To observe SpA targeting to the cross-wall, 2 ml mid-log phase S. aureus culture (OD600 0.8) were centrifuged at 18,000 × g for 5 min, supernatant removed, bacteria washed once in 2 ml PBS and suspended in 1 ml PBS containing 0.5 mg/ml trypsin (Sigma). After incubation at 37°C for 1 hr, staphylococci were washed twice with PBS, suspended in fresh TSB containing 2.5 mg/ml soybean trypsin inhibitor (Sigma) and incubated at 37°C for 20 min with rotation. 250 µl of the cell suspension was removed and immediately mixed with fixation solution (2.5% paraformaldehyde and 0.006% glutaraldehyde in PBS). The cells were fixed for 20 min at room temperature, washed three times with PBS and applied to poly-L-lysine coated 8-well glass slides (MP Biomedicals) for 5 min. Excess and non-adherent cells were washed away with PBS. Immobilized cells were blocked with 3% BSA in PBS for 45 min and incubated with SpA-specific mouse hybridoma monoclonal antibody 5A10 (Kim et al., 2010) (diluted 1:4000 in 3% BSA) for 1 hr. Cells were washed eight times with PBS and further incubated in the dark with Alexa Fluor 647 conjugated anti-mouse IgG (1:500 in 3% BSA) (Invitrogen, Carlsbad, CA) for 1 hr. Cells were washed 10 times with PBS and incubated with 1 µg/ml BODIPY-FL vancomycin (ThermoFisher) for 10 min in the dark followed by washing five times with PBS. A drop of SlowFade Gold reagent (Molecular Probes) was applied to samples before sealing coverslips with nail polish. Fluorescent images were visualized and captured on a Leica SP5 Tandem Scanner Spectral 2-Photon Confocal microscope with 100 × oil objective. Identical settings and exposure times were applied to all samples.

To image SpA display on the staphylococcal surface, 1 ml of mid-log phase S. aureus cultures were centrifuged at 18,000 × g for 5 min and supernatant removed. Bacteria were washed once in 2 ml PBS and suspended in 1 ml PBS and mixed with fixation solution. Cells were fixed for 20 min at room temperature, washed three times with PBS and applied to poly-L-lysine coated 8-well glass slides (MP Biomedicals) for 5 min, stained with vancomycin and αSpA antibodies and analyzed by fluorescence microscopy.

To localize intracellular SpA, 2 ml of mid-log phase staphylococcal cultures were centrifuged at 18,000 × g for 5 min and supernatant removed. Bacteria were washed once in 2 ml PBS and suspended in 1 ml PBS, 0.5 mg/ml trypsin (Sigma). After incubation at 37°C for 1 hr, staphylococcal cells were washed twice with PBS and fixed with fixation solution. The cells were fixed for 15 min at room temperature and 30 min on ice, washed three times with PBS and suspended in 1 ml GTE buffer [50 mM glucose, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA]. Lysostaphin (10 µg/ml) was added and 50 µl cell suspensions were immediately applied to poly-L-lysine coated 8-well glass slides and incubated for 1 min. Non-adherent cells were removed and PBS, 0.2% Triton X-100 was applied to samples for 10 s. Excessive liquid was aspirated and slides were air-dried. Dried slides were immediately dipped in methanol at −20°C for 5 min, and in acetone at −20°C for 30 s and then allowed to dry completely. Afterwards, the cells on the slides were re-hydrated with PBS for 5 min, blocked with 3% BSA, stained with the membrane dye Nile red (Sigma) and rabbit antibodies specific for SpA followed by Alexa-Fluor-488 conjugated goat-anti-rabbit-IgG and analyzed by fluorescence microscopy as described above.

To visualize the sub-cellular localization of SecA-sfGFP, samples from 3 and 6 hr growth cultures were removed as described above. Bacteria were sedimented by centrifugation and washed twice in PBS. Cells were stained with 1 µg/µl FM4-64FX (Molecular Probes) for 10 min in the dark and were then fixed with fixation solution for 20 min. After washing twice with PBS, cells were applied to poly-L-lysine coated coverslips, and incubated for 5 min. After removing excess bacterial cells, SlowFade Gold reagent was added to the samples and the coverslips were sealed onto glass slides. Samples were visualized under Leica SP8 3D, 3-color Stimulated Emission Depletion (STED) laser scanning confocal microscope with 100×/1.45 oil objective. Images were captured with identical settings. Deconvolution images with identical parameters were generated by using a HyVolution module installed on the microscope.

All the images were analyzed in Image J software (Schneider et al., 2012). To quantify the frequency of SpA cross-wall localization at 20 min regeneration after trypsin digestion, numbers of diplococci and numbers of cross-wall localized SpA were counted manually using the cell counter tool in Image J. Diplococci were defined as two daughter cells that had divided and formed a cross-wall but had not yet separated. Cross-wall localized SpA signals were defined as lines at the cross-wall of diplococci. Diplococci were counted in vancomycin stained images and cross-wall localized SpA was counted in merged images. The frequency was determined by dividing cross-wall localized SpA by the number of diplococci. An example of the counting method is displayed in the Source file to Figure 2e. At least two random images were acquired per sample for each experiment. Three or more independent experiments were performed and data from more than 200 diplococci were analyzed for statistically significant differences using one-way ANOVA with Dunnett's multiple comparison test comparing staphylococci expressing wild-type and spa variants (Figure 2e). In Figure 6c and Figure 10c the Tukey's multiple comparison test was used to analyze differences between multiple groups.

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