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As a fundamental biological process, DNA replication ensures the accurate copying of genetic information. However, the impact of this process on cellular plasticity in multicellular organisms remains elusive. Here, we find that reducing the level or activity of a replication component, DNA Polymerase α (Polα), facilitates cell reprogramming in diverse stem cell systems across species. In Drosophila male and female germline stem cell lineages, reducing Polα levels using heterozygotes significantly enhances fertility of both sexes, promoting reproductivity during aging without compromising their longevity. Consistently, in C. elegans the pola heterozygous hermaphrodites exhibit increased fertility without a reduction in lifespan, suggesting that this phenomenon is conserved. Moreover, in male germline and female intestinal stem cell lineages of Drosophila , polα heterozygotes exhibit increased resistance to tissue damage caused by genetic ablation or pathogen infection, leading to enhanced regeneration and improved survival during post-injury recovery, respectively. Additionally, fine tuning of an inhibitor to modulate Polα activity significantly enhances the efficiency of reprogramming human embryonic fibroblasts into induced pluripotent cells. Together, these findings unveil novel roles of a DNA replication component in regulating cellular reprogramming potential, and thus hold promise for promoting tissue health, facilitating post-injury rehabilitation, and enhancing healthspan., Competing Interests: Competing Interest Statement: We have a provisional patent application through the Johns Hopkins Technology Ventures.

Chromobacterium subtsugae exhibits toxicity to Drosophila melanogaster , providing a new infection model to study host homeostasis. Previous studies using pathogen models have proven to be a useful tool to understand host physiology. Here, we report on the whole-genome sequences of these microbes obtained from short and long reads., Competing Interests: The authors declare no conflict of interest.

The practice of designating two or more authors as equal contributors (ECs) on a scientific publication is increasingly common as a form of sharing credit. However, EC authors are often unclearly attributed on curriculum vitae (CVs) or citation engines, and it is unclear how research teams determine author order within an EC listing. In response to studies showing that male authors were more likely to be placed first in an EC listing, the American Society for Microbiology (ASM) required that authors explain the reasons for author order beginning in 2020. In this study, we analyze data from over 2,500 ASM publications to see how this policy affected gender bias and how research teams are making decisions on author order. Data on publications from 2018 to 2021 show that gender bias was largely nonsignificant both before and after authors were asked by ASM to provide an EC statement. The most likely reasons for EC order included alphabetical order, seniority, and chance, although there were differences for publications from different geographic regions. However, many research teams used unique methods in order selection, highlighting the importance of EC statements to provide clarity for readers, funding agencies, and tenure committees., Importance: First-author publications are important for early career scientists to secure funding and educational opportunities. However, an analysis published in eLife in 2019 noted that female authors are more likely to be placed second even when both authors report they have contributed equally. American Society for Microbiology announced in response that they would require submissions to include a written justification of author order. In this paper, we analyze the resultant data and show that laboratories are most likely to use some combination of alphabetical order, seniority, and chance to determine author order. However, the prevalence of these methods varies based on the research team's geographic location. These findings highlight the importance of equal contributor statements to provide clarity for readers, funding agencies, and tenure committees. Furthermore, this work is critically important for understanding how these decisions are made and provides a glimpse of the sociology of science., Competing Interests: A.C. serves on the advisory board for Sab Therapeutics and has stock options in the company.

Microbial invasions underlie host-microbe interactions resulting in pathogenesis and probiotic colonization. In this study, we explore the effects of the microbiome on microbial invasion in Drosophila melanogaster. We demonstrate that gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival and lead to a reduction in microbial burden during infection. Using a microbial interaction assay, we report that L. plantarum inhibits the growth of invasive bacteria, while A. tropicalis reduces this inhibition. We further show that inhibition by L. plantarum is linked to its ability to acidify its environment via lactic acid production by lactate dehydrogenase, while A. tropicalis diminishes the inhibition by quenching acids. We propose that acid from the microbiome is a gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods and findings described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing [Tn-Seq, also known as insertion sequencing (INSeq)] to identify genes in P. aeruginosa that contribute to fitness during the colonization of Drosophila melanogaster . Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies on P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila , demonstrate the power of Tn-Seq in the fly model system and advance the existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats.IMPORTANCE Drosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from the analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies-and advances in high-throughput sequencing of transposon mutants-support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable the pathogen P. aeruginosa to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics., Competing Interests: The authors declare no conflict of interest.

