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A general approach to explore prokaryotic protein glycosylation reveals the unique surface layer modulation of an anammox bacterium


The enormous chemical diversity and strain variability of prokaryotic protein glycosylation makes their large-scale exploration exceptionally challenging. Therefore, despite the universal relevance of protein glycosylation across all domains of life, the understanding of their biological significance and the evolutionary forces shaping oligosaccharide structures remains highly limited. Here, we report on a newly established mass binning glycoproteomics approach that establishes the chemical identity of the carbohydrate components and performs untargeted exploration of prokaryotic oligosaccharides from large-scale proteomics data directly. We demonstrate our approach by exploring an enrichment culture of the globally relevant anaerobic ammonium-oxidizing bacterium Ca. Kuenenia stuttgartiensis. By doing so we resolve a remarkable array of oligosaccharides, which are produced by two seemingly unrelated biosynthetic routes, and which modify the same surface-layer protein simultaneously. More intriguingly, the investigated strain also accomplished modulation of highly specialized sugars, supposedly in response to its energy metabolism—the anaerobic oxidation of ammonium—which depends on the acquisition of substrates of opposite charges. Ultimately, we provide a systematic approach for the compositional exploration of prokaryotic protein glycosylation, and reveal a remarkable example for the evolution of complex oligosaccharides in bacteria.

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Fig. 1: The mass binning glycoproteomics approach to explore prokaryotic protein glycosylation.
Fig. 2: Carbohydrate profiles obtained from the anammox enrichment cultures (and reference samples) using the MS2 mass binning approach.
Fig. 3: Outline of identified sugar components and observed oligosaccharide profiles for the Ca. Kuenenia stuttgartiensis enrichment.
Fig. 4: Glycosylated proteins and strains present in the explored anammox enrichment cultures.
Fig. 5: Physiology of the Ca. Kuenenia stuttgartiensis surface layer protein (SLP) and oligosaccharides.

Data availability

The mass spectrometry proteomics raw data have been deposited in the ProteomeXchange consortium database with the dataset identifier PXD021600. Raw sequencing data are available through the NCBI Sequence Read Archive (SRA) under accession number: SRR12344472. The MAGs are available at GenBank under accession numbers JACFMP000000000 to JACFOJ000000000. The BioProject accession number is PRJNA647942.


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We acknowledge Laura van Niftrik for reading the manuscript and providing constructive feedback. We further would like to acknowledge Claire Chassagne for discussions on surface charges, Guylaine Nuijten and Katinka van de Pas-Schoonen for anammox biomass sampling, technical assistance and reactor care, and Ben Abbas for the support with DNA extraction. The authors acknowledge the SIAM consortium and the TU Delft for startup funding. Additionally, SL was supported by a NWO VIDI grant (016.Vidi.189.050), and ML was supported by a Marie Skłodowska-Curie Individual Fellowship (752992), and a VENI grant from the Dutch Research Council (NWO, VI.Veni.192.252).

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Pabst, M., Grouzdev, D.S., Lawson, C.E. et al. A general approach to explore prokaryotic protein glycosylation reveals the unique surface layer modulation of an anammox bacterium. ISME J (2021).

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