Biology subjects

Klassen, J. L.

Publications and source records attributed to Klassen, J. L..

10 recordsLinked to original sources

bioRxiv2026

Ant abaecin-2 is a context-dependent copper-binding effector that can be either inhibitory or protective

Host defense peptides (HDPs) are important components of the innate immune system that are used to combat pathogens and often rely on metal binding for their function. However, controlling trace nutrients such as transition metals may have other roles in host-symbiont interactions beyond poisoning harmful pathogens. This study characterizes the evolution, structural properties, and biochemical activity of the novel hymenopteran HDP abaecin-2. In myrmicine ants such as the fungus-growing tribe Attini, abaecin-2 has evolved to include an Amino-Terminal Cu(II) and Ni(II)-binding (ATCUN) motif, which we hypothesize may bind copper, a trace nutrient that is enriched in attine ant colonies. Combined results from mass spectrometry, competitive binding assays, circular dichroism, and NMR indicate that the abaecin-2 peptide lacks a defined secondary structure and can associate with up to 2 Cu(II) ions, one strongly bound at the ATCUN motif and another weakly bound, likely at a conserved histidine residue. Despite its copper-binding activity, abaecin-2 alone does not exhibit antibacterial activity against Escherichia coli or Bacillus subtilis (models for bacteria that live in ant fungus gardens). However, it synergizes with a model pore-forming peptide cecropin A to inhibit the growth of E. coli, similar to the related peptide abaecin-1. The copper-binding activity conferred by the ATCUN motif also protects copper-sensitive E. coli from excess copper toxicity. The dual, context-dependent inhibitory and protective roles we propose for abaecin-2 indicate that this previously under-characterized HDP may be used by attine ants to regulate both harmful and beneficial symbionts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/719391v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1298f41org.highwire.dtl.DTLVardef@17c16d9org.highwire.dtl.DTLVardef@1c03081org.highwire.dtl.DTLVardef@b0a5a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Untrimmed ITS2 metabarcode sequences cause artificially reduced abundances of specific fungal taxa

Advances in DNA metabarcoding have greatly expanded our knowledge of microbial communities in recent years. Pipelines and parameters have been tested extensively for bacterial metabarcoding using the 16S rRNA gene and best practices are largely established. For fungal metabarcoding using the ITS gene, however, only a few studies have considered how such pipelines and parameters can affect community prediction. Here we report a novel bias uncovered during ITS2 sequencing of Trichoderma-infected ant fungus gardens and confirmed using mock communities. Abnormally low forward read quality caused Trichoderma ITS2 reads to be computationally filtered before and during read pair merging, thus almost entirely eliminating Trichoderma ASVs from the resulting fungal community profiles. Sliding window quality trimming before filtering allowed most of these reads to pass filtering and merge successfully, producing community profiles that now correlated with visual signs of Trichoderma infection and matched the composition of the mock communities. Applying such sliding window trimming to a previously generated environmental ITS2 dataset increased the detected fungal diversity and again overcame read quality biases against Trichoderma to instead detect it in nearly every sample and often at high relative abundances. This analysis additionally identified a similar, but distinct, bias against a second fungal genus Meyerozyma. The prevalence of such quality biases against other fungal ITS sequences is unknown but may be widespread. We therefore advocate for routine use of sliding window quality trimming as a best practice in ITS2 metabarcoding analysis. ImportanceMetabarcode sequencing produces DNA abundance profiles that are presumed to reflect the actual microbial composition of the samples that they analyze. However, this assumption is not always tested, and taxon-specific biases are often not apparent, especially for low-abundance taxa in complex communities. Here we identified ITS2 read quality aberrations that caused dramatic reductions in the relative abundances of specific taxa in multiple datasets characterizing ant fungus gardens. Such taxon-specific biases in read quality may be widespread in other environments and for other fungal taxa, thereby causing incorrect descriptions of these mycobiomes.

microbiology↗

bioRxiv2024

Isolation and characterization of mollicute symbionts from a fungus-growing ant reveals genome reduction and host specialization