Host-microbe interactions constitute dynamical systems that can be represented by mathematical formulations that determine their dynamic nature and are categorized as deterministic, stochastic, or chaotic. Knowing the type of dynamical interaction is essential for understanding the system under study. Very little experimental work has been done to determine the dynamical characteristics of host-microbe interactions, and its study poses significant challenges. The most straightforward experimental outcome involves an observation of time to death upon infection. However, in measuring this outcome, the internal parameters and the dynamics of each particular host-microbe interaction in a population of interactions are hidden from the experimentalist. To investigate whether a time-to-death (time-to-event) data set provides adequate information for searching for chaotic signatures, we first determined our ability to detect chaos in simulated data sets of time-to-event measurements and successfully distinguished the time-to-event distribution of a chaotic process from a comparable stochastic one. To do so, we introduced an inversion measure to test for a chaotic signature in time-to-event distributions. Next, we searched for chaos in the time-to-death of Caenorhabditis elegans and Drosophila melanogaster infected with Pseudomonas aeruginosa or Pseudomonas entomophila , respectively. We found suggestions of chaotic signatures in both systems but caution that our results are preliminary and highlight the need for more fine-grained and larger data sets in determining dynamical characteristics. If validated, chaos in host-microbe interactions would have important implications for the occurrence and outcome of infectious diseases, the reproducibility of experiments in the field of microbial pathogenesis, and the prediction of microbial threats.IMPORTANCEIs microbial pathogenesis a predictable scientific field? At a time when we are dealing with coronavirus disease 2019, there is intense interest in knowing about the epidemic potential of other microbial threats and new emerging infectious diseases. To know whether microbial pathogenesis will ever be a predictable scientific field requires knowing whether a host-microbe interaction follows deterministic, stochastic, or chaotic dynamics. If randomness and chaos are absent from virulence, there is hope for prediction in the future regarding the outcome of microbe-host interactions. Chaotic systems are inherently unpredictable, although it is possible to generate short-term probabilistic models, as is done in applications of stochastic processes and machine learning to weather forecasting. Information on the dynamics of a system is also essential for understanding the reproducibility of experiments, a topic of great concern in the biological sciences. Our study finds preliminary evidence for chaotic dynamics in infectious diseases., Competing Interests: The authors declare no conflict of interest.

Lactiplantibacillus plantarum and Acetobacter tropicalis are bacterial symbionts commonly isolated from decaying fruits and from the microbiome of Drosophila melanogaster . Studies have shown that these organisms interact synergistically, imparting beneficial effects on the host. Here, we report whole-genome sequences of these microbes obtained from long and short reads., Competing Interests: The authors declare no conflict of interest.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti, is lacking. In the current study, we utilized solid-state nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and transmission electron microscopy to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti. No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector., (© 2023. The Author(s).)

Most insects exhibit high reproductive capacity, which demands large amounts of energy, including macronutrients and micronutrients. Interestingly, many proteins involved in oogenesis depend on metals ions, in particular iron (Fe), zinc (Zn), and copper (Cu). Mechanisms by which metal ions influence reproduction have been described in Drosophila melanogaster, but remain poorly understood in hematophagous insects where blood meals include significant ingestion of metal ions. Moreover, there is evidence that some proteins involved in reproduction and immunity could have dual function in both processes. This review highlights the importance of metal ions in the reproduction of non-hematophagous and hematophagous insects. In addition, we discuss how insects optimize physiological processes using proteins involved in crosstalk between reproductive physiology and immunity, which is a double-edge sword in allocating their functions to protect the insect and ensure reproduction., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

A fundamental and highly contested issue in microbiome research is whether internal organs such as the liver, brain, placenta, pancreas, and others are sterile and privileged or harbor a detectable and functional microbial biomass. In this issue of the JCI, Leinwand, Paul, et al. addressed this question using an array of diverse techniques and reported that normal healthy liver possesses a microbiome that is selectively recruited from the gut. They further showed that liver-enriched microbes contributed to shaping the immune network of this organ. Here, we attempt to put their findings into the context of other organs, discuss the technical challenges of defining such microbial communities, and provide some perspective about the road ahead for the field.

The COVID-19 pandemic has caused unprecedented disruptions to healthcare, including direct impacts on service delivery related to autism spectrum disorder (ASD). Caregiver-mediated tele-assessment offers an opportunity to continue services while adhering to social distancing guidelines. The present study describes a model of tele-assessment for ASD in young children, implemented in direct response to disruptions in care caused by the COVID-19 pandemic. We present preliminary data on the outcomes and provider perceptions of tele-assessments, together with several lessons learned during the period of initial implementation., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations., Competing Interests: The authors have declared that no competing interests exist.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery., (Copyright © 2021 Hurley et al.)

