Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

Antimicrobial Peptides and Copper(II) Ions: Novel Therapeutic Opportunities.

The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.

North American Fireflies Host Low Bacterial Diversity.

Although there are numerous studies of firefly mating flashes, lantern bioluminescence, and anti-predation lucibufagin metabolites, almost nothing is known about their microbiome. We therefore used 16S rRNA community amplicon sequencing to characterize the gut and body microbiomes of four North American firefly taxa: Ellychnia corrusca, the Photuris versicolor species complex, Pyractomena borealis, and Pyropyga decipiens. These firefly microbiomes all have very low species diversity, often dominated by a single species, and each firefly type has a characteristic microbiome. Although the microbiomes of male and female fireflies did not differ from each other, Ph. versicolor gut and body microbiomes did, with their gut microbiomes being enriched in Pseudomonas and Acinetobacter. Ellychnia corrusca egg and adult microbiomes were unique except for a single egg microbiome that shared a community type with E. corrusca adults, which could suggest microbial transmission from mother to offspring. Mollicutes that had been previously isolated from fireflies were common in our firefly microbiomes. These results set the stage for further research concerning the function and transmission of these bacterial symbionts.

Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing.

BACKGROUND: Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated. RESULTS: We sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions. CONCLUSIONS: Genome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore cost-effectively advance studies of microbial evolution and genome-driven drug discovery.

Propagating annotations of molecular networks using in silico fragmentation.

The annotation of small molecules is one of the most challenging and important steps in untargeted mass spectrometry analysis, as most of our biological interpretations rely on structural annotations. Molecular networking has emerged as a structured way to organize and mine data from untargeted tandem mass spectrometry (MS/MS) experiments and has been widely applied to propagate annotations. However, propagation is done through manual inspection of MS/MS spectra connected in the spectral networks and is only possible when a reference library spectrum is available. One of the alternative approaches used to annotate an unknown fragmentation mass spectrum is through the use of in silico predictions. One of the challenges of in silico annotation is the uncertainty around the correct structure among the predicted candidate lists. Here we show how molecular networking can be used to improve the accuracy of in silico predictions through propagation of structural annotations, even when there is no match to a MS/MS spectrum in spectral libraries. This is accomplished through creating a network consensus of re-ranked structural candidates using the molecular network topology and structural similarity to improve in silico annotations. The Network Annotation Propagation (NAP) tool is accessible through the GNPS web-platform https://gnps.ucsd.edu/ProteoSAFe/static/gnps-theoretical.jsp.

Isolation, Biosynthesis and Chemical Modifications of Rubterolones A-F: Rare Tropolone Alkaloids from Actinomadura sp. 5-2.

The discovery of six new, highly substituted tropolone alkaloids, rubterolones A-F, from Actinomadura sp. 5-2, isolated from the gut of the fungus-growing termite Macrotermes natalensis is reported. Rubterolones were identified by using fungus-bacteria challenge assays and a HRMS-based dereplication strategy, and characterised by NMR and HRMS analyses and by X-ray crystallography. Feeding experiments and subsequent chemical derivatisation led to a first library of rubterolone derivatives (A-L). Genome sequencing and comparative analyses revealed their putative biosynthetic pathway, which was supported by feeding experiments. This study highlights how gut microbes can present a prolific source of secondary metabolites.

Comparison of Xenorhabdus bovienii bacterial strain genomes reveals diversity in symbiotic functions.

