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

Bi-Jing Xiong,Dietmar Schlosser,Lukas Y Wick,Christian Dusny,Jens Appel,Hauke Harms,Lin Wang,Kristin Lindstaedt

doi:10.1038/s43705-021-00075-3

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.

Highlights

Despite the high motility of protons, pH gradients over even short distances may be both driver and the result of localised microbial activity in spatially structured microhabitats [1,2,3,4,5] such as the mycosphere, biofilms [6], biocrusts [7], soil-biochar surfaces [8] or the rhizosphere [9]
Using the Tat pathway [19], the ratiometric pH-sensitive GFP variant reporter protein pHluorin2 [20, 21] was translocated to the periplasm of Synechocystis sp
The 510 nm emission intensity ratio from two excitations (RI510–475/I510–395, abbreviated as RI475/I395) increases in response to decreasing environmental pH. pHluorin2 is a wellknown pH sensor that has been introduced to cells and tissues for sensing intracellular pH [23,24,25], yet to our knowledge has never been applied to report extracellular pH

Summary

Introduction

Despite the high motility of protons, pH gradients over even short distances (μm-mm) may be both driver and the result of localised microbial activity in spatially structured microhabitats [1,2,3,4,5] such as the mycosphere, biofilms [6], biocrusts [7], soil-biochar surfaces [8] or the rhizosphere [9]. Techniques involving nanoparticles [12, 13], needle-type microelectrodes [14, 15], or planar optodes [7] have been developed for the in vivo analysis of pH. Commercial microelectrodes typically have needle tips of ~8 μm diameter and allow for pH analysis at approximately the 10 μm scale. They are, though, highly invasive, mainly when applied for mapping the pH distribution PCC6803_peripHlu) that allows for spatial and temporal in vivo analysis of environmental pH at the single-cell scale (~3 μm).

Methods

Results

Conclusion