Drosophila melanogaster gut microbes play important roles in host nutritional physiology. However, these associations are often indirect, and studies typically are in the context of specialized nutritional conditions, making it difficult to discern how microbiome-mediated impacts translate to physiologically relevant conditions, in the laboratory or nature. In this study, we quantified changes in dietary nutrients due to D. melanogaster gut bacteria on three artificial diets and a natural diet of grapes. We show that under all four diet conditions, bacteria altered the protein, carbohydrates, and moisture of the food substrate. An in-depth analysis of one diet revealed that bacteria also increased the levels of tryptophan, an essential amino acid encountered scarcely in nature. These nutrient changes result in an increased protein-to-carbohydrate (P:C) ratio in all diets, which we hypothesized to be a significant determinant of microbiome-mediated host nutritional physiology. To test this, we compared life history traits of axenic flies reared on the three artificial diets with increased P:C ratios or continuous bacterial inoculation. We found that while on some diets, an environment of nutritional plenitude had impacts on life history, it did not fully explain all microbiome-associated phenotypes. This suggests that other factors, such as micronutrients and feeding behavior, likely also contribute to life history traits in a diet-dependent manner. Thus, while some bacterial impacts on nutrition occur across diets, others are dictated by unique dietary environments, highlighting the importance of diet-microbiome interactions in D. melanogaster nutritional physiology. IMPORTANCE Both in the laboratory and in nature, D. melanogaster -associated microbes serve as nutritional effectors, either through the production of metabolites or as direct sources of protein biomass. The relationship between the microbiome and the resulting host nutritional physiology is significantly impacted by diet composition, yet studies involving D. melanogaster are performed using a wide range of artificial diets, making it difficult to discern which aspects of host-microbe interactions may be universal or diet dependent. In this study, we utilized three standard D. melanogaster diets and a natural grape diet to form a comprehensive understanding of the quantifiable nutritional changes mediated by the host microbial community. We then altered these artificial diets based on the observed microbe-mediated changes to demonstrate their potential to influence host physiology, allowing us to identify nutritional factors whose effects were either universal for the three artificial diets or dependent on host diet composition., (Copyright © 2020 Lesperance and Broderick.)

Enterobacteria, including Escherichia coli , bloom to high levels in the gut during inflammation and strongly contribute to the pathology of inflammatory bowel diseases. To survive in the inflamed gut, E. coli must tolerate high levels of antimicrobial compounds produced by the immune system, including toxic metals like copper and reactive chlorine oxidants such as hypochlorous acid (HOCl). Here, we show that extracellular copper is a potent detoxifier of HOCl and that the widely conserved bacterial HOCl resistance enzyme RclA, which catalyzes the reduction of copper(II) to copper(I), specifically protects E. coli against damage caused by the combination of HOCl and intracellular copper. E. coli lacking RclA was highly sensitive to HOCl when grown in the presence of copper and was defective in colonizing an animal host. Our results indicate that there is unexpected complexity in the interactions between antimicrobial toxins produced by innate immune cells and that bacterial copper status is a key determinant of HOCl resistance and suggest an important and previously unsuspected role for copper redox reactions during inflammation. IMPORTANCE During infection and inflammation, the innate immune system uses antimicrobial compounds to control bacterial populations. These include toxic metals, like copper, and reactive oxidants, including hypochlorous acid (HOCl). We have now found that RclA, a copper(II) reductase strongly induced by HOCl in proinflammatory Escherichia coli and found in many bacteria inhabiting epithelial surfaces, is required for bacteria to resist killing by the combination of intracellular copper and HOCl and plays an important role in colonization of an animal host. This finding indicates that copper redox chemistry plays a critical and previously underappreciated role in bacterial interactions with the innate immune system., (Copyright © 2020 Derke et al.)

Nutrition is a major factor influencing many aspects of Drosophila melanogaster physiology. However, a wide range of diets, many of which are termed "standard" in the literature, are utilized for D. melanogaster research, leading to inconsistencies in reporting of nutrition-dependent phenotypes across the field. This is especially evident in microbiome studies, as diet has a pivotal role in microbiome composition and resulting host-microbe interactions. Here, we performed a meta-analysis of diets used in fly microbiome research and provide a web-based tool for researchers to determine the nutritional content of diets of interest. While our meta-analysis primarily focuses on microbiome studies, our goal in developing these resources is to aid the broader community in contextualizing past and future studies across the scope of D. melanogaster research to better understand how individual lab diets can contribute to observed phenotypes., (Copyright © 2020 Lesperance, Broderick.)

Drosophila melanogaster harbors a simple gut microbial community, or microbiome, that regulates several facets of its physiology. As a result, the host employs multiple mechanisms of maintaining control over its microbiome in an effort to promote overall organismal homeostasis. Perturbations to the balance between microbiome and host can result in states of instability or disease, making maintenance of microbial homeostasis a fundamental physiologic aspect of D. melanogaster biology. While the interactions between microbes and their hosts can be direct, particularly in the context of immunity and gut renewal, effects resulting from indirect interactions, such as those between microbiota members, can be equally as important. This review highlights the major ways, in which D. melanogaster regulates microbial homeostasis, the consequences of disruptions to homeostasis, and the different mechanisms, by which the microbiome interacts with its host., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an a ntibiotic- i nduced r esponse ( air ) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules. IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration., (Copyright © 2020 Lozano et al.)