Two mollicute species belonging to the Mesoplasma and Spiroplasma genera have been detected in several species of fungus-growing ants using molecular methods. However, their ecological roles remain largely only inferred from metagenomic data. To better understand their diversity and specialization, we cultured both of these Mesoplasma and Spiroplasma symbionts from the fungus-growing ant Trachymyrmex septentrionalis, providing the first isolated mollicutes from any fungus-growing ant species. The genomes of our isolates and related metagenome-assembled genomes (MAGs) from T. septentrionalis fungus gardens comprise two unique phylogenetic lineages compared to previously described Mesoplasma and Spiroplasma species, and from related MAGs previously sequenced from the leaf-cutting ant Acromyrmex echinatior. This suggests that the T. septentrionalis symbionts comprise undescribed species with distinct host specificities. Mesoplasma genomes and MAGs also demonstrate regional specificity with their T. septentrionalis ant hosts. Both Mesoplasma and Spiroplasma strains from T. septentrionalis can catabolize glucose and fructose; both sugars are common in the ants diet. Similarly, both these Mesoplasma and Spiroplasma can catabolize arginine but only Mesoplasma can catabolize N-acetylglucosamine, which could both produce ammonia for the ants or fungus garden. Based on our genomic and phenotypic analyses, we describe these T. septentrionalis symbionts as Mesoplasma whartonense sp. nov. and Spiroplasma attinicola sp. nov., providing insight into their genomic and phenotypic diversity, and cultures to facilitate future studies of these common but poorly understood members of the fungus-growing ant symbiosis.

microbiology↗

bioRxiv2022

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 Kochs postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish Trichoderma spp. as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant non-cultivar 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 new 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. Significance StatementAn extended defense response may exist in any relationship where one partner benefits from defending a mutualistic partner. Such a response is observed in the fungus-growing ant symbiosis, where ants must identify and remove pathogens of their symbiotic fungus gardens. Here we describe the fungal pathogen Trichoderma and its associated metabolites, which induce Trachymyrmex septentrionalis ant weeding behavior. Ants removed fungus garden pieces inoculated with Trichoderma spores or peptaibol-rich Trichoderma extracts, and peptaibols as a class cued ant defensive behavior, allowing T. septentrionalis to differentiate healthy from diseased fungus gardens. Extended defense responses mediated by chemical cues may be underappreciated mechanisms that structure symbiotic interactions.

microbiology↗

bioRxiv2021

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 USA 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 USA revealed a conserved T. septentrionalis worker ant microbiome, and 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, 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 that these microbiomes are further modified by developmental differences between insect castes and the different environmental conditions experienced by each colony. IMPORTANCEThis study investigates microbiome assembly in the fungus-growing ant Trachymyrmex septentrionalis, showing how colony, caste, and lab adaptation influences 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 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.

microbiology↗

bioRxiv2020

The ant fungus garden acts as an external digestive system

Most animals digest their food within their own bodies, but some do not. Many species of ants grow fungus gardens that pre-digest food as an essential step of the ants nutrient uptake. To better understand this digestion process, we generated a 3D molecular map of an Atta texana fungus garden, revealing chemical modifications mediated by the fungus garden as plant material passes through.

systems biology↗

bioRxiv2020

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.

microbiology↗

bioRxiv2019

MASST: A Web-based Basic Mass Spectrometry Search Tool for Molecules to Search Public Data.

CorrespondenceWe introduce a web-enabled small-molecule mass spectrometry (MS) search engine. To date, no tool can query all the public small-molecule tandem MS data in metabolomics repositories, greatly limiting the utility of these resources in clinical, environmental and natural product applications. Therefore, we introduce a Mass Spectrometry Search Tool (MASST) (https://proteosafe-extensions.ucsd.edu/masst/), that enables the discovery of molecular relationships among accessible public metabolomics and natural product tandem mass spectrometry data (MS/MS).

bioinformatics↗

bioRxiv2019

Biogeography and Microscale Diversity Shape the Biosynthetic Potential of Fungus-growing Ant-associated Pseudonocardia

The geographic and phylogenetic scale of ecologically relevant microbial diversity is still poorly understood. Using a model mutualism, fungus-growing ants and their defensive bacterial associate Pseudonocardia, we analyzed genetic diversity and biosynthetic potential in 46 strains isolated from ant colonies in a 20km transect near Barro Colorado Island in Panama. Despite an average pairwise core genome similarity of greater than 99%, population genomic analysis revealed several distinct bacterial populations matching ant host geographic distribution. We identified both genetic diversity signatures and divergent genes distinct to each lineage. We also identify natural product biosynthesis clusters specific to isolation locations. These geographic patterns were observable despite the populations living in close proximity to each other and provides evidence of ongoing genetic exchange. Our results add to the growing body of literature suggesting that variation in traits of interest can be found at extremely fine phylogenetic scales.

microbiology↗

bioRxiv2019

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 nests 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 S. griseus XylebKG-1 and the fungal symbionts from X. saxesenii revealed strong inhibitory activity of the actinobacterium towards 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.

microbiology↗