Abstract
Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.
Highlights
Bacteroidetes is a phylum of Gram-negative bacteria that colonize diverse ecological niches
To determine how prevalent these FucT orthologs are in Bacteroidetes species, we searched the genomes of 35 diverse Bacteroidetes strains for orthologs
By analysis of mutants unable to synthesize GDP-L-fucose, we showed that L-Fuc is a component of the outer glycan of B. fragilis and is present in the O-linked glycans of diverse other Bacteroidetes species [7,16]
Summary
Bacteroidetes is a phylum of Gram-negative bacteria that colonize diverse ecological niches. Within this phylum are members of the order Bacteroidales, which include abundant anaerobic gut symbionts such as Bacteroides species that provide benefits to their host [1,2], as well as pathogenic anaerobic species such as the periodontal pathogens Tannerella forsythia and Porphyromonas gingivalis [3]. Flavobacteriales and Sphingobacteriales are other orders of this phylum that are generally aerobes or facultative anaerobes and Biomolecules 2021, 11, 1795. Biomolecules 2021, 11, 1795 typically colonize diverse environmental and non-mammalian host ecosystems. Members of this phylum have a tremendous capability to degrade high molecular-weight polysaccharides using dedicated polysaccharide utilization loci, many of which are shared between distantly related members [4].
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