The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by β-lactam antibiotics or inhibitors of WTA biosynthesis.

beta-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of beta-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of beta-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30 degrees C, but not at 37 degrees C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with beta-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by beta-lactams or by inhibiting an early step in WTA biosynthesis. The finding that beta-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that beta-lactams do not kill S. aureus simply by weakening the cell wall. Author summary The bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all beta-lactam antibiotics has made treatment increasingly difficult. beta-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of beta-lactams. Without beta-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to beta-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that beta-lactam antibiotics can prevent lysis of a mutant with deregulated activity of autolytic enzymes involved in daughter cell splitting, emphasizes the idea that beta-lactams interfere with the coordination between cell division and daughter cell splitting, and do not kill S. aureus simply by weakening the cell wall.