To test a possible bridging effect of mycelial networks, plasmid donor and recipient were inoculated on individual agar pieces separated by an air-filled gap. Environments allowing for low, medium and high mobility of bacteria were simulated using semi- solid media prepared with varying agar concentrations 20 and HGT events evaluated by flow cytometry and microscopy. The effect of transport networks was studied by adding either mycelia or glass fibres as abiotic surrogates to exclude other than physical effects. The oomycete Pythium ultimum was inoculated on the donor side.
The presence of all three cell types — donor, wildtype and resulting transconjugant — in the vicinity of the hyphae was visualized by epifluorescence microscopy. In control setups consisting of separated systems without mycelial structures, no transconjugants were detected. Emerging transconjugants green are visible along the mycelium. Outlined area is shown in detail in b.
HGT frequencies in systems with or without transport networks were quantified on semi- solid ABC-media 21 containing varying amounts of agar to influence bacterial mobility. High mobility agar containing 0. The media were either overgrown with P. The resulting transconjugants in this case constitutively express YFP and additionally started expressing GFP, allowing them to be differentiated via microscopy and quantified by flow cytometry After the incubation, the mycelia-covered area between the inoculation spots of donor and recipient colonies displayed numerous transconjugants Fig.
The HGT promoting effect of the mycelium was especially notable at the equidistant contact zone between donor and recipient colonies Fig. Here, the transconjugants appeared along and in between the hyphal network structures of P. White arrows indicate inoculation points of P. Zone in the dashed white box was used as an input for the simulation model in Fig.
The dashed white line represents the equal distance between both inoculation points E. Transconjugants can be seen emerging along the network structures. Images were taken 3 days after incubation. While comparably low transconjugant numbers were likewise found in high mobility systems with mycelia 2. Thus, the beneficial effect of mycelia was especially pronounced in low and medium mobility systems, i. The same trend was even more pronounced in systems supplemented with glass fibres as abiotic, i.
In presence of glass fibres, systems showed significantly higher amounts of transconjugants than without transport vectors in all three cases relative transconjugant fractions of 1. Even in high mobility systems with glass fibres there was a notable increase in transconjugants, compared to systems with mycelia or without transport vectors. Diamonds represent single data points; black lines show arithmetic mean, grey boxes show upper and lower quartile. To further investigate the underlying effects, an individual-based model was employed cf. Supplementary Information for details.
Model parameters were chosen according to the laboratory microcosm or literature values except for the characterization of the exchange flux of bacterial cells between the agar phase and the liquid film on the hyphal surface. This flux, defined by the attachment and detachment parameters k attach and k detach , was systematically varied, in effect allowing bacterial cells to utilize the hyphal network to different extents. As bacterial migration in our planar experimental setup is mainly following along a horizontal plane, either within the agar layer or along the flat hyphal network on top the agar, we discretized the agar layer as a two-dimensional grid on which cells as part of opposing fronts of donor- and recipient cells randomly explore the system.
The structure of a mycelium of P.
In order to assess the drivers for the HGT promoting effect of hyphae, a detailed analysis of the proposed driving parameters of HGT along hyphae was performed. Interestingly, conjugation rates depended only to a minor extent on the relative bacterial mobility on hyphal surfaces relative to the agar Fig. Simulation results of a the spatial distribution of transconjugants and b the role of hyphal attractiveness on conjugation rate in low mobility systems with mycelia.
As attractiveness increases more bacteria populated the hyphal network color and more conjugation events occured. D agar was set to 1.
In this study we give experimental and theoretical evidence that mycelia-based dispersal promotes HGT due to an elevated contact probability between bacteria. Here, we show for the first time that the narrow liquid films along continuous surfaces of hyphae and glass fibres promote close cell-to-cell contact by increased settling of initially spatially separate conjugation partners along the network structures 10 Fig. While previous microbial conjugation models assumed well-mixed populations 26 , 27 or considered space explicitly in up to three dimensions 28 , we used a quasi three-dimensional model to corroborate our findings.
We propose two driving mechanisms for the increased contact probabilities in the hyphosphere: Reduction of the space available for bacterial dispersal and, secondly, reduction of the degree of freedom of bacterial mobility. Such reasoning becomes clear by comparing a scenario in which bacterial cells populate a cube of water with a scenario in which cells only populate a water film surrounding hyphae forming a network within a cube of the same volume.
Even in case of dense networks, the latter scenario results in a clearly reduced volume available for bacterial dispersal. In contrast to the water cube scenario, hyphae additionally represent defined corridors for dispersal, and cells migrating along a hypha can overcome longer linear distances than in the water cube in a given time span. Presuming bacterial mobility within the liquid film around hyphae resembles a two- rather than a three-dimensional movement, the calculated increase of contact probability of two cells amounts to a factor of approximately see Supplement for derivation ; i.
This effect may become even larger if thin water films reduce the randomness of bacterial movement 29 or even prevent a reversal of the direction of movement, i.
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Indeed, fewer transconjugants were detected when transport networks were added to high mobility systems; here hyphae are losing their exclusivity as transport paths and their ability to confine the movement of the cells with the effect of reduced frequencies of bacterial encounter and HGT.
