Microbial strains

A bacterial reporter system consisting of two Pseudomonas strains was used to visualize HGT events as described by Seoane21. Pseudomonas putida KT2442::dsRed-lacIq(pWW0::P lac -gfp) was used as plasmid donor, expressing DsRed while at the same time repressing plasmid-encoded GFP by the simultaneous expression of LacIq. Pseudomonas putida KT2440::yfp acted as potential plasmid recipient, constitutively expressing YFP21. The resulting transconjugant Pseudomonas putida KT2440::yfp(pWW0:: P lac -gfp) simultaneously expresses YFP and GFP, detectable with appropriate filter sets for yellow and green fluorescence, indicating successful plasmid transfer. Donor cells were grown on Brunner-Medium47 supplemented with 10 mM m-toluic acid (mTol) to select for the pWW0 plasmid. Recipient and wildtype cells were grown on R2A medium48.

Transconjugant cells were created by cross-streaking donor and recipient cells on R2A media supplemented with 20 mM mTol and 18 g/L agar and cultivation for 24 hrs at 30 °C. After selection for plasmid harbouring cells on Minimal-Medium-Agar with 20 mM mTol, colonies were microscopically checked for their fluorescence signals. 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.

The oomycete Pythium ultimum was grown at room temperature on plates of AB-medium21 supplemented with 10 mM Na-citrate (ABC) and 18 g/L agar.

Epifluorescence microscopy

For epifluorescence microscopy, the P. putida KT2442 donor strain was used in combination with the P. putida KT2440 wildtype-strain as potential plasmid recipient. To observe the influence of mycelial networks on spatially isolated donor and wildtype colonies, individual ABC agar pieces (18 g/L agar) were inoculated with 1 μL overnight cultures adjusted to an optical density of 0.05. As mycelial organism, P. ultimum was additionally inoculated on the donor side. The agar pieces were inverted and placed side by side in a 50-mm glass bottom dish (Ibidi, Munich, Germany) with an air gap of about 400 μm between them. Setups were incubated at room temperature for 3 days and examined with an AZ100M fluorescence microscope (Nikon, Düsseldorf, Germany), using distinct filtersets for DsRed (F46-005) and GFP (F46-002, both AHF, Tübingen, Germany). Images were captured with the Nikon NIS-Elements software and annotated with Adobe Photoshop (Adobe Systems, San Jose, CA, USA).

Microcosm Setups

Depending on the agar concentration, the used media generally permitted three dimensional movements of motile bacteria. Liquid (3 g agar/L) and semi-solid agar (5 g agar/L) were assumed to allow nearly unhindered and hampered movement, respectively, whereas movement in solid agar (18 g agar/L) was regarded negligible13,38.

To investigate the effect of alternative transport routes provided by mycelial networks, microcosms were set up in triplicates in 12-well plates (Greiner Bio-One, Frickenhausen, Germany) by filling each well with 2 mL AB-medium supplemented with 10 mM Na-citrate and the agar concentrations of choice. Solid and semi-solid agar were left to dry for 24 h, liquid agar for 1 h. P. ultimum was inoculated on the side of each well and incubated at room temperature for 3 days until a complete, planar mycelial network had established on the agar in each well. Setups with abiotic transport networks were prepared in parallel by using glass fibres with diameter of ≈12 μm, cut to a length of 2 cm (P-D Glasseiden, Oschatz, Germany). Before covering the agar with approximately 300 fibres for each well, the glass fibres were cleaned by heating to 600 °C for 5 h49. All wells were subsequently inoculated with 1 μL each of overnight cultures of donor and recipient, adjusted to an optical density of 3.75 at a separation distance of 7 mm. After incubation for 3 days at room temperature, each microcosm was resuspended in 2 mL of phosphate buffered saline (PBS) to harvest the cells by vortexing and ultrasonication as described by Kohlmeier et al.19. 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. putida donor and wildtype strain were examined after 3 days of incubation. Images were taken with an AZ100M fluorescence microscope (Nikon, Düsseldorf, Germany), using the NIS-Elements software and distinct filtersets for DsRed (F46-005) and GFP (F46-002, both AHF, Tübingen, Germany). Adobe Photoshop (Adobe Systems, San Jose, CA, USA) was used to annotate images and reduce noise. 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.

Flow Cytometric Analysis

Flow cytometric measurements were carried out using a MoFlo cell sorter (DakoCytomation, Fort Collins, CO, USA) equipped with a water-cooled argon-ion laser (Innova 70C, Coherent, Santa Clara, CA, USA). 488 nm light of 400 mW was used to analyze the forward light scatter (FSC) and the side light scatter (SSC), and to excite GFP, YFP, and dsRed. Fluorescences were detected after passage of the optical filters 510/10 (GFP), 542/27 (YFP), and 600/37 (dsRed), respectively (Semrock Inc., Rochester, NY, USA). Data recording and subsequent data analyses were made applying Summit software v4.3 (DakoCytomation, Fort Collins, CO, USA).

For absolute quantification, cell numbers per mL were determined using reference microspheres (FluoSpheres polystyrene microspheres, 1.0 μm, yellow-green fluorescent, 505/515, Molecular Probes, Eugene, OR, USA) of known concentration as described previously50. Prior to measurement, the cell suspensions were filtered through a 50-μm CellTrics filter (Sysmex Partec, Görlitz, Germany) to exclude debris.

The identification and quantification of GFP/YFP fluorescent transconjugants within a cell suspension containing a multitude of YFP fluorescent recipient and dsRed fluorescent donor cells was done as follows: Pure cultures of recipient, donor, and transconjugant cells were analyzed by flow cytometry. On an FL1 (GFP fluorescence) vs. FL2 (YFP fluorescence) dot plot, three areas (gates) were defined with the premises that gate 1 exclusively includes transconjugant cells, gate 2 exclusively those of the recipient strain, and gate 3 those of the FL1/FL2 non-fluorescent donor strain. Based on this definition, all conjugational samples were analyzed. If a cell had displayed a gate 1 specific FL1/FL2 fluorescence it was classified as a transconjugant. The total number of transconjugants per sample was calculated by means of the above mentioned yellow-green fluorescent, 1 μm microspheres.

The transconjugant cell numbers within each replicate series (n = 6) were tested for skewness51 and outliers were detected using Grubbs’ test (α = 0.05). The results of the replicate series for low, medium, and high mobility systems without transport networks were compared to the respective systems with transport networks, using a two-sided Student’s t-test to test for significant differences (significance levels: *p = 0.05; **p = 0.005; ***p = 0.001).

Individual-based Model

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 earlier52) 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

with b agar and b hyphae denoting microbial cells on the respective layer and rate parameters k attach and k detach describing bacterial attachment and detachment from fungal hyphae. Following Levin26, 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

with rate parameter γ53. 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.