Activity-Based Tracking of Glycan Turnover in Microbiomes

Abstract

Glycans shape microbiomes in the ocean and the gut, driving key steps in the global carbon cycle and human health. Yet, our ability to track microbial glycan turnover across microbiomes is limited, as identifying active degraders without prior genomic knowledge remains a key challenge. Here, we introduce an activity-based fluorescence resonance energy transfer (FRET) probe that enables direct visualization and quantification of glycan metabolism in complex microbial communities. As a proof of concept, we investigated α-mannan degradation, a prominent polysaccharide in algal blooms. Using automated glycan assembly, we synthesized a mannan hexasaccharide bearing a fluorescein-rhodamine FRET pair. The probe was validated using a recombinantly expressed endo-α-mannanase (GH76) from Salegentibacter sp. Hel_I_6. It was shown to function in cell lysates, pure cultures, and complex microbiomes (via plate assays and microscopy). This probe enabled spatiotemporal visualization of in situ α-mannan turnover in a marine microbiome. Glycan FRET probes are versatile tools for tracking glycan degradation across biological scales from single enzymes to microbiomes.

1

Structure-guided design of a FRET mannan probe. (a) GH76 from sp. Hel_I_6 (PDB ID: 6SHD) represented using rainbow protein structure. (b) Solved crystal structure of GH76 in complex with a mannan tetrasaccharide, aromatic side chains, and arrow provide orientation. (c) Alternative view of the active site, highlighting the tunnel-like topology and solvent-exposed region that provide good exit vectors for the fluorophore and quencher. (d) Conformational diversity of 1,6-linked mannans, ranging from 21 to 28 nm in end-to-end length. (e) Fluorescein–rhodamine FRET pair has proven biocompatibility in microbial communities, good optical properties, and function within the length of the mannan hexasaccharide.

2

Automated glycan assembly of the fluorescence quenched mannan probe. (a) Automated assembly of a fluorescent quenched probe. (b) Building blocks and resin were required for synthesis. (c) Left: crude ELSD trace of hexasaccharide after automated glycan assembly (SI Method 1, YMC-Diol-300). Right: MALDI-TOF of hexasaccharide, calculated C₁₉₀H₁₈₆N₂NaO₄₀ [M + Na]⁺ 3158.2480 m/z, found 3158.134 m/z. For full spectrum, see Figure S2. (d) UV-trace (566 nm) of purified 3 on an HPLC (SI Method 2, Luna C5 column).

3

Biochemical characterization of GH76 from sp. Hel_I_6. (a) Quenching efficiency of the fluorescence-quenched mannan probe (68.9%) quantified by comparing the slopes of linear regression of fluorescein and probe emission intensities. Dotted line depicts linear regression analysis with individual data points shown. (b) Incubation of 10 nM FRET mannan probe with 50 nM GH76 results in a time-dependent fluorescence increase. Fluorescence at 520 nm indicates glycan hydrolysis (Excitation 495 nm). (c) Increasing concentration of GH76 results in increased fluorescence intensity when incubated with 10 nM probe at 500 s. Shown is the mean, and error bars represent standard deviation from the mean (n = 3).

4

Detection of mannan degradation in marine bacterial lysates and cultures using FRET probes. (a) Fluorescence signal following incubation of mannan probe (1 μM) with cell lysates from sp. Hel_I_6 (SB), (GF), Hel1_33_131 (FH), and (FA). The probes were excited at 495 nm, and emission was detected at 520 nm. Cell lysates were prepared using BugBuster reagent. A negative control (Ctrl) of heat-killed cells (95 °C for 30 min) of each strain were incubated with 1 μM mannan probe. The background fluorescence of each strain was recorded and annotated as strain acronym alone (e.g., SB alone). Experiments were performed as independent triplicates (n = 3) and error bars represent the standard deviation of the mean. (b) Representative microscopy of four species of marine bacteria. Green (FITC) corresponds to mannan probe signal, and blue (DAPI) labels the cell nuclei. Merge is the overlay of FITC and DAPI signals. Scale bar: 2 μM.

5

Visualization of α-mannan utilization in a complex microbial community. (a) Workflow for sensing α-mannan carbon in microbial communities. (b) Quantification of nonspecific (0.5 g/L yeast extract) and specific (2 g/L α-mannan) culture conditions on the hydrolysis of the mannan probe in a marine microbial community. (c) Shown is a representative microscopy image of a selective hydrolysis of the mannan probe in a marine microbiome from the nonspecific enrichment sample at 7 h. Green (FITC) indicates mannan probe hydrolysis, and blue (DAPI) labels cell nuclei. Merge is overlay of FITC and DAPI signals. Constant exposure times were used, and fluorescence signal thresholds were determined from control cells not exposed to FRET probe. Experiment is representative of duplicate. Scale bar: 2 μm.