Figure 5. Cell localization and growth of Bacteroides on complex AGPs.

a, Western blot detection of BT0264 and a known surface enzyme (BT4662)³⁰ in LA-AGP/heparin cultured B. thetaiotaomicron after treatment of the bacterial cells with proteinase K (PK+) or untreated (PK-). Purified recombinant BT0264 was also subjected to proteinase treatment to verify the enzyme is sensitive to the proteinase. The data show that the enzyme is resistant to the proteinase and thus is not located on the cell surface. The blot is an example of biological replicates where n=3. Wild type B. thetaiotaomicron (Bt) and B. thetaiotaomicron expressing Baccell00844 (Bt::Baccell00844) were cultured in 0.2 ml of minimal medium containing AGPs under anaerobic conditions. b, growth was assessed on GA-AGP and GA-AGP pre-treated with BT0264 [GA-AGP(BT0264)] or Baccell00844 [GA-AGP(Baccell00844)]. In c growth was evaluated on wheat AGP (WH-AGP). In b and c error bars report standard errors of the mean of biological replicates (n = 4). d, HPAEC analysis of the products generated by recombinant Baccell00844 (1 μM) incubated with β-1,3-galactan for 16 h using standard conditions. The chromatographs are examples of biological replicates (n = 2). e, Bt, Bt::Baccell00844 and B. cellulosilyticus (Baccell) cells derived from cultured grown on GA-AGP were incubated with 0.5% β1,3-galactan for 16 h in phosphate buffered saline in aerobic conditions for 16 h. Under these conditions substrate is only available to the surface enzymes. Products released from the glycan was evaluated by TLC. The example is from biological replicates n =3. f, Venn diagram of the number of proteins identified in the surfome, the surfome and total proteome, and total proteome. Baccell00844 was unique to the surfome fraction. The 46 proteins detected only in the surfome are described in Supplementary Table 5.