Phylogenetic and Functional Analysis of -Glucanases in Tannerella forsythia Reveals Distinct GH16 Family Glycoside Hydrolase Lineages in Oral Bacteria

Tannerella forsythia is an asaccharolytic anaerobic bacterium whose presence in oral biofilms is associated with poorer outcomes for periodontitis. T. forsythia expresses a β-glucanase enzyme, TfGlcA, that is upregulated on contact with the pathobiont Fusobacterium nucleatum , supporting the latter’s growth through release of glucose oligomers. Here, we systematically characterize the enzymatic properties of TfGlcA and investigate the extent to which its genomic architecture is common among oral β-glucanases (also in Glycoside Hydrolase Family 16 Subfamily 3, GH16_3). First, we evaluated the substrate specificity, kinetics, and structural features of TfGlcA. TfGlcA displayed highest activity toward laminarin, followed by lichenin, with minimal activity toward yeast β-glucan, and no detectable activity against chitin or carboxymethyl cellulose. These findings on proxy-substrates indicate strong preference for β1,3-linked glucose polysaccharides, enriched in dietary plant cell walls. Second, we investigated the phylogenetic diversity and genome-neighborhoods of oral GH16_3s. GH16_3 genes were found in 15% of oral bacterial species, representing phylogenetically diverse taxa. A subset of genes, including TfGlcA, occur in polysaccharide utilization loci (PUL), with the TfGlcA PUL overall unique in its association with an adjacent extracytoplasmic function sigma/anti-sigma factor regulatory module. A second GH16_3 in T. forsythia, GlcB, falls within a large clade of sequences dominated by Bacteroidota and lacking PUL architecture. This work represents the first systematic characterization of substrate specificity and kinetics of an oral β(1,3) glucanase and suggests that T. forsythia plays a distinctive role in shaping public-goods nutrient dynamics in the periodontal niche.