Plants expend significant resources to select and maintain rhizosphere communities that benefit their growth and protect them from pathogens. A better understanding of assembly and function of rhizosphere microbial communities will provide new avenues for improving crop production. Secretion of antibiotics is one means by which bacteria interact with neighboring microbes and sometimes change community composition. In our analysis of a taxonomically diverse consortium from the soybean rhizosphere, we found that Pseudomonas koreensis selectively inhibits growth of Flavobacterium johnsoniae and other members of the Bacteroidetes grown in soybean root exudate. A genetic screen in P. koreensis identified a previously uncharacterized biosynthetic gene cluster responsible for the inhibitory activity. Metabolites were isolated based on biological activity and were characterized using tandem mass spectrometry, multidimensional nuclear magnetic resonance, and Mosher ester analysis, leading to the discovery of a new family of bacterial tetrahydropyridine alkaloids, koreenceine A to D (metabolites 1 to 4). Three of these metabolites are analogs of the plant alkaloid γ-coniceine. Comparative analysis of the koreenceine cluster with the γ-coniceine pathway revealed distinct polyketide synthase routes to the defining tetrahydropyridine scaffold, suggesting convergent evolution. Koreenceine-type pathways are widely distributed among Pseudomonas species, and koreenceine C was detected in another Pseudomonas species from a distantly related cluster. This work suggests that Pseudomonas and plants convergently evolved the ability to produce similar alkaloid metabolites that can mediate interbacterial competition in the rhizosphere. IMPORTANCE The microbiomes of plants are critical to host physiology and development. Microbes are attracted to the rhizosphere due to massive secretion of plant photosynthates from roots. Microorganisms that successfully join the rhizosphere community from bulk soil have access to more abundant and diverse molecules, producing a highly competitive and selective environment. In the rhizosphere, as in other microbiomes, little is known about the genetic basis for individual species' behaviors within the community. In this study, we characterized competition between Pseudomonas koreensis and Flavobacterium johnsoniae , two common rhizosphere inhabitants. We identified a widespread gene cluster in several Pseudomonas spp. that is necessary for the production of a novel family of tetrahydropyridine alkaloids that are structural analogs of plant alkaloids. We expand the known repertoire of antibiotics produced by Pseudomonas in the rhizosphere and demonstrate the role of the metabolites in interactions with other rhizosphere bacteria., (Copyright © 2019 Lozano et al.)

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P aeruginosa PGPR2 for root colonization., (Copyright © 2019 Sivakumar et al.)

The quest to manipulate microbiomes has intensified, but many microbial communities have proven to be recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing an understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa, the Firmicutes , Proteobacteria , and Bacteroidetes We chose Bacillus cereus as a model rhizosphere firmicute and characterized 20 other candidates, including "hitchhikers" that coisolated with B. cereus from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers, Pseudomonas koreensis from the top tier of the competition network and Flavobacterium johnsoniae from the bottom of the network, to represent the Proteobacteria and Bacteroidetes , respectively. The model community has several emergent properties, induction of dendritic expansion of B. cereus colonies by either of the other members, and production of more robust biofilms by the three members together than individually. Moreover, P. koreensis produces a novel family of alkaloid antibiotics that inhibit growth of F. johnsoniae , and production is inhibited by B. cereus We designate this community THOR, because the members are t he h itchhikers o f the r hizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior. IMPORTANCE The manipulation and engineering of microbiomes could lead to improved human health, environmental sustainability, and agricultural productivity. However, microbiomes have proven difficult to alter in predictable ways, and their emergent properties are poorly understood. The history of biology has demonstrated the power of model systems to understand complex problems such as gene expression or development. Therefore, a defined and genetically tractable model community would be useful to dissect microbiome assembly, maintenance, and processes. We have developed a tractable model rhizosphere microbiome, designated THOR, containing Pseudomonas koreensis , Flavobacterium johnsoniae , and Bacillus cereus , which represent three dominant phyla in the rhizosphere, as well as in soil and the mammalian gut. The model community demonstrates emergent properties, and the members are amenable to genetic dissection. We propose that THOR will be a useful model for investigations of community-level interactions., (Copyright © 2019 Lozano et al.)

We analyzed 2898 scientific papers published between 1995 and 2017 in which two or more authors shared the first author position. For papers in which the first and second authors made equal contributions, mixed-gender combinations were most frequent, followed by male-male and then female-female author combinations. For mixed-gender combinations, more male authors were in the first position, although the disparity decreased over time. For papers in which three or more authors made equal contributions, there were more male authors than female authors in the first position and more all-male than all-female author combinations. The gender inequalities observed among authors who made equal contributions are not consistent with random or alphabetical ordering of authors. These results raise concerns about female authors not receiving proper credit for publications and suggest a need for journals to request clarity on the method used to decide author order among those who contributed equally., Competing Interests: NB, AC No competing interests declared, (© 2019, Broderick and Casadevall.)

The last two decades have seen an explosion in research about the beneficial microbial communities associated with plants and animals. Initially this explosion was driven by technological advances that enabled explorations of microbiomes on unprecedented scales. Increasingly, the drive is coming from conceptual advances that are the fruit of research investments into experimental systems to probe the functions of these beneficial microbes and their mechanisms of action. The Conference on Beneficial Microbes has been one of the premiere venues for this research. The 7th Conference on Beneficial Microbes was held July 8-11, 2018, at the University of Wisconsin-Madison Memorial Union. The 308 attendees-representing academia, industry, journals, and funding agencies-participated in an intense 4-day meeting encompassing research frontiers in beneficial microbiology and microbiome science., (Copyright © 2018 American Society for Microbiology.)

Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies ( Glossina spp.) house maternally transmitted symbionts that regulate the development and function of their host's immune system. Herein we demonstrate that the obligate mutualist, Wigglesworthia , up-regulates expression of odorant binding protein six in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor lozenge and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval Drosophila's indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems., Competing Interests: The authors declare that no competing interests exist.

Animals host multi-species microbial communities (microbiomes) whose properties may result from inter-species interactions; however, current understanding of host-microbiome interactions derives mostly from studies in which elucidation of microbe-microbe interactions is difficult. In exploring how Drosophila melanogaster acquires its microbiome, we found that a microbial community influences Drosophila olfactory and egg-laying behaviors differently than individual members. Drosophila prefers a Saccharomyces - Acetobacter co-culture to the same microorganisms grown individually and then mixed, a response mainly due to the conserved olfactory receptor, Or42b. Acetobacter metabolism of Saccharomyces- derived ethanol was necessary, and acetate and its metabolic derivatives were sufficient, for co-culture preference. Preference correlated with three emergent co-culture properties: ethanol catabolism, a distinct volatile profile, and yeast population decline. Egg-laying preference provided a context-dependent fitness benefit to larvae. We describe a molecular mechanism by which a microbial community affects animal behavior. Our results support a model whereby emergent metabolites signal a beneficial multispecies microbiome., Competing Interests: The authors declare that no competing interests exist.

Drosophila melanogaster lives, breeds and feeds on fermenting fruit, an environment that supports a high density, and often a diversity, of microorganisms. This association with such dense microbe-rich environments has been proposed as a reason that D. melanogaster evolved a diverse and potent antimicrobial peptide (AMP) response to microorganisms, especially to combat potential pathogens that might occupy this niche. Yet, like most animals, D. melanogaster also lives in close association with the beneficial microbes that comprise its microbiota, or microbiome, and recent studies have shown that antimicrobial peptides (AMPs) of the epithelial immune response play an important role in dictating these interactions and controlling the host response to gut microbiota. Moreover, D. melanogaster also eats microbes for food, consuming fermentative microbes of decaying plant material and their by-products as both larvae and adults. The processes of nutrient acquisition and host defence are remarkably similar and use shared functions for microbe detection and response, an observation that has led to the proposal that the digestive and immune systems have a common evolutionary origin. In this manner, D. melanogaster provides a powerful model to understand how, and whether, hosts differentiate between the microbes they encounter across this spectrum of associations.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'., (© 2016 The Author(s).)

The dearth of new antibiotics in the face of widespread antimicrobial resistance makes developing innovative strategies for discovering new antibiotics critical for the future management of infectious disease. Understanding the genetics and evolution of antibiotic producers will help guide the discovery and bioengineering of novel antibiotics. We discovered an isolate in Alaskan boreal forest soil that had broad antimicrobial activity. We elucidated the corresponding antimicrobial natural products and sequenced the genome of this isolate, designated Streptomyces sp. 2AW. This strain illustrates the chemical virtuosity typical of the Streptomyces genus, producing cycloheximide as well as two other biosynthetically unrelated antibiotics, neutramycin, and hygromycin A. Combining bioinformatic and chemical analyses, we identified the gene clusters responsible for antibiotic production. Interestingly, 2AW appears dissimilar from other cycloheximide producers in that the gene encoding the polyketide synthase resides on a separate part of the chromosome from the genes responsible for tailoring cycloheximide-specific modifications. This gene arrangement and our phylogenetic analyses of the gene products suggest that 2AW holds an evolutionarily ancestral lineage of the cycloheximide pathway. Our analyses support the hypothesis that the 2AW glutaramide gene cluster is basal to the lineage wherein cycloheximide production diverged from other glutarimide antibiotics. This study illustrates the power of combining modern biochemical and genomic analyses to gain insight into the evolution of antibiotic-producing microorganisms.

Historically, the digestive and immune systems were viewed and studied as separate entities. However, there are remarkable similarities and shared functions in both nutrient acquisition and host defense. Here, I propose a common origin for both systems. This association provides a new prism for viewing the emergence and evolution of host defense mechanisms.

