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Page 10 of 15 Alekseeva et al. Microbiome Res Rep 2023;2:10 https://dx.doi.org/10.20517/mrr.2023.06

Figure 5. Three-dimensional models of ΔFN3.1 proteins from different groups of B. longum subsp. longum using the trRosetta server: (A)
group 1 (do not contain amino acid substitutions in comparison to the reference strain B. longum GT15); (B) group 2 (contained a single
substitution 43 A→V); (C) group 3 (contained four substitutions 43 A→V, 51 A→T, 417 P→Q and 424 A→T); (D) group 4 (contained
three substitutions: 111 T→I, 417 P→Q and 424 A→T).

The PFNA operon is found only in bifidobacteria. Other bacteria (including commensal bacteria of the
human gastrointestinal tract) contain proteins that carry FN3 domains. Only the FN3 domains containing
annotated MCRs exhibit similarities with the FN3 domains of human proteins. Our bioinformatics analysis
of the sequenced genomes of bacterial strains belonging to the families Lactobacillaceae, Bacteroidaceae, and
Clostridioides revealed the presence of a number of proteins containing a single FN3 domain, most often in
the C-terminal or the central region of the protein. We were able to divide the identified proteins into two
groups: Group 1: proteins containing annotated MCRs in the FN3 domains [Supplementary Table 2]; and
Group 2: proteins lacking MCRs in the FN3 domains [Supplementary Table 3].

Group 1 encompassed GHs, which represent a common group of enzymes (EC 3.2.1.) that hydrolyze the
glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate
moiety. Bacteria of the Lactobacillaceae and Bacteroidaceae families contained proteins belonging to the GH
family 31 (alpha-glucosidase and alpha-xylosidase) containing a single FN3 domain each, in which MCRs
were found. Bacteria belonging to Bacteroidaceae possessed proteins of the GH family 43 (1,4-beta-
xylanase), while bacteria belonging to Clostridioides contained proteins of the GH family 18 and family 20b
(hyaluronoglucosaminidase).

Proteins lacking MCRs in FN3 domains were assigned to Group 2. Proteins originating from Bacteroidaceae
belonged to the GH family 20 (beta-hexosaminidase) and to the GH family 92 (alpha-mannosidase) and
proteins of Clostridoides belonged to the GH family 65 (maltose phosphorylase). Bacterial proteins