In the outgoing phase, the RUMIC project investigated whether glycans found in human and livestock diets could be fluorescently labelled and whether the produced fluorescently labelled polysaccharides (FLAPS) enable visualisation of selective glycan metabolism in rumen isolates and complex rumen microbiomes.
Dr Greta Reintjes, in collaboration with the Abbott Lab of the Lethbridge Research and Development Center, Agriculture and Agri-Food Canada, successfully labelled two prominent dietary glycans, yeast-mannan (YM) and rhamnogalacturonan-II (RGII), and incubated these with the model gut microbe; Bacteroidetes thetaiotaomicron VPI-5482 (B.theta). For the first time, they showed that FLAPS can be used to visualise the selective glycan metabolism of B.theta. They demonstrated the differential uptake of FLAPS by individual cells through super-resolution microscopy and flow cytometry. Furthermore, investigations with genetically modified B.theta strains showed that FLAPS uptake was highly specific and dependent on the presence of polysaccharide utilisation loci (PULs).
This investigation of the RUMIC project highlighted that FLAPS are a novel functional tool that can be applied to rapidly assign metabolic phenotypes in rumen microorganisms and can be used to decipher glycan-microbial interactions in microbiomes.
Hehemann, J.H. Reintjes, G., et al., (2019) 'Single cell fluorescence imaging of glycan uptake by intestinal bacteria', ISME J, 13(7), pp. 1883-1889.
https://doi.org/10.1038/s41396-019-0406-z(se abrirá en una nueva ventana) In a further investigation, Dr Reintjes tested whether the novel FLAPS could be used to identify individual cell physiologies in bovine B.theta strains. The bovine B.theta isolates consume YM using the same proteins encoded on PULs as the model B.theta strain. Compellingly, however, the bovine B.theta strains showed two distinct growth phenotypes on YM for which there were no distinct genotypic explanations. Using FLAPS, Dr Reintjes could visualise the metabolism of YM by individual bovine B.theta strains and identify strain-level variability in the carbohydrate utilisation systems of the two foraging phenotypes. Furthermore, this new next-generation physiology approach combined with "omics" approaches enabled her to characterise microbial adaptations to a prebiotic in the rumen ecosystem.
Klassen, L., Reintjes, G., et al., (2021) 'Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria', Microbiome, 9(1), pp. 23.
https://doi.org/10.1186/s40168-020-00975-x(se abrirá en una nueva ventana)In the return phase, Dr Reintjes used culture-independent approaches to identify prebiotic-microbial interactions in simplified artificial and complex rumen communities. Benefiting from the expertise of the Lethbridge Research and Development Center, Dr Reintjes worked with an in vitro artificial rumen system that simulates the microbial rumen processes in a regulated environment (rumen-simulated technique) and live animal feed trials. FLAPS were applied to the complex communities in these trials to illuminate the functional potential encoded within each microbial community. Dr Reintjes found that both artificial and native rumen microbiomes respond rapidly to the addition of prebiotics and that there is a functional adaptation to specific prebiotics from agricultural and seaweed sources. Furthermore, the metabolically active cells were functionally sorted from these complex microbial communities, as proposed in the RUMIC project. The sorted microbes could be directly identified using 16S rRNA sequencing and characterised using proteomic analysis.
Klassen, L., Reintjes, G., et al., (2023). Fluorescence-activated cell sorting and fermentation analysis to study rumen microbiome responses to administered live microbial and yeast cell wall derived prebiotics. Frontiers in Microbiology 13. DOI 10.3389/fmicb.2022.1020250
Finally, the new FLAPS methodological developments achieved in the outgoing phase in the Abbott Lab of the Lethbridge Research and Development Center, Agriculture and Agri-Food Canada and return phase at the Max Planck Institute for Marine Microbiology, Bremen, Germany, are available here: Klassen, L., et al.,(2021) 'Approaches to Investigate Selective Dietary Polysaccharide Utilization by Human Gut Microbiota at a Functional Level', Frontiers in Microbiology, 12(308).
https://doi.org/10.3389/fmicb.2021.632684(se abrirá en una nueva ventana)Visualisation of Carbohydrate Uptake Using Fluorescent Polysaccharides” in Carbohydrate-Protein Interactions: Methods and Protocols. Springer US. (2023) Editors Abbott, D.W. and Zandberg, W.F
This work could not have been achieved without access to the outstanding facilities, excellent supervision and tremendous day-to-day support from the Abbott Lab at the Lethbridge Research and Development Center, Agriculture and Agri-Food Canada and the Max Planck Institute for Marine Microbiology, Bremen, Germany. Thank you for being so supportive.
