The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by β-lactam antibiotics or inhibitors of WTA biosynthesis
Fig 5
Aberrant progression of septal PG synthesis in S. aureus clpX cells is rescued by oxacillin.
S. aureus wild-type (SA564) and clpX cells were grown at 30°C in the absence (A and C) or presence of 0.05 μg ml-1 oxacillin (B and C), and PG synthesis was followed by sequentially labeling with NADA for 10 min, followed by washing and labeling with HADA for additional 10 min before SR-SIM imaging. In order to improve contrast, the NADA signal is displayed in magenta, while the HADA signal is displayed in cyan. In the absence of oxacillin (A) septal PG synthesis progressed predictably in wild-type cells and in some clpX cells (= non-overlapping septal NADA and HADA signals, marked with green arrows). In contrast, some clpX cells that initiated septum formation during the first period of labeling showed co-localization of NADA and HADA signals in an early septum ingrowth + HADA signal in the peripheral wall (examples are marked with white arrows), and in some of these cells splitting of the premature septum was observed (examples marked with red arrows). Enlarged examples are depicted in the lower panel. (B) In the presence of sub-lethal concentrations of oxacillin, some wild-type cells displayed overlapping NADA and HADA septal signals (examples displayed in middle panel), a phenotype that was not observed for clpX cells grown in the presence of oxacillin. (C) To examine progression of septal PG synthesis in clpX cells displaying premature septal split, 50 cells from each of three biological replicates (grown +/- oxacillin) that initiated septum formation during incubation with NADA, and displayed premature splitting were randomly selected. PG synthesis was followed by assessing HADA incorporation. (i-iii) show examples and distribution of the three phenotypes observed. (i) show the number of cells where septum synthesis was continued; (ii) shows the number of cells that did not continue septum synthesis and instead displayed a HADA signal in the peripheral wall; (iii) shows the number of cells where no HADA signal was detected. Numbers are given as the mean and SD of the three biological replicates. Scale bars, 0.5 μm. *** P < 0.001; statistical analysis was performed using the chi square test for independence.