Enterococcus faecalis is a commensal and pathogen of humans and insects. In Manduca sexta, E. faecalis is an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigate E. faecalis factors required for commensalism, we identified E. faecalis genes that are upregulated in the gut of M. sexta using recombinase-based in vivo expression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designated pglA. A pglA deletion mutant was impaired in both pathogenesis and gut persistence in M. sexta and produced enhanced biofilms compared with the wild type in an in vitro polystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants for E. faecalis commensal and pathogenic interactions with M. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to prevent E. faecalis infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The increasing prevalence of antibiotic-resistant bacteria is a global threat to public health. Agricultural use of antibiotics is believed to contribute to the spread of antibiotic resistance, but the mechanisms by which many agricultural practices influence resistance remain obscure. Although manure from dairy farms is a common soil amendment in crop production, its impact on the soil microbiome and resistome is not known. To gain insight into this impact, we cultured bacteria from soil before and at 10 time points after application of manure from cows that had not received antibiotic treatment. Soil treated with manure contained a higher abundance of β-lactam-resistant bacteria than soil treated with inorganic fertilizer. Functional metagenomics identified β-lactam-resistance genes in treated and untreated soil, and indicated that the higher frequency of resistant bacteria in manure-amended soil was attributable to enrichment of resident soil bacteria that harbor β-lactamases. Quantitative PCR indicated that manure treatment enriched the blaCEP-04 gene, which is highly similar (96%) to a gene found previously in a Pseudomonas sp. Analysis of 16S rRNA genes indicated that the abundance of Pseudomonas spp. increased in manure-amended soil. Populations of other soil bacteria that commonly harbor β-lactamases, including Janthinobacterium sp. and Psychrobacter pulmonis, also increased in response to manure treatment. These results indicate that manure amendment induced a bloom of certain antibiotic-resistant bacteria in soil that was independent of antibiotic exposure of the cows from which the manure was derived. Our data illustrate the unintended consequences that can result from agricultural practices, and demonstrate the need for empirical analysis of the agroecosystem.

Unlabelled: To elucidate mechanisms underlying the complex relationships between a host and its microbiota, we used the genetically tractable model Drosophila melanogaster. Consistent with previous studies, the microbiota was simple in composition and diversity. However, analysis of single flies revealed high interfly variability that correlated with differences in feeding. To understand the effects of this simple and variable consortium, we compared the transcriptome of guts from conventionally reared flies to that for their axenically reared counterparts. Our analysis of two wild-type fly lines identified 121 up- and 31 downregulated genes. The majority of these genes were associated with immune responses, tissue homeostasis, gut physiology, and metabolism. By comparing the transcriptomes of young and old flies, we identified temporally responsive genes and showed that the overall impact of microbiota was greater in older flies. In addition, comparison of wild-type gene expression with that of an immune-deficient line revealed that 53% of upregulated genes exerted their effects through the immune deficiency (Imd) pathway. The genes included not only classic immune response genes but also those involved in signaling, gene expression, and metabolism, unveiling new and unexpected connections between immunity and other systems. Given these findings, we further characterized the effects of gut-associated microbes on gut morphology and epithelial architecture. The results showed that the microbiota affected gut morphology through their impacts on epithelial renewal rate, cellular spacing, and the composition of different cell types in the epithelium. Thus, while bacteria in the gut are highly variable, the influence of the microbiota at large has far-reaching effects on host physiology., Importance: The guts of animals are in constant association with microbes, and these interactions are understood to have important roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruit fly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well-established and genetically tractable invertebrate model., (Copyright © 2014 Broderick et al.)

Intestinal homeostasis is achieved, in part, by the integration of a complex set of mechanisms that eliminate pathogens and tolerate the indigenous microbiota. Drosophila melanogaster feeds on microorganism-enriched matter and therefore has developed efficient mechanisms to control ingested microorganisms. Regulatory mechanisms ensure an appropriate level of immune reactivity in the gut to accommodate the presence of beneficial and dietary microorganisms, while allowing effective immune responses to clear pathogens. Maintenance of D. melanogaster gut homeostasis also involves regeneration of the intestine to repair damage associated with infection. Entomopathogenic bacteria have developed common strategies to subvert these defence mechanisms and kill their host.

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.

There is growing interest in using Drosophila melanogaster to elucidate mechanisms that underlie the complex relationships between a host and its microbiota. In addition to the many genetic resources and tools Drosophila provides, its associated microbiota is relatively simple (1-30 taxa), in contrast to the complex diversity associated with vertebrates (> 500 taxa). These attributes highlight the potential of this system to dissect the complex cellular and molecular interactions that occur between a host and its microbiota. In this review, we summarize what is known regarding the composition of gut-associated microbes of Drosophila and their impact on host physiology. We also discuss these interactions in the context of their natural history and ecology and describe some recent insights into mechanisms by which Drosophila and its gut microbiota interact.

