The role of GpsB in Staphylococcus aureus cell morphogenesis.

For decades, cells of the Gram-positive bacterial pathogen Staphylococcus aureus were thought to lack a dedicated elongation machinery. However, S. aureus cells were recently shown to elongate before division, in a process that requires a shape elongation division and sporulation (SEDS)/penicillin-binding protein (PBP) pair for peptidoglycan synthesis, consisting of the glycosyltransferase RodA and the transpeptidase PBP3. In ovococci and rod-shaped bacteria, the elongation machinery, or elongasome, is composed of various proteins besides a dedicated SEDS/PBP pair. To identify proteins required for S. aureus elongation, we screened the Nebraska Transposon Mutant Library, which contains transposon mutants in virtually all non-essential staphylococcal genes, for mutants with modified cell shape. We confirmed the roles of RodA/PBP3 in S. aureus elongation and identified GpsB, SsaA, and RodZ as additional proteins involved in this process. The gpsB mutant showed the strongest phenotype, mediated by the partial delocalization from the division septum of PBP2 and PBP4, two penicillin-binding proteins that synthesize and cross-link peptidoglycan. Increased levels of these PBPs at the cell periphery versus the septum result in higher levels of peptidoglycan insertion/crosslinking throughout the entire cell, possibly overriding the RodA/PBP3-mediated peptidoglycan synthesis at the outer edge of the septum and/or increasing stiffness of the peripheral wall, impairing elongation. Consequently, in the absence of GpsB, S. aureus cells become more spherical. We propose that GpsB has a role in the spatio-temporal regulation of PBP2 and PBP4 at the septum versus cell periphery, contributing to the maintenance of the correct cell morphology in S. aureus. IMPORTANCE: Staphylococcus aureus is a Gram-positive clinical pathogen, which is currently the second cause of death by antibiotic-resistant infections worldwide. For decades, S. aureus cells were thought to be spherical and lack the ability to undergo elongation. However, super-resolution microscopy techniques allowed us to observe the minor morphological changes that occur during the cell cycle of this pathogen, including cell elongation. S. aureus elongation is not required for normal growth in laboratory conditions. However, it seems to be essential in the context of some infections, such as osteomyelitis, during which S. aureus cells apparently elongate to invade small channels in the bones. In this work, we uncovered new determinants required for S. aureus cell elongation. In particular, we show that GpsB has an important role in the spatio-temporal regulation of PBP2 and PBP4, two proteins involved in peptidoglycan synthesis, contributing to the maintenance of the correct cell morphology in S. aureus.

EzrA contributes to the regulation of cell size in Staphylococcus 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.

New role of the disulfide stress effector YjbH in ß-lactam susceptibility of Staphylococcus aureus.

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 ß-lactam antibiotics, and this phenotypic change was associated with higher penicillin-binding protein 4 levels and increased peptidoglycan cross-linking. Of note, the impact of YjbH on ß-lactam susceptibility still was observed when the four cysteines of YjbH were mutated, indicating that the roles of YjbH in disulfide stress and ß-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 ß-lactam resistance mechanisms of S. aureus are closely linked.

Absence of nucleoid occlusion effector Noc impairs formation of orthogonal FtsZ rings during Staphylococcus aureus cell division.

The 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.

Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis.

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.

Fluorescent reporters for studies of cellular localization of proteins in Staphylococcus aureus.

We have constructed a set of plasmids that allow expression, from their native chromosomal loci, of Staphylococcus aureus proteins fused to one of four different fluorescent proteins (green fluorescent protein [GFP], cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], 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.