investigating-bacterial-fungal-interactions-using-fungal-highway

Investigating Bacterial-Fungal Interactions using Fungal Highway Columns in Diverse Environments and Substrates

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and ...

Los Alamos National Laboratory

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans. There is evidence that  could contribute to IBD. Here, we present the complete genome sequence of a strain designated CavFT-hAR46, which was isolated from a gut intramural cavernous fistulous tract (CavFT) microlesion in a CD patient.

Complete Genome Sequence of a Parabacteroides distasonis Strain (CavFT hAR46) Isolated from a Gut Wall-Cavitating Microlesion in a Patient with Severe Crohn's Disease.

 is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies  as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive  diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness.

Development of Anti- Human Antibodies with Features Required for Diagnostic and Therapeutic Applications.

Bacteria-fungi interactions (BFIs) are essential in ecosystem functioning. These interactions are modulated not only by local nutritional conditions but also by the physicochemical constraints and 3D structure of the environmental niche. In soils, the unsaturated and complex nature of the substrate restricts the dispersal and activity of bacteria. Under unsaturated conditions, some bacteria engage with filamentous fungi in an interaction (fungal highways) in which they use fungal hyphae to disperse. Based on a previous experimental device to enrich pairs of organisms engaging in this interaction in soils, we present here the design and validation of a modified version of this sampling system constructed using additive printing. The 3D printed devices were tested using a novel application in which a target fungus, the common coprophilous fungus Coprinopsis cinerea, was used as bait to recruit and identify bacterial partners using its mycelium for dispersal. Bacteria of the genera Pseudomonas, Sphingobacterium and Stenotrophomonas were highly enriched in association with C. cinerea. Developing and producing these new easy-to-use tools to investigate how bacteria overcome dispersal limitations in cooperation with fungi is important to unravel the mechanisms by which BFIs affect processes at an ecosystem scale in soils and other unsaturated environments.

Democratization of fungal highway columns as a tool to investigate bacteria associated with soil fungi.

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

Widespread bacterial diversity within the bacteriome of fungi.

Public sequencing databases are invaluable resources to biological researchers, but assessing data veracity as well as the curation and maintenance of such large collections of data can be challenging. Genomes of eukaryotic organelles, such as chloroplasts and other plastids, are particularly susceptible to assembly errors and misrepresentations in these databases due to their close evolutionary relationships with bacteria, which may co-occur within the same environment, as can be the case when sequencing plants. Here, based on sequence similarities with bacterial genomes, we identified several suspicious chloroplast assemblies present in the National Institutes of Health (NIH) Reference Sequence (RefSeq) collection. Investigations into these chloroplast assemblies reveal examples of erroneous integration of bacterial sequences into chloroplast ribosomal RNA (rRNA) loci, often within the rRNA genes, presumably due to the high similarity between plastid and bacterial rRNAs. The bacterial lineages identified within the examined chloroplasts as the most likely source of contamination are either known associates of plants, or co-occur in the same environmental niches as the examined plants. Modifications to the methods used to process untargeted 'raw' shotgun sequencing data from whole genome sequencing efforts, such as the identification and removal of bacterial reads prior to plastome assembly, could eliminate similar errors in the future.

Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes.

Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial-fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence  hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial-fungal relationships.

Advances and Challenges in Fluorescence  Hybridization for Visualizing Fungal Endobacteria.

Cohort-based learning for microbiome research community standards.

As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how "microbiomes within microbiomes" affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales. Video Abstract.

The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research.

