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5 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Signatures of Mollicutes-related endobacteria in publicly available Mucoromycota genomes

ABSTRACT Mucoromycota fungi and their Mollicutes-related endobacteria (MRE) are an ideal system for studying bacterial–fungal interactions and evolution due to the long-term and intimate nature of their interactions. However, methods for detecting MRE face specific challenges due to the poor representation of MRE in sequencing databases coupled with the high sequence divergence of their genomes, making traditional similarity searches unreliable. This has precluded estimations on the diversity of MRE associated with Mucoromycota. To determine the prevalence of previously undetected MRE in fungal genome sequences, we scanned 389 Mucoromycota genome assemblies available from the National Center for Biotechnology Information for the presence of MRE sequences using publicly available tools to map contigs from fungal assemblies to publicly available MRE genomes. We demonstrate a higher diversity of MRE genomes than previously described in Mucoromycota and a lack of cophylogeny between MRE and the majority of their fungal hosts. This supports the late invasion hypothesis regarding MRE acquisition across most of the examined fungal families. In contrast with other Mucoromycota lineages, MRE from the Gigasporaceae displayed some degree of cophylogeny with their hosts, which may indicate that horizontal transmission is restricted between members of this family or that transmission is strictly vertical. These results underscore the need for a refined process to capture sequencing data from potential fungal endosymbionts to discern their evolution and transmission. Screens of fungal genomes for MRE can help improve the quality of fungal genome assemblies while identifying new MRE lineages to further test hypotheses on their origin and evolution. IMPORTANCE Mollicutes-related endobacteria (MRE) are obligate intracellular bacteria found within Mucoromycota fungi. Despite their frequent detection, MRE roles in host functioning are still unknown. Comparative genomic investigations can improve our understanding of the impact of MRE on their fungal hosts by identifying similarities and differences in MRE genome evolution. However, MRE genomes have only been assembled from a small fraction of Mucoromycota hosts. Here, we demonstrate that MRE can be present yet undetected in publicly available Mucoromycota genome assemblies. We use these newfound sequences to assess the broader diversity of MRE and their phylogenetic relationships with respect to their hosts. We demonstrate that publicly available tools can be used to extract novel MRE sequences from assembled fungal genomes leading to insights on MRE evolution. This work contributes to a greater understanding of the fungal microbiome, which is crucial to improving knowledge on the dynamics and impacts of fungi in microbial ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria

We report that 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 in situhybridization (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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparative genomics of Mollicutes-related endobacteria supports a late invasion into Mucoromycota fungi

Abstract Diverse members of early-diverging Mucoromycota, including mycorrhizal taxa and soil-associated Mortierellaceae, are known to harbor Mollicutes-related endobacteria (MRE). It has been hypothesized that MRE were acquired by a common ancestor and transmitted vertically. Alternatively, MRE endosymbionts could have invaded after the divergence of Mucoromycota lineages and subsequently spread to new hosts horizontally. To better understand the evolutionary history of MRE symbionts, we generated and analyzed four complete MRE genomes from two Mortierellaceae genera: Linnemannia (MRE-L) and Benniella (MRE-B). These genomes include the smallest known of fungal endosymbionts and showed signals of a tight relationship with hosts including a reduced functional capacity and genes transferred from fungal hosts to MRE. Phylogenetic reconstruction including nine MRE from mycorrhizal fungi revealed that MRE-B genomes are more closely related to MRE from Glomeromycotina than MRE-L from the same host family. We posit that reductions in genome size, GC content, pseudogene content, and repeat content in MRE-L may reflect a longer-term relationship with their fungal hosts. These data indicate Linnemannia and Benniella MRE were likely acquired independently after their fungal hosts diverged from a common ancestor. This work expands upon foundational knowledge on minimal genomes and provides insights into the evolution of bacterial endosymbionts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Effect of Endosymbiotic Bacteria on Fungal Resistance Toward Heavy Metals

Most studies on metal removal or tolerance by fungi or bacteria focus on single isolates, without taking into consideration that some fungi in nature may be colonized by endobacteria. To address this knowledge gap, we investigated the tolerance and removal of diverse metals with two fungal species: Linnemannia elongata containing Burkholderia-related endobacteria and Benniella erionia containing Mollicute-related endobacteria. Isogenic lines of both species were generated with antibiotic treatments to remove their respective endobacteria. Experiments involved comparing the isogenic lines and wild type fungi in relation to the minimum inhibitory concentration for the metals, the fungal ability to remove these different metals via atomic adsorption spectroscopy, and the interaction of the metals with specific functional groups of the fungi and fungi-bacteria to determine the role of the bacteria via attenuated total reflection fourier transformed infrared (ATR-FTIR). Finally, we determined the influence of different metal concentrations, associated with moderate and high fungal growth inhibition, on the presence of the endobacteria inside the fungal mycelium via quantitative real-time PCR. Results showed that the presence of the endosymbiont increased B. erionia resistance to Mn 2+ and increased the removal of Fe 2+ compared to isogenic lines. The absence of the endosymbiont in L. elongata increased the fungal resistance toward Fe 2+ and improved the removal of Fe 2+ . Furthermore, when the bacterial endosymbiont was present in L. elongata , a decrease in the fungal resistance to Ca 2+ , Fe 2+ , and Cr 6+ was noticeable. In the ATR-FTIR analysis, we determined that C-H and C = O were the major functional groups affected by the presence of Cu 2+ , Mn 2+ , and Fe 2+ for L. elongata and in the presence of Cu 2+ and Ca 2+ for B. eronia . It is noteworthy that the highest concentration of Pb 2+ led to the loss of endobacteria in both L. elongata and B. eronia , while the other metals generally increased the concentration of endosymbionts inside the fungal mycelium. From these results, we concluded that bacterial endosymbionts of fungi can play a fundamental role in fungal resistance to metals. This study provides the first step toward a greater understanding of symbiotic interactions between bacteria and fungi in relation to metal tolerance and remediation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

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

Abstract 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.

59 BASIC BIOLOGICAL SCIENCES↗