Background: Gut homeostasis is central to whole organism health, and its disruption is associated with a broad range of pathologies. Following damage, complex physiological events are required in the gut to maintain proper homeostasis. Previously, we demonstrated that ingestion of a nonlethal pathogen, Erwinia carotovora carotovora 15, induces a massive increase in stem cell proliferation in the gut of Drosophila. However, the precise cellular events that occur following infection have not been quantitatively described, nor do we understand the interaction between multiple pathways that have been implicated in epithelium renewal., Results: To understand the process of infection and epithelium renewal in more detail, we performed a quantitative analysis of several cellular and morphological characteristics of the gut. We observed that the gut of adult Drosophila undergoes a dynamic remodeling in response to bacterial infection. This remodeling coordinates the synthesis of new enterocytes, their proper morphogenesis and the elimination of damaged cells through delamination and anoikis. We demonstrate that one signaling pathway, the epidermal growth factor receptor (EGFR) pathway, is key to controlling each of these steps through distinct functions in intestinal stem cells and enterocytes. The EGFR pathway is activated by the EGF ligands, Spitz, Keren and Vein, the latter being induced in the surrounding visceral muscles in part under the control of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Additionally, the EGFR pathway synergizes with the JAK/STAT pathway in stem cells to promote their proliferation. Finally, we show that the EGFR pathway contributes to gut morphogenesis through its activity in enterocytes and is required to properly coordinate the delamination and anoikis of damaged cells. This function of the EGFR pathway in enterocytes is key to maintaining homeostasis, as flies lacking EGFR are highly susceptible to infection., Conclusions: This study demonstrates that restoration of normal gut morphology following bacterial infection is a more complex phenomenon than previously described. Maintenance of gut homeostasis requires the coordination of stem cell proliferation and differentiation, with the incorporation and morphogenesis of new cells and the expulsion of damaged enterocytes. We show that one signaling pathway, the EGFR pathway, is central to all these stages, and its activation at multiple steps could synchronize the complex cellular events leading to gut repair and homeostasis.

Trees in the genus Populus can provide substantial commercial and ecological benefits, including sustainable alternatives to traditional forestry. Realization of this potential requires intensive management, but damage by defoliating insects can severely limit productivity in such systems. Two approaches to limiting these losses include cultivation of poplar varieties with inherent resistance to pests and application of microbial pesticides. Little is known about the interaction between host resistance and the ability of poplars to support the efficacy of biocontrol agents. The research described here was conducted to survey the effect of hybrid poplar clones on gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), a pest on these trees. We assessed the effect of various poplar clones on larval performance and susceptibility to Bacillus thuringiensis subsp. kurstaki. Larvae were reared from hatching on the foliage of 25 hybrid poplar clones and we monitored larval survival, development time, and weight at fourth instar. Eight of these clones showed high resistance against gypsy moth. The remaining clones showed high variation in their effect on larval performance. We evaluated the susceptibility of third-instar larvae to B. thuringiensis subsp. kurstaki when reared on the 17 remaining clones. There was a significant effect of poplar clone on time to death after ingestion of B. thuringiensis subsp. kurstaki. The susceptibility of gypsy moth larvae to B. thuringiensis on various clones was not correlated with the effects of these clones on larval performance in the absence of B. thuringiensis, suggesting this interaction is more complex than merely reflecting higher mortality to previously stressed larvae.

Background: The gut comprises an essential barrier that protects both invertebrate and vertebrate animals from invasion by microorganisms. Disruption of the balanced relationship between indigenous gut microbiota and their host can result in gut bacteria eliciting host responses similar to those caused by invasive pathogens. For example, ingestion of Bacillus thuringiensis by larvae of some species of susceptible Lepidoptera can result in normally benign enteric bacteria exerting pathogenic effects., Results: We explored the potential role of the insect immune response in mortality caused by B. thuringiensis in conjunction with gut bacteria. Two lines of evidence support such a role. First, ingestion of B. thuringiensis by gypsy moth larvae led to the depletion of their hemocytes. Second, pharmacological agents that are known to modulate innate immune responses of invertebrates and vertebrates altered larval mortality induced by B. thuringiensis. Specifically, Gram-negative peptidoglycan pre-treated with lysozyme accelerated B. thuringiensis-induced killing of larvae previously made less susceptible due to treatment with antibiotics. Conversely, several inhibitors of the innate immune response (eicosanoid inhibitors and antioxidants) increased the host's survival time following ingestion of B. thuringiensis., Conclusions: This study demonstrates that B. thuringiensis infection provokes changes in the cellular immune response of gypsy moth larvae. The effects of chemicals known to modulate the innate immune response of many invertebrates and vertebrates, including Lepidoptera, also indicate a role of this response in B. thuringiensis killing. Interactions among B. thuringiensis toxin, enteric bacteria, and aspects of the gypsy moth immune response may provide a novel model to decipher mechanisms of sepsis associated with bacteria of gut origin.

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK-STAT (Janus kinase-signal transducers and activators of transcription) and JNK (c-Jun NH(2) terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity.

