Host and nonhost bacteria support bacteriophage dissemination along mycelia and abiotic dispersal networks.

Bacteriophages play a crucial role in shaping bacterial communities, yet the mechanisms by which nonmotile bacteriophages interact with their hosts remain poorly understood. This knowledge gap is especially pronounced in structured environments like soil, where spatial constraints and air-filled zones hinder aqueous diffusion. In soil, hyphae of filamentous microorganisms form a network of 'fungal highways' (FHs) that facilitate the dispersal of other microorganisms. We propose that FHs also promote bacteriophage dissemination. Viral particles can diffuse in liquid films surrounding hyphae or be transported by infectable (host) or uninfectable (nonhost) bacterial carriers coexisting on FH networks. To test this, two bacteriophages that infect Pseudomonas putida DSM291 (host) but not KT2440 (nonhost) were used. In the absence of carriers, bacteriophages showed limited diffusion on 3D-printed abiotic networks, but diffusion was significantly improved in Pythium ultimum-formed FHs when the number of connecting hyphae exceeded 20. Transport by both host and nonhost carriers enhanced bacteriophage dissemination. Host carriers were five times more effective in transporting bacteriophages, particularly in FHs with over 30 connecting hyphae. This study enhances our understanding of bacteriophage dissemination in nonsaturated environments like soils, highlighting the importance of biotic networks and bacterial hosts in facilitating this process.

Complete genome sequence of Thermaerobacter composti strain Ins1, a spore-forming filamentous bacterium isolated from a deep geothermal reservoir.

We report the complete genome sequence of Thermaerobacter composti strain Ins1, a gram-positive filamentous spore-forming bacterium, isolated from deep geothermal fluids used for electricity production. This is the first complete (circular) genome assigned to the species Thermaerobacter composti.

Gamification as a tool to teach key concepts in microbiology to bachelor-level students in biology: a case study using microbial interactions and soil functioning.

Microbiology is a difficult topic to teach given that the objects of study are mostly invisible to the learner. The majority of university students beginning their training in biology are more interested in natural objects that can be seen with the naked eye. Nonetheless, micro-organisms are key components of the biosphere and a good microbiological background is required for a thorough training in natural sciences. Lectures are still a common teaching format in universities. However, it is a passive learning format and no longer considered the most adequate approach in most teaching situations. Instead, alternatives consisting of more active teaching formats have been recognized to better motivate students to acquire and consolidate knowledge. In addition, transferable skills, such as effective communication, critical thinking and time management, are acquired simultaneously. A similar engagement can be obtained using games as part of the teaching experience. In this study, we designed a card game to teach key concepts in basic bacteriology and mycology to bachelor-level students. The first task consists of creating and designing microbial characters based on a list of species. This proved very useful for second-year bachelor students in terms of grasping concepts such as cell morphologies, taxonomy and life cycles. In the second task, third-year students used the characters created in the second-year class to develop a game based on an ecological function, namely forest litter degradation. In addition, they also considered experimental validation of the microbial activities and incorporated knowledge acquired in other fields.

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

Members of the fungal genus Morchella 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 Morchella isolates grown in vitro. These investigations included diverse representatives from both Elata and Esculenta Morchella clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual Morchella 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 Pseudomonas and Ralstonia constituted the core bacterial associates of Morchella mycelia and sclerotia, while other genera (e.g., Pedobacter spp., Deviosa spp., and Bradyrhizobium spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of Pseudomonas as a key member of the bacteriome was supported by the isolation of several Pseudomonas strains from mycelia during in vitro cultivation. Four of the six mycelial-derived Pseudomonas 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 Morchella isolates. Genome sequences obtained from these Pseudomonas 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 Pseudomonas spp. are frequently associated with Morchella and these associations may greatly impact fungal physiology.

Fungal drops: a novel approach for macro- and microscopic analyses of fungal mycelial growth.

This study presents an inexpensive approach for the macro- and microscopic observation of fungal mycelial growth. The 'fungal drops' method allows to investigate the development of a mycelial network in filamentous microorganisms at the colony and hyphal scales. A heterogeneous environment is created by depositing 15-20 µl drops on a hydrophobic surface at a fixed distance. This system is akin to a two-dimensional (2D) soil-like structure in which aqueous-pockets are intermixed with air-filled pores. The fungus (spores or mycelia) is inoculated into one of the drops, from which hyphal growth and exploration take place. Hyphal structures are assessed at different scales using stereoscopic and microscopic imaging. The former allows to evaluate the local response of regions within the colony (modular behaviour), while the latter can be used for fractal dimension analyses to describe the hyphal network architecture. The method was tested with several species to underpin the transferability to multiple species. In addition, two sets of experiments were carried out to demonstrate its use in fungal biology. First, mycelial reorganization of Fusarium oxysporum was assessed as a response to patches containing different nutrient concentrations. Second, the effect of interactions with the soil bacterium Pseudomonas putida on habitat colonization by the same fungus was assessed. This method appeared as fast and accessible, allowed for a high level of replication, and complements more complex experimental platforms. Coupled with image analysis, the fungal drops method provides new insights into the study of fungal modularity both macroscopically and at a single-hypha level.