Simulation experiments corroborated the effect. They indicated that the volume reduction effect leading to higher bacterial densities in the vicinity of mycelial networks is the dominant cause for increased HGT activity in the experimental system. Mycelial organisms have been shown to internally translocate compounds from nutrient-rich to nutrient-poor regions; thereby not only facilitating their own growth 33 , 34 , but also allowing surrounding microorganisms to thrive on exuded compounds Mycelia thereby facilitate the access of bacteria to suitable microhabitats for growth 20 , enable efficient contaminant biodegradation 38 or increase the functional stability in systems exposed to osmotic stress Our data emphasize the physical role of mycelia in enabling bacterial transport and concentration.
The concentrating effect of mycelia will be particularly pronounced when the liquid film associated with the mycelium represents a substantial part of the habitable space; for instance in unsaturated soil or when mycelium-associated liquid films are likely to gather bacteria in situations of receding soil water, thus concentrating bacteria in the hyphosphere.
megaplasmid pAO1; horizontal gene transfer — Institut für Biochemie und Molekularbiologie
Xerophilic fungi are furthermore able to grow at low water activities of only 0. Earlier studies have shown that bacterial dispersal along mycelia depends both on the physico-chemical surface properties of the hyphae and the motility of the bacteria: Although most effective dispersal was found by flagella-driven swimming along hydrophilic mycelia 19 , transport of non-flagellated bacteria along hydrophilic hyphae 18 , 44 , 45 or of flagellated bacteria along more hydrophobic surfaces has been described It is reasonable to assume that the hyphal benefit for bacterial HGT is not restricted to the ideal situation chosen in this experiment, yet rather may vary depending on the bacteria and fungi and their possible antagonistic effects The role of mycelia as preferential dispersal pathways and promotors of HGT between bacteria hence also may be a driving factor of the evolution of bacterial diversity and may have led to significant increase of prokaryotic diversity after the appearance of the mycelial fungi in microbial evolution.
A bacterial reporter system consisting of two Pseudomonas strains was used to visualize HGT events as described by Seoane Pseudomonas putida KT yfp acted as potential plasmid recipient, constitutively expressing YFP The resulting transconjugant Pseudomonas putida KT yfp pWW P lac -gfp simultaneously expresses YFP and GFP, detectable with appropriate filter sets for yellow and green fluorescence, indicating successful plasmid transfer. Recipient and wildtype cells were grown on R2A medium Transconjugant cells were selected based on the presence of yellow and green fluorescence and the absence of red fluorescence.
Presence of the plasmid in the transconjugant cells was confirmed by plasmid isolation and subsequent gel electrophoresis, as described in the Supplementary Information.
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For epifluorescence microscopy, the P. As mycelial organism, P.
Depending on the agar concentration, the used media generally permitted three dimensional movements of motile bacteria. Flow cytometry was used to quantify the cells according to their fluorescence signal. To microscopically observe the influence of the mycelial network on transconjugant formation in the microcosms, parallel setups with a combination of P.
To reveal localization of transconjugants along mycelial network structures, a white overlay of the mycelial network was created by applying a signal intensity threshold to a transmission light image and modest manual corrections. Data recording and subsequent data analyses were made applying Summit software v4. For absolute quantification, cell numbers per mL were determined using reference microspheres FluoSpheres polystyrene microspheres, 1. Based on this definition, all conjugational samples were analyzed.
Microbial cells populated a two-dimensional grid of two layers in the model. The bottom layer was continuous and represented the agar, while the top layer was obtained by digitizing a microscopic image of actual fungal hyphae to represent the hyphal network. Microbial cells performed random walks as implemented earlier 52 on both layers with prescribed diffusion constants D agar and D hyphae , and could switch layers at locations where a fungal grid cell was present on top the agar grid cell according to. Following Levin 26 , plasmid transfer between the plasmid bearing donor population X D and plasmid-free recipient population X R giving rise to the transconjugant population X T was modeled within individual grid cells according to.
The model was implemented as a stochastic, individual-based model, initialized with a population of donor cells on one domain boundary and a population of recipient cells on the opposing boundary, with keeping cell numbers constant along both boundaries during the simulation. A total simulation time of 5 minutes was enough for population fronts to mix well, but short compared to the typical lag-phase between transconjugant events. Hence, all cells were allowed to only take part in one HGT event, requiring to consider only three cell types in the model: donor cells, recipient cells, and HGT-wise inactive cells.
Further modeling details are provided in the Supplementary Information. The simulation source code in Standard C is available from the authors on request. How to cite this article : Berthold, T. Mycelia as a focal point for horizontal gene transfer among soil bacteria. The authors thank Barth F. Boronin G. Author Contributions T. All authors revised the manuscript and Supplementary Information. National Center for Biotechnology Information , U.
Sci Rep. Published online Nov 4. Wick a, 1. Lukas Y.
Author information Article notes Copyright and License information Disclaimer. Received Jun 8; Accepted Oct This work is licensed under a Creative Commons Attribution 4. This article has been cited by other articles in PMC. Abstract Horizontal gene transfer HGT is a main mechanism of bacterial evolution endowing bacteria with new genetic traits. View access options below.
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