Purification and Characterization of Membrane Vesicles

Membrane vesicles were purified from the strains that overexpress either FtsH or SaeQ. To overexpress FtsH, the vector pYJ335 was PCR-amplified with the primers P371/372, while the ftsH gene including the promoter sequence was amplified with the primers P39/370 (Supplementary Table 8). The plasmid was assembled with LIC as described above. The plasmid was inserted into E. coli DH5a, and then into S. aureus RN4220. Finally, the plasmid was transduced by ϕ85 into wild type of S. aureus Newman. For SaeQ protein, we used the NMΔftsH strain with pYJ-SaeQ-his plasmid. The membrane vesicles were prepared from the test strains as described previously^{30 (link)}. The protein concentration in the membrane vesicle suspension was determined by the bicinchoninic acid assay (Yeasen Bio, China). The membrane vesicles were stored at -80 °C until use.