Table 1.
The PGH complement of L. casei BL23 predicted in silico on whole genome sequence.
Table 2.
Proteomic identification by 1D SDS-PAGE and LC-MS/MS of the PGHs present in L. casei BL23 extracts and estimation of relative amounts by PAI calculation.
Figure 1.
Analysis of purified recombinant Lc-p75.
(A) SDS-PAGE after Coomassie blue staining and (B) zymogram assay.
Figure 2.
RP-HPLC separation profile of muropeptides obtained from L. casei BL23 PG.
PG was digested by mutanolysin (A) or by mutanolysin and recombinant Lc-p75 (B). Numbers correspond to the main muropeptides which amounts changed between Panel A and B. Letters indicate new peaks present in Panel B and absent in Panel A. Complete annotation of the chromatograms is presented in Figure S3.
Figure 3.
Schematic structure of L. casei BL23 peptidoglycan and site of cleavage determined for Lc-p75.
GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid. MurNAc may be O-acetylated (O-Ac) or not in PG.
Table 3.
Main muropeptides from L. casei BL23 PG hydrolyzed by Lc-p75 and main products of digestion.
Table 4.
Lc-p75 activity on purified muropeptides selected as substrates.
Figure 4.
Phenotype comparison between wild type L. casei BL23, Lc-p75-negative mutant and complemented Lc-p75 mutant.
Pictures of wild type L. casei (A, C, F, H), Lc-p75-negative mutant (B, D, I) and complemented Lc-p75 mutant (E). Colony morphology (A, B), phase contrast microscopy (C, D, E) fluorescence microscopy with merged FM-4-64 (red) and DAPI (blue) staining (F, G) and transmission electron microscopy (H, I).
Figure 5.
Indirect immunofluorescence localization of Strep-tagged Lc-p75 in L. casei.
Strep-tagged Lc-p75 was localized in overexpressing strain (A) and in complemented negative mutant (B) with monoclonal antibody directed against Strep-tag as first antibody.
Figure 6.
Detection of Lc-p75 in culture supernatant.
Analysis by SDS-PAGE (A, B) and zymogram assay (C, D) of the culture supernatant of wild type BL23 (A, C) and negative mutant (B, D) grown on AOAC medium. Lc-p75 is indicated by an arrow.