. Individual wild apes cultivate more phyla, classes, orders, families, genera, and species of bacteria than do individual humans across a range of societies. These results indicate that humanity has experienced a depletion of the gut flora since diverging from Pan.The human microbiome is shaped by host genetics, environment, and lifestyle (1–3); thus, humanity''s unique evolutionary and cultural histories must have altered our associations with microorganisms (4). Despite intensive investigation of the microbiomes of humans spanning a range of geographic locations and cultures (5–7), how the composition of the microbiome has changed since humans diverged from other species, and since human populations diverged from one another, remains unclear, owing to a lack of knowledge about the microbiomes of ancestral hominid populations.Understanding how the composition of the human microbiome has changed over evolutionary time requires the inclusion of the microbiomes of phylogenetic outgroups (i.e., the African apes) into analyses of human microbiomes. Previous comparisons of the gut microbiomes of humans and the African apes have been restricted to just a few individuals per host species (8), precluding detection of the precise compositional differences that distinguish the microbiomes of the host species. Comparing the microbiomes of populations of chimpanzees, bonobos, gorillas, and humans while considering the phylogenetic relatedness among the hosts can reveal how the composition of the microbiome has changed since the host species diversified.Here we used a phylogenetic approach to identify the shifts in the composition of the microbiome that occurred along the lineages leading to the extant species of Homo and Pan. This analysis shows that humans across a range of cultures and geographies harbor microbiomes that are disproportionately divergent from those within wild apes. In particular, among the living hominid species, humans harbor uncharacteristically low levels of microbial diversity within their gut microbiomes.     

Antagonistic pleiotropic effects reduce the potential adaptive value of the FRIGIDA locus

Although the occurrence of epistasis and pleiotropy is widely accepted at the molecular level, its effect on the adaptive value of fitness-related genes is rarely investigated in plants. Knowledge of these features of a gene is critical to understand the molecular basis of adaptive evolution. Here we investigate the importance of pleiotropy and epistasis in determining the adaptive value of a candidate gene using the gene FRI (FRIGIDA), which is thought to be the major gene controlling flowering time variation in Arabidopsis thaliana. The effect of FRI on flowering time was analyzed in an outbred population created by randomly mating 19 natural accessions of A. thaliana. This unique population allows the estimation of FRI effects independent of any linkage association with other loci due to demographic processes or to coadapted genes. It also allows for the estimation of pleiotropic effects of FRI on fitness and inflorescence architecture. We found that FRI explains less variation in flowering time than previously observed among natural accessions, and interacts epistatically with the FLC locus. Although early flowering plants produce more fruits under spring conditions, and nonfunctional alleles of FRI were associated with early flowering, variation at FRI was not associated with fitness. We show that nonfunctional FRI alleles have negative pleiotropic effects on fitness by reducing the numbers of nodes and branches on the inflorescence. We propose that these antagonistic pleiotropic effects reduce the adaptive value of FRI, and helps explain the maintenance of alternative life history strategies across natural populations of A. thaliana.     

Genetic activation of BK currents in vivo generates bidirectional effects on neuronal excitability

Large-conductance calcium-activated potassium channels (BK) are potent negative regulators of excitability in neurons and muscle, and increasing BK current is a novel therapeutic strategy for neuro- and cardioprotection, disorders of smooth muscle hyperactivity, and several psychiatric diseases. However, in some neurons, enhanced BK current is linked with seizures and paradoxical increases in excitability, potentially complicating the clinical use of agonists. The mechanisms that switch BK influence from inhibitory to excitatory are not well defined. Here we investigate this dichotomy using a gain-of-function subunit (BK^R207Q) to enhance BK currents. Heterologous expression of BK^R207Q generated currents that activated at physiologically relevant voltages in lower intracellular Ca²⁺, activated faster, and deactivated slower than wild-type currents. We then used BK^R207Q expression to broadly augment endogenous BK currents in vivo, generating a transgenic mouse from a circadian clock-controlled Period1 gene fragment (Tg-BK^R207Q). The specific impact on excitability was assessed in neurons of the suprachiasmatic nucleus (SCN) in the hypothalamus, a cell type where BK currents regulate spontaneous firing under distinct day and night conditions that are defined by different complements of ionic currents. In the SCN, Tg-BK^R207Q expression converted the endogenous BK current to fast-activating, while maintaining similar current-voltage properties between day and night. Alteration of BK currents in Tg-BK^R207Q SCN neurons increased firing at night but decreased firing during the day, demonstrating that BK currents generate bidirectional effects on neuronal firing under distinct conditions.