Background: Gut microbiota contribute to the health of their hosts, and alterations in the composition of this microbiota can lead to disease. Previously, we demonstrated that indigenous gut bacteria were required for the insecticidal toxin of Bacillus thuringiensis to kill the gypsy moth, Lymantria dispar. B. thuringiensis and its associated insecticidal toxins are commonly used for the control of lepidopteran pests. A variety of factors associated with the insect host, B. thuringiensis strain, and environment affect the wide range of susceptibilities among Lepidoptera, but the interaction of gut bacteria with these factors is not understood. To assess the contribution of gut bacteria to B. thuringiensis susceptibility across a range of Lepidoptera we examined larval mortality of six species in the presence and absence of their indigenous gut bacteria. We then assessed the effect of feeding an enteric bacterium isolated from L. dispar on larval mortality following ingestion of B. thuringiensis toxin., Results: Oral administration of antibiotics reduced larval mortality due to B. thuringiensis in five of six species tested. These included Vanessa cardui (L.), Manduca sexta (L.), Pieris rapae (L.) and Heliothis virescens (F.) treated with a formulation composed of B. thuringiensis cells and toxins (DiPel), and Lymantria dispar (L.) treated with a cell-free formulation of B. thuringiensis toxin (MVPII). Antibiotics eliminated populations of gut bacteria below detectable levels in each of the insects, with the exception of H. virescens, which did not have detectable gut bacteria prior to treatment. Oral administration of the Gram-negative Enterobacter sp. NAB3, an indigenous gut resident of L. dispar, restored larval mortality in all four of the species in which antibiotics both reduced susceptibility to B. thuringiensis and eliminated gut bacteria, but not in H. virescens. In contrast, ingestion of B. thuringiensis toxin (MVPII) following antibiotic treatment significantly increased mortality of Pectinophora gossypiella (Saunders), which was also the only species with detectable gut bacteria that lacked a Gram-negative component. Further, mortality of P. gossypiella larvae reared on diet amended with B. thuringiensis toxin and Enterobacter sp. NAB3 was generally faster than with B. thuringiensis toxin alone., Conclusion: This study demonstrates that in some larval species, indigenous gut bacteria contribute to B. thuringiensis susceptibility. Moreover, the contribution of enteric bacteria to host mortality suggests that perturbations caused by toxin feeding induce otherwise benign gut bacteria to exert pathogenic effects. The interaction between B. thuringiensis and the gut microbiota of Lepidoptera may provide a useful model with which to identify the factors involved in such transitions.

Little is known about the significance of insects as environmental reservoirs of antibiotic-resistant bacteria. We characterized the antibiotic resistome of the microbial community in gypsy moth larval midguts by applying functional metagenomics to cultured isolates. The minimum inhibitory concentrations of 12 antibiotics were determined for 44 cultured isolates, and antibiotic resistance genes were selected from metagenomic libraries derived from DNA extracted from a pool of the isolates. Six unique clones were identified. Two were highly resistant to penicillin-type beta-lactams, two were moderately resistant to erythromycin, and two were moderately resistant to a range of antibiotics, including erythromycin, carbenicillin, and chloramphenicol. Sequence analysis predicted that the active genes encoded efflux pumps, a transcriptional activator of efflux pump protein expression, and an extended-spectrum class A beta-lactamase. Insect guts are a reservoir of antibiotic resistance genes with the potential for dissemination.

Although Drosophila systemic immunity is extensively studied, little is known about the fly's intestine-specific responses to bacterial infection. Global gene expression analysis of Drosophila intestinal tissue to oral infection with the Gram-negative bacterium Erwinia carotovora revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. Ingestion of bacteria had a dramatic impact on the physiology of the gut that included modulation of stress response and increased stem cell proliferation and epithelial renewal. Our data suggest that gut homeostasis is maintained through a balance between cell damage due to the collateral effects of bacteria killing and epithelial repair by stem cell division. The Drosophila gut provides a powerful model to study the integration of stress and immunity with pathways associated with stem cell control, and this study should prove to be a useful resource for such further studies.

Bacillus thuringiensis is the most widely applied biological insecticide and is used to manage insects that affect forestry and agriculture and transmit human and animal pathogens. This ubiquitous spore-forming bacterium kills insect larvae largely through the action of insecticidal crystal proteins and is commonly deployed as a direct bacterial spray. Moreover, plants engineered with the cry genes encoding the B. thuringiensis crystal proteins are the most widely cultivated transgenic crops. For decades, the mechanism of insect killing has been assumed to be toxin-mediated lysis of the gut epithelial cells, which leads to starvation, or B. thuringiensis septicemia. Here, we report that B. thuringiensis does not kill larvae of the gypsy moth in the absence of indigenous midgut bacteria. Elimination of the gut microbial community by oral administration of antibiotics abolished B. thuringiensis insecticidal activity, and reestablishment of an Enterobacter sp. that normally resides in the midgut microbial community restored B. thuringiensis-mediated killing. Escherichia coli engineered to produce the B. thuringiensis insecticidal toxin killed gypsy moth larvae irrespective of the presence of other bacteria in the midgut. However, when the engineered E. coli was heat-killed and then fed to the larvae, the larvae did not die in the absence of the indigenous midgut bacteria. E. coli and the Enterobacter sp. achieved high populations in hemolymph, in contrast to B. thuringiensis, which appeared to die in hemolymph. Our results demonstrate that B. thuringiensis-induced mortality depends on enteric bacteria.