BACKGROUND: Xenorhabdus bacteria engage in a beneficial symbiosis with Steinernema nematodes, in part by providing activities that help kill and degrade insect hosts for nutrition. Xenorhabdus strains (members of a single species) can display wide variation in host-interaction phenotypes and genetic potential indicating that strains may differ in their encoded symbiosis factors, including secreted metabolites. METHODS: To discern strain-level variation among symbiosis factors, and facilitate the identification of novel compounds, we performed a comparative analysis of the genomes of 10 Xenorhabdus bovienii bacterial strains. RESULTS: The analyzed X. bovienii draft genomes are broadly similar in structure (e.g. size, GC content, number of coding sequences). Genome content analysis revealed that general classes of putative host-microbe interaction functions, such as secretion systems and toxin classes, were identified in all bacterial strains. In contrast, we observed diversity of individual genes within families (e.g. non-ribosomal peptide synthetase clusters and insecticidal toxin components), indicating the specific molecules secreted by each strain can vary. Additionally, phenotypic analysis indicates that regulation of activities (e.g. enzymes and motility) differs among strains. CONCLUSIONS: The analyses presented here demonstrate that while general mechanisms by which X. bovienii bacterial strains interact with their invertebrate hosts are similar, the specific molecules mediating these interactions differ. Our data support that adaptation of individual bacterial strains to distinct hosts or niches has occurred. For example, diverse metabolic profiles among bacterial symbionts may have been selected by dissimilarities in nutritional requirements of their different nematode hosts. Similarly, factors involved in parasitism (e.g. immune suppression and microbial competition factors), likely differ based on evolution in response to naturally encountered organisms, such as insect hosts, competitors, predators or pathogens. This study provides insight into effectors of a symbiotic lifestyle, and also highlights that when mining Xenorhabdus species for novel natural products, including antibiotics and insecticidal toxins, analysis of multiple bacterial strains likely will increase the potential for the discovery of novel molecules.

Gene fragmentation in bacterial draft genomes: extent, consequences and mitigation.

UNLABELLED: Ongoing technological advances in genome sequencing are allowing bacterial genomes to be sequenced at ever-lower cost. However, nearly all of these new techniques concomitantly decrease genome quality, primarily due to the inability of their relatively short read lengths to bridge certain genomic regions, e.g., those containing repeats. Fragmentation of predicted open reading frames (ORFs) is one possible consequence of this decreased quality. In this study we quantify ORF fragmentation in draft microbial genomes and its effect on annotation efficacy, and we propose a solution to ameliorate this problem. RESULTS: A survey of draft-quality genomes in GenBank revealed that fragmented ORFs comprised > 80% of the predicted ORFs in some genomes, and that increased fragmentation correlated with decreased genome assembly quality. In a more thorough analysis of 25 Streptomyces genomes, fragmentation was especially enriched in some protein classes with repeating, multi-modular structures such as polyketide synthases, non-ribosomal peptide synthetases and serine/threonine kinases. Overall, increased genome fragmentation correlated with increased false-negative Pfam and COG annotation rates and increased false-positive KEGG annotation rates. The false-positive KEGG annotation rate could be ameliorated by linking fragmented ORFs using their orthologs in related genomes. Whereas this strategy successfully linked up to 46% of the total ORF fragments in some genomes, its sensitivity appeared to depend heavily on the depth of sampling of a particular taxon's variable genome. CONCLUSIONS: Draft microbial genomes contain many ORF fragments. Where these correspond to the same gene they have particular potential to confound comparative gene content analyses. Given our findings, and the rapid increase in the number of microbial draft quality genomes, we suggest that accounting for gene fragmentation and its associated biases is important when designing comparative genomic projects.

Microbial strain prioritization using metabolomics tools for the discovery of natural products.

Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.

Trachymyrmex septentrionalis Ant Microbiome Assembly Is Unique to Individual Colonies and Castes.

Within social insect colonies, microbiomes often differ between castes due to their different functional roles and between colony locations. Trachymyrmex septentrionalis fungus-growing ants form colonies throughout the eastern United States and northern Mexico that include workers, female and male alates (unmated reproductive castes), larvae, and pupae. How T. septentrionalis microbiomes vary across this geographic range and between castes is unknown. Our sampling of individual ants from colonies across the eastern United States revealed a conserved T. septentrionalis worker ant microbiome and revealed that worker ant microbiomes are more conserved within colonies than between them. A deeper sampling of individual ants from two colonies that included all available castes (pupae, larvae, workers, and female and male alates), from both before and after adaptation to controlled laboratory conditions, revealed that ant microbiomes from each colony, caste, and rearing condition were typically conserved within but not between each sampling category. Tenericute bacterial symbionts were especially abundant in these ant microbiomes and varied widely in abundance between sampling categories. This study demonstrates how individual insect colonies primarily drive the composition of their microbiomes and shows that these microbiomes are further modified by developmental differences between insect castes and the different environmental conditions experienced by each colony. IMPORTANCE This study investigates microbiome assembly in the fungus-growing ant Trachymyrmex septentrionalis, showing how colony, caste, and lab adaptation influence the microbiome and revealing unique patterns of mollicute symbiont abundance. We find that ant microbiomes differ strongly between colonies but less so within colonies. Microbiomes of different castes and following lab adaptation also differ in a colony-specific manner. This study advances our understanding of the nature of individuality in social insect microbiomes and cautions against the common practice of only sampling a limited number of populations to understand microbiome diversity and function.