Members of the fungal genus  are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing to characterize bacterial communities associated with sexual fruiting bodies from wild specimens, as well as vegetative mycelium and sclerotia obtained from  isolates grown . These investigations included diverse representatives from both Elata and Esculenta  clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual  isolates or specimens. However, specific bacterial taxa were frequently detected in association with certain structures, providing support for an associated core bacterial community. Bacteria from the genus  and  constituted the core bacterial associates of  mycelia and sclerotia, while other genera (e.g.,  spp.,  spp., and  spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of  as a key member of the bacteriome was supported by the isolation of several  strains from mycelia during  cultivation. Four of the six mycelial-derived  isolates shared 16S rDNA sequence identity with amplicon sequences recovered directly from the examined fungal structures. Distinct interaction phenotypes (antagonistic or neutral) were observed in confrontation assays between these bacteria and various  isolates. Genome sequences obtained from these  isolates revealed intriguing differences in gene content and annotated functions, specifically with respect to toxin-antitoxin systems, cell adhesion, chitinases, and insecticidal toxins. These genetic differences correlated with the interaction phenotypes. This study provides evidence that  spp. are frequently associated with  and these associations may greatly impact fungal physiology.

Associated bacterial communities, confrontation studies, and comparative genomics reveal important interactions between  with  spp.

Comparative analysis of (meta)genomes necessitates aggregation, integration, and synthesis of well-annotated data using standards. The Genomic Standards Consortium (GSC) collaborates with the research community to develop and maintain the Minimum Information about any (x) Sequence (MIxS) reporting standard for genomic data. To facilitate the use of the GSC's MIxS reporting standard, we provide a description of the structure and terminology, how to navigate ontologies for required terms in MIxS, and demonstrate practical usage through a soil metagenome example.

A Practical Approach to Using the Genomic Standards Consortium MIxS Reporting Standard for Comparative Genomics and Metagenomics.

Diverse and complex microbiomes are found in virtually every environment on Earth. Bacteria and fungi often co-dominate environmental microbiomes, and there is growing recognition that bacterial-fungal interactions (BFI) have significant impacts on the functioning of their associated microbiomes, environments, and hosts. Investigating BFI  remains a challenge, particularly when attempting to examine interactions at multiple scales of system complexity. Fabricated devices can provide control over both biotic composition and abiotic factors within an experiment to enable the characterization of diverse BFI phenotypes such as modulation of growth rate, production of biomolecules, and alterations to physical movements. Engineered devices ranging from microfluidic chips to simulated rhizosphere systems have been and will continue to be invaluable to BFI research, and it is anticipated that such devices will continue to be developed for diverse applications in the field. This will allow researchers to address specific questions regarding the nature of BFI and how they impact larger microbiome and environmental processes such as biogeochemical cycles, plant productivity, and overall ecosystem resilience. Devices that are currently used for experimental investigations of bacteria, fungi, and BFI are discussed herein along with some of the associated challenges and several recommendations for future device design and applications.

Article	Year
Complete Genome Sequence of a Parabacteroides distasonis Strain (CavFT hAR46) Isolated from a Gut Wall-Cavitating Microlesion in a Patient with Severe Crohn's Disease. Microbiology resource announcements\| PubMed ID: 31488526	2019
Development of Anti- Human Antibodies with Features Required for Diagnostic and Therapeutic Applications. ImmunoTargets and therapy\| PubMed ID: 33294421	2020
Democratization of fungal highway columns as a tool to investigate bacteria associated with soil fungi. FEMS microbiology ecology\| PubMed ID: 33440006	2021
Widespread bacterial diversity within the bacteriome of fungi. Communications biology\| PubMed ID: 34621007	2021
Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes. Frontiers in genetics\| PubMed ID: 35096026	2021
Advances and Challenges in Fluorescence Hybridization for Visualizing Fungal Endobacteria. Frontiers in microbiology\| PubMed ID: 35722318	2022
Cohort-based learning for microbiome research community standards. Nature microbiology\| PubMed ID: 37069400	2023
The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research. Microbiome\| PubMed ID: 37626434	2023
Associated bacterial communities, confrontation studies, and comparative genomics reveal important interactions between with spp. Frontiers in fungal biology\| PubMed ID: 38155707	2023
A Practical Approach to Using the Genomic Standards Consortium MIxS Reporting Standard for Comparative Genomics and Metagenomics. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 38819573	2024
Fabricated devices for performing bacterial-fungal interaction experiments across scales. Frontiers in microbiology\| PubMed ID: 39171270	2024

Julia M. Kelliher

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