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

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.

Nutrients and flow shape the cyclic dominance games between Escherichia coli strains.

Evolutionary game theory has provided various models to explain the coexistence of competing strategies, one of which is the rock-paper-scissors (RPS) game. A system of three Escherichia coli strains-a toxin-producer, a resistant and a sensitive-has become a classic experimental model for studying RPS games. Previous experimental and theoretical studies, however, often ignored the influence of ecological factors such as nutrients and toxin dynamics on the evolutionary game dynamics. In this work, we combine experiments and modelling to study how these factors affect competition dynamics. Using three-dimensional printed mini-bioreactors, we tracked the frequency of the three strains in different culturing media and under different flow regimes. Although our experimental system fulfilled the requirements of cyclic dominance, we did not observe clear cycles or long-term coexistence between strains. We found that both nutrients and flow rates strongly impacted population dynamics. In our simulations, we explicitly modelled the release, removal and diffusion of toxin. We showed that the amount of toxin that is retained in the system is a simple indicator that can predict competition outcomes across broad parameter space. Moreover, our simulation results suggest that high rates of toxin diffusion might have prevented cyclic patterns from emerging in our experimental system. This article is part of the theme issue 'Half a century of evolutionary games: a synthesis of theory, application and future directions'.

Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria.

The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores; Actinobacteria, forming exospores; Cyanobacteria, forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes.

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.

Spatial scales of competition and a growth-motility trade-off interact to determine bacterial coexistence.

The coexistence of competing species is a long-lasting puzzle in evolutionary ecology research. Despite abundant experimental evidence showing that the opportunity for coexistence decreases as niche overlap increases between species, bacterial species and strains competing for the same resources are commonly found across diverse spatially heterogeneous habitats. We thus hypothesized that the spatial scale of competition may play a key role in determining bacterial coexistence, and interact with other mechanisms that promote coexistence, including a growth-motility trade-off. To test this hypothesis, we let two Pseudomonas putida strains compete at local and regional scales by inoculating them either in a mixed droplet or in separate droplets in the same Petri dish, respectively. We also created conditions that allow the bacterial strains to disperse across abiotic or fungal hyphae networks. We found that competition at the local scale led to competitive exclusion while regional competition promoted coexistence. When competing in the presence of dispersal networks, the growth-motility trade-off promoted coexistence only when the strains were inoculated in separate droplets. Our results provide a mechanism by which existing laboratory data suggesting competitive exclusion at a local scale is reconciled with the widespread coexistence of competing bacterial strains in complex natural environments with dispersal.

Design and construction of 3D printed devices to investigate active and passive bacterial dispersal on hydrated surfaces.

BACKGROUND: To disperse in water-unsaturated environments, such as the soil, bacteria rely on the availability and structure of water films forming on biotic and abiotic surfaces, and, especially, along fungal mycelia. Dispersal along such "fungal highways" may be driven both by mycelial physical properties and by interactions between bacteria and fungi. However, we still do not have a way to disentangle the biotic and abiotic elements. RESULTS: We designed and 3D printed two devices establishing stable liquid films that support bacteria dispersal in the absence of biotic interactions. The thickness of the liquid film determined the presence of hydraulic flow capable of transporting non-motile cells. In the absence of flow, only motile cells can disperse in the presence of an energy source. Non-motile cells could not disperse autonomously without flow but dispersed as "hitchhikers" when co-inoculated with motile cells. CONCLUSIONS: The 3D printed devices can be used as an abiotic control to study bacterial dispersal on hydrated surfaces, such as plant roots and fungal hyphae networks in the soil. By teasing apart the abiotic and biotic dimensions, these 3D printed devices will stimulate further research on microbial dispersal in soil and other water-unsaturated environments.

Importance of appropriate genome information for the design of mating type primers in black and yellow morel populations.

Morels are highly prized edible fungi where sexual reproduction is essential for fruiting-body production. As a result, a comprehensive understanding of their sexual reproduction is of great interest. Central to this is the identification of the reproductive strategies used by morels. Sexual reproduction in fungi is controlled by mating-type (MAT) genes and morels are thought to be mainly heterothallic with two idiomorphs, MAT1-1 and MAT1-2. Genomic sequencing of black (Elata clade) and yellow (Esculenta clade) morel species has led to the development of PCR primers designed to amplify genes from the two idiomorphs for rapid genotyping of isolates from these two clades. To evaluate the design and theoretical performance of these primers we performed a thorough bioinformatic investigation, including the detection of the MAT region in publicly available Morchella genomes and in-silico PCR analyses. All examined genomes, including those used for primer design, appeared to be heterothallic. This indicates an inherent fault in the original primer design which utilized a single Morchella genome, as the use of two genomes with complementary mating types would be required to design accurate primers for both idiomorphs. Furthermore, potential off-targets were identified for some of the previously published primer sets, but verification was challenging due to lack of adequate genomic information and detailed methodologies for primer design. Examinations of the black morel specific primer pairs (MAT11L/R and MAT22L/R) indicated the MAT22 primers would correctly target and amplify the MAT1-2 idiomorph, but the MAT11 primers appear to be capable of amplifying incorrect off-targets within the genome. The yellow morel primer pairs (EMAT1-1 L/R and EMAT1-2 L/R) appear to have reporting errors, as the published primer sequences are dissimilar with reported amplicon sequences and the EMAT1-2 primers appear to amplify the RNA polymerase II subunit (RPB2) gene. The lack of the reference genome used in primer design and descriptive methodology made it challenging to fully assess the apparent issues with the primers for this clade. In conclusion, additional work is still required for the generation of reliable primers to investigate mating types in morels and to assess their performance on different clades and across multiple geographical regions.

