Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

Abstract

