Illuminate the hidden: in vivo mapping of microscale pH in the mycosphere using a novel whole-cell biosensor

Abstract

The pH of an environment is both a driver and the result of diversity and functioning of microbial habitats such as the area affected by fungal hyphae (mycosphere). Here we used a novel pH-sensitive bioreporter, Synechocystis sp. PCC6803_peripHlu, and ratiometric fluorescence microscopy, to spatially and temporally resolve the mycosphere pH at the micrometre scale. Hyphae of the basidiomycete Coprionopsis cinerea were allowed to overgrow immobilised and homogeneously embedded pH bioreporters in an agarose microcosm. Signals of >700 individual cells in an area of 0.4 × 0.8 mm were observed over time and used to create highly resolved (3 × 3 µm) pH maps using geostatistical approaches. C. cinerea changed the pH of the agarose from 6.9 to ca. 5.0 after 48 h with hyphal tips modifying pH in their vicinity up to 1.8 mm. pH mapping revealed distinct microscale spatial variability and temporally stable gradients between pH 4.4 and 5.8 over distances of ≈20 µm. This is the first in vivo mapping of a mycosphere pH landscape at the microscale. It underpins the previously hypothesised establishment of pH gradients serving to create spatially distinct mycosphere reaction zones.

Impact of fungal hyphae on growth and dispersal of obligate anaerobic bacteria in aerated habitats

“…3). This suggests that hyphal oxygen consumption affected similar regions as described for pH changes (31), enzymatic activity (48) or the dispersal of bacteria (29, 49). Air-exposed hyphae of C. cinerea by this means also enabled the dispersal of C. acetobutylicum and the colonization of anoxic habitats separated by ambient air (Fig.…”

“…1a) that was inversely placed on an oxygen optode (Ø 18 mm, SF-RPSu4, PreSens, Regensburg, Germany; Fig. 1b) as described earlier (31). Briefly, 400 μL of aerated SM824 medium (1.5% low-melt agarose, Karl Roth, Karlsruhe, Germany) were placed on a circular cover slide (Ø 18 mm, ibidi, Gräfelfing, Germany), immediately covered by a second cover slide, and allowed to cool for 10 min.…”

“…As fungi may both form and bridge anoxic microsites, we here assess to which degree hyphae thriving in oxic habitats enable spore germination, vegetative growth, and dispersal of strictly anaerobic bacteria. Towards this aim, we determined spatial and temporal oxygen profiles around the filamentous mycelia of the fast-growing (~100 μm per h (31)) aerobic soil fungus Coprinopsis cinerea. Planar optodes and custom-made micrometer-sized oxygen-sensitive beads determined mycelial oxygen distribution.…”

pH Distribution along Growing Fungal Hyphae at Microscale

“…As well as oxygen availability [ 6 , 7 ], pH is an important driver for hyphal-bound microbial activity [ 1 ] such as bacterial motility [ 8 ]; degradation of organic matter, lignin and lignocellulose; the mobilisation and transport of nutrients [ 9 , 10 ]; mineral weathering [ 11 ]; and soil structure changes. Fungal mycelia typically modulate the environmental pH in their hyphosphere [ 12 , 13 ]. However, knowledge of the microscale pH of dynamically changing hyphal surfaces still is limited and often inferred only after destructive sampling [ 11 ].…”

“…Using a whole-cell bacterial pH bioreporter ( Synechocystis sp. PCC6803_peripHlu) that allows for spatial and temporal in vitro analysis of environmental pH at the single-cell scale (~3 µm), recent work [ 12 ] mapped spatially distinct and temporally stable gradients between pH 4.4 and 5.8 in habitats during hyphal colonisation. The pH bioreporter is based on a periplasm-localised, ratiometric pH-sensitive green fluorescent protein (GFP), pHluorin2, that displays varying bimodal excitation at 395 nm and 475 nm with maximum emission at 510 nm, thus reporting the local environmental pH [ 12 ].…”