Fig. 7.
LtaS functions as a full-length enzyme but not as a split enzyme. (A) Schematic representation of S. aureus strains used for separate expression of the 5TM domain and the eLtaS domain. The secreted eLtaS variant (Sec-eLtaS) was expressed from a chromosomal integration vector by the addition of Atet, and the 5TM domain of LtaS (5TM), an inactive full-length LtaS variant (LtaST300A), or WT LtaS (LtaS; positive control) was expressed from a multicopy plasmid. A strain containing the empty plasmid vector pCN34 (no insert) was used as a negative control. (B) Bacterial growth curves. The growth of the S. aureus strains described in panel A was monitored by determining OD600 readings at the indicated time points. Cultures were back diluted 1:100 in fresh medium at the 4-h time point to maintain them in the logarithmic growth phase. LTA (C) and LtaS (D) were detected by Western blotting. Samples were prepared and analyzed by Western blotting as described in the legend to Fig. 3. The anti-LtaS Western blot with cell samples was exposed twice as long as the Western blot with supernatant samples. A star indicates the full-length LtaS protein, and a square indicates the cleaved eLtaS protein. S. aureus strains ANG1226, ANG1227, ANG1228, and ANG1690 were used for this experiment.