SummaryThe Gram-positive pathogen Staphylococcus aureus divides by synthesizing the septum in three orthogonal planes over three consecutive division cycles. This process has to be tightly coordinated with chromosome segregation to avoid bisection of the nucleoid by the septum. Here we show that deletion of the nucleoid occlusion effector Noc in S. aureus results in the formation of Z-rings over the nucleoid, as well as in DNA breaks, indicating that Noc has an important role as an antiguillotine checkpoint that prevents septa from forming over the DNA. Furthermore, Noc deleted cells show multiple Z-rings which are no longer placed in perpendicular planes. We propose that the axis of chromosome segregation has a role in determining the placement of the division septum. This is achieved via the action of Noc which restricts the placement of the division septum to one of an infinite number of potential division planes that exist in S. aureus.

EzrA is a negative regulator of FtsZ in Bacillus subtilis, involved in the coordination between cell growth and cell division and in the control of the cell elongation–division cycle. We have now studied the role of the Staphylococcus aureus homologue of the B. subtilis EzrA protein and shown that it is not essential for cell viability. EzrA conditional and null mutants have an overall increase of the average cell size, compared to wild type strains. In the larger ezrA mutant S. aureus cells, cell division protein FtsZ and the cell wall synthesizing Penicillin Binding Proteins (PBPs) are not properly localized. This suggests that there may be a maximum cell diameter that allows formation of a Z-ring capable of recruiting the other components of the divisome and of driving cytokinesis. We propose that the major role of EzrA in S. aureus is in cell size homeostasis.

The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.

, and mCherry), using two different resistance markers (kanamycin and erythromycin). We have also constructed a plasmid that allows expression of proteins from the ectopic spa locus in the S. aureus chromosome. This toolbox can be used for studies of the localization of proteins in S. aureus, a prominent pathogen in both health care and community settings.

Staphylococcus aureus is exposed to multiple antimicrobial compounds, including oxidative burst products and antibiotics. The various mechanisms and regulatory pathways governing susceptibility or resistance are complex and only superficially understood. Bacillus subtilis recently has been shown to control disulfide stress responses by the thioredoxin-related YjbH protein, which binds to the transcriptional regulator Spx and controls its degradation via the proteasome-like ClpXP protease. We show that the S. aureus YjbH homolog has a role in susceptibility to the disulfide stress-inducing agent diamide that is similar to that in B. subtilis, and we demonstrate that the four cysteine residues in YjbH are required for this activity. In addition, the inactivation of YjbH led to moderate resistance to oxacillin and other ␤-lactam antibiotics, and this phenotypic change was associated with higher penicillin-binding protein 4 levels and increased peptidoglycan crosslinking. Of note, the impact of YjbH on ␤-lactam susceptibility still was observed when the four cysteines of YjbH were mutated, indicating that the roles of YjbH in disulfide stress and ␤-lactam resistance rely on different types of interactions. These data suggest that the ClpXP adaptor YjbH has more target proteins than previously thought, and that oxidative burst and ␤-lactam resistance mechanisms of S. aureus are closely linked.Staphylococcus aureus is a major human pathogen causing a wide spectrum of diseases that range from mild skin infections to life-threatening septicemia, pneumonia, and toxic-shock syndrome (27). ␤-Lactam antibiotics such as oxacillin are among to the most effective drugs against S. aureus infections, but their usefulness is continuously decreasing because ␤-lactam-resistant strains, particularly methicillin-resistant S. aureus (MRSA), are spreading in hospitals and, more recently, in the community at large, alarming international health authorities (11). Resistance is based on ␤-lactamases (penicillin-resistant S. aureus) or on an alternative peptidoglycan-biosynthetic enzyme, the penicillin-binding protein 2a (MRSA) (5, 14). In addition, mutations in a variety of proteins affecting cell wall biosynthesis and turnover can affect ␤-lactam susceptibility, leading either to hypersensitivity or to moderate levels of resistance (3,5,9,17,24,36). In many cases it remains unclear how the mutated proteins affect staphylococcal ␤-lactam resistance, and the underlying mechanisms remain only superficially understood.During the process of infection, S. aureus is engulfed by phagocytic cells such as neutrophils and macrophages, and the bacteria are exposed to the microbicidal activity of these cells.One of the most powerful antimicrobial mechanisms is based on the production of reactive oxygen species (ROS) or nitrogen species (RNS) by phagocyte NADPH oxidase and nitric oxide synthase, respectively (19,29). In addition, ROS also are generated during incomplete electron transfer in the bacterial respiratory chain (21). Therefore, even nonpath...