FIG 4.

Impact of lipoprotein expression and localization on S. aureus biofilm development. (A) LAC strains were constructed with the pEPSA5 empty vector (EV) or various lipoproteins. Biofilms were grown in 48-well plates in TSB plus 0.4% glucose and xylose at a range of concentrations (0 to 0.6%) to induce protein expression. Error bars are the standard deviation for 12 wells (three experiments with four wells per lipoprotein). Statistics for two-way analysis of variance are P values. **, P ≤ 0.005; ***, P ≤ 0.0005; ****, P ≤ 0.00005. (B) Confocal microscope images of flow cell biofilms for the strain with the empty vector (left), SaeP-expressing strains (center), and DsbA-expressing strains (right). Biofilms were grown for 2.5 days in 2% TSB supplemented with 0.2% glucose, 1 μg/ml of chloramphenicol, and 0.1% xylose. (C) SCAM analysis of native SaeP in strain LAC shows that cysteine residue C133 is modified without lysostaphin pretreatment, indicating that SaeP is located on the cell surface. Protein expression was evaluated by T7 immunoblotting, while cysteine labeling was analyzed using Strep-HRP. (D) Western blot analyses performed with whole-cell lysates and bacterial supernatants of LAC expressing WT SaeP-T7 or the C21A mutant of SaeP-T7 show that the WT protein is associated with cells, whereas the C21A mutant is found in the supernatant, confirming that the C21A mutant is no longer a lipoprotein. (E) Biofilm assays, performed as described in the legend to panel A, of WT and C21A mutation-expressing strains (compared to strains expressing DsbA as a control) show that the mutant no longer supports enhanced biofilm accumulation. Error bars are the standard deviation for three replicate wells. Statistics for two-way analysis of variance are P values. ****, P ≤ 0.00005.