Deciphering the Microbiome: Integrating Theory, New Technologies, and Inclusive Science.

The diversity and functional significance of microbiomes have become increasingly clear through the extensive sampling of Earth's many habitats and the rapid adoption of new sequencing technologies. However, much remains unknown about what makes a "healthy" microbiome, how to restore a disrupted microbiome, and how microbiomes assemble. In December 2019, we convened a workshop that focused on how to identify potential "rules of life" that govern microbiome structure and function. This collection of mSystems Perspective pieces reflects many of the main challenges and opportunities in the field identified by both in-person and virtual workshop participants. By borrowing conceptual and theoretical approaches from other fields, including economics and philosophy, these pieces suggest new ways to dissect microbiome patterns and processes. The application of conceptual advances, including trait-based theory and community coalescence, is providing new insights on how to predict and manage microbiome diversity and function. Technological and analytical advances, including deep transfer learning, metabolic models, and advances in analytical chemistry, are helping us sift through complex systems to pinpoint mechanisms of microbiome assembly and dynamics. Integration of all of these advancements (theory, concepts, technology) across biological and spatial scales is providing dramatically improved temporal and spatial resolution of microbiome dynamics. This integrative microbiome research is happening in a new moment in science where academic institutions, scientific societies, and funding agencies must act collaboratively to support and train a diverse and inclusive community of microbiome scientists.

Draft genome sequence of Streptomyces sp. strain Wigar10, isolated from a surface-sterilized garlic bulb.

Streptomyces sp. strain Wigar10 was isolated from a surface-sterilized garlic bulb (Allium sativum var. Purple Stripe). Its genome encodes several novel secondary metabolite biosynthetic gene clusters and provides a genetic basis for further investigation of this strain's chemical biology and potential for interaction with its garlic host.

Genome sequence of Streptomyces griseus strain XylebKG-1, an ambrosia beetle-associated actinomycete.

Streptomyces griseus strain XylebKG-1 is an insect-associated strain of the well-studied actinobacterial species S. griseus. Here, we present the genome of XylebKG-1 and discuss its similarity to the genome of S. griseus subsp. griseus NBRC13350. XylebKG-1 was isolated from the fungus-cultivating Xyleborinus saxesenii system. Given its similarity to free-living S. griseus subsp. griseus NBRC13350, comparative genomics will elucidate critical components of bacterial interactions with insects.

Characterization of Hymenobacter isolates from Victoria Upper Glacier, Antarctica reveals five new species and substantial non-vertical evolution within this genus.

We isolated several Hymenobacter-like strains from Victoria Upper Glacier, Antarctica, basal ice that diverged substantially from currently defined Hymenobacter species according to their 16S rRNA and gyrB gene phylogenies. All strains were psychrotolerant, heterotrophic aerobes which grew preferentially on low salt and low nutrient strength agar. Further phenotypic and chemotaxonomic characterization of these isolates supported their assignment as five novel species: H. algoricola sp. nov., H. antarcticus sp. nov., H. elongatus sp. nov., H. fastidiosus sp. nov., and H. glaciei sp. nov. Remarkable among these data was the prevalence of horizontal gene transfers and phenotypic variation, even between apparently closely related strains. These results suggest extensive non-vertical evolution within the genus Hymenobacter, and may reflect evolutionary trajectories resulting from dormancy, e.g., during interment in glacial ice.

Chemical Gradients of Plant Substrates in an Atta texana Fungus Garden.

Many ant species grow fungus gardens that predigest food as an essential step of the ants' nutrient uptake. These symbiotic fungus gardens have long been studied and feature a gradient of increasing substrate degradation from top to bottom. To further facilitate the study of fungus gardens and enable the understanding of the predigestion process in more detail than currently known, we applied recent mass spectrometry-based approaches and generated a three-dimensional (3D) molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments to compare with lab-maintained ecosystems. IMPORTANCE The study of complex ecosystems requires an understanding of the chemical processes involving molecules from several sources. Some of the molecules present in fungus-growing ants' symbiotic system originate from plants. To facilitate the study of fungus gardens from a chemical perspective, we provide a molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments.

Pseudonocardia Symbionts of Fungus-Growing Ants and the Evolution of Defensive Secondary Metabolism.

Actinobacteria belonging to the genus Pseudonocardia have evolved a close relationship with multiple species of fungus-growing ants, where these bacteria produce diverse secondary metabolites that protect the ants and their fungal mutualists from disease. Recent research has charted the phylogenetic diversity of this symbiosis, revealing multiple instances where the ants and Pseudonocardia have formed stable relationships in which these bacteria are housed on specific regions of the ant's cuticle. Parallel chemical and genomic analyses have also revealed that symbiotic Pseudonocardia produce diverse secondary metabolites with antifungal and antibacterial bioactivities, and highlighted the importance of plasmid recombination and horizontal gene transfer for maintaining these symbiotic traits. Here, we propose a multi-level model for the evolution of Pseudonocardia and their secondary metabolites that includes symbiont transmission within and between ant colonies, and the potentially independent movement and diversification of their secondary metabolite biosynthetic genes. Because of their well-studied ecology and experimental tractability, Pseudonocardia symbionts of fungus-growing ants are an especially useful model system to understand the evolution of secondary metabolites, and also comprise a significant source of novel antibiotic and antifungal agents.

Draft Genome Sequence of Spiroplasma platyhelix ATCC 51748, Isolated from a Dragonfly.

Spiroplasma platyhelix is a helical bacterium belonging to the class Mollicutes First isolated from a Pachydiplax longipennis dragonfly, it has the smallest reported Spiroplasma genome size of 740 kbp. Here, we report the genome sequence of S. platyhelix ATCC 51748.

Cycloheximide-Producing Streptomyces Associated With Xyleborinus saxesenii and Xyleborus affinis Fungus-Farming Ambrosia Beetles.

Symbiotic microbes help a myriad of insects acquire nutrients. Recent work suggests that insects also frequently associate with actinobacterial symbionts that produce molecules to help defend against parasites and predators. Here we explore a potential association between Actinobacteria and two species of fungus-farming ambrosia beetles, Xyleborinus saxesenii and Xyleborus affinis. We isolated and identified actinobacterial and fungal symbionts from laboratory reared nests, and characterized small molecules produced by the putative actinobacterial symbionts. One 16S rRNA phylotype of Streptomyces (XylebKG-1) was abundantly and consistently isolated from the galleries and adults of X. saxesenii and X. affinis nests. In addition to Raffaelea sulphurea, the symbiont that X. saxesenii cultivates, we also repeatedly isolated a strain of Nectria sp. that is an antagonist of this mutualism. Inhibition bioassays between Streptomyces griseus XylebKG-1 and the fungal symbionts from X. saxesenii revealed strong inhibitory activity of the actinobacterium toward the fungal antagonist Nectria sp. but not the fungal mutualist R. sulphurea. Bioassay guided HPLC fractionation of S. griseus XylebKG-1 culture extracts, followed by NMR and mass spectrometry, identified cycloheximide as the compound responsible for the observed growth inhibition. A biosynthetic gene cluster putatively encoding cycloheximide was also identified in S. griseus XylebKG-1. The consistent isolation of a single 16S phylotype of Streptomyces from two species of ambrosia beetles, and our finding that a representative isolate of this phylotype produces cycloheximide, which inhibits a parasite of the system but not the cultivated fungus, suggests that these actinobacteria may play defensive roles within these systems.

Broadening Participation in Scientific Conferences during the Era of Social Distancing.

Virtual conferences can offer significant benefits but require considerable planning and creativity to be successful. Here we describe the successes and failures of a hybrid in-person/virtual conference model. The COVID-19 epidemic presents the scientific community with an opportunity to pioneer novel models that effectively engage virtual participants to advance conference goals.