Complete Genome Sequences of the Soil Oxalotrophic Bacterium Cupriavidus oxalaticus Strain Ox1 and Its Derived mCherry-Tagged Strain.

Here, we report the complete genome sequences of the soil oxalotrophic bacterium Cupriavidus oxalaticus Ox1 and a derived mCherry-tagged strain. The genome size is approximately 6.69 Mb, with a GC content of 66.9%. The genome sequence of C. oxalaticus Ox1 contains a complete operon for the degradation and assimilation of oxalate.

Fungi-on-a-Chip: microfluidic platforms for single-cell studies on fungi.

This review highlights new advances in the emerging field of 'Fungi-on-a-Chip' microfluidics for single-cell studies on fungi and discusses several future frontiers, where we envisage microfluidic technology development to be instrumental in aiding our understanding of fungal biology. Fungi, with their enormous diversity, bear essential roles both in nature and our everyday lives. They inhabit a range of ecosystems, such as soil, where they are involved in organic matter degradation and bioremediation processes. More recently, fungi have been recognized as key components of the microbiome in other eukaryotes, such as humans, where they play a fundamental role not only in human pathogenesis, but also likely as commensals. In the food sector, fungi are used either directly or as fermenting agents and are often key players in the biotechnological industry, where they are responsible for the production of both bulk chemicals and antibiotics. Although the macroscopic fruiting bodies are immediately recognizable by most observers, the structure, function, and interactions of fungi with other microbes at the microscopic scale still remain largely hidden. Herein, we shed light on new advances in the emerging field of Fungi-on-a-Chip microfluidic technologies for single-cell studies on fungi. We discuss the development and application of microfluidic tools in the fields of medicine and biotechnology, as well as in-depth biological studies having significance for ecology and general natural processes. Finally, a future perspective is provided, highlighting new frontiers in which microfluidic technology can benefit this field.

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

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 situ 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.

Recent Advances in Fungal Infections: From Lung Ecology to Therapeutic Strategies With a Focus on Aspergillus spp.

Fungal infections are estimated to be the main cause of death for more than 1.5 million people worldwide annually. However, fungal pathogenicity has been largely neglected. This is notably the case for pulmonary fungal infections, which are difficult to diagnose and to treat. We are currently facing a global emergence of antifungal resistance, which decreases the chances of survival for affected patients. New therapeutic approaches are therefore needed to face these life-threatening fungal infections. In this review, we will provide a general overview on respiratory fungal infections, with a focus on fungi of the genus Aspergillus. Next, the immunological and microbiological mechanisms of fungal pathogenesis will be discussed. The role of the respiratory mycobiota and its interactions with the bacterial microbiota on lung fungal infections will be presented from an ecological perspective. Finally, we will focus on existing and future innovative approaches for the treatment of respiratory fungal infections.

Improved methods to assess the effect of bacteria on germination of fungal spores.

Bacterial-fungal interactions (BFI) play a major role on ecosystem functioning and might be particularly relevant at a specific development stage. For instance, in the case of biological control of fungal pathogens by bacteria, a highly relevant kind of BFI, in-vitro experiments often assess the impact of a bacterium on the inhibition of actively growing mycelia. However, this fails to consider other stages of plant infection such as the germination of a spore or a sclerotium. This study aims to present novel experimental platforms for in-vitro experiments with fungal spores, in order to assess the effect of bacteria on germination and fungal growth control, to recover the metabolites produced in the interaction, and to enhance direct visualisation of BFI. Botrytis cinerea, a phytopathogenic fungus producing oxalic acid (OA) as pathogenicity factor, was used as model. Given that oxalotrophic bacteria have been shown previously to control the growth of B. cinerea, the oxalotrophic bacteria Cupriavidus necator and Cupriavidus oxalaticus were used as models. The experiments performed demonstrated the suitability of the methods and confirmed that both bacteria were able to control the growth of B. cinerea, but only in media in which soluble OA was detected by the fungus. The methods presented here can be easily performed in any microbiology laboratory and are not only applicable to screen for potential biocontrol agents, but also to better understand BFI.

Spores-on-a-chip: new frontiers for spore research.

In recent years, microfluidic technologies have become widespread in biological science. However, the suitability of this technique for understanding different aspects of spore research has hardly been considered. Herein, we review recent developments in 'spores-on-a-chip' technologies, highlighting how they could be exploited to drive new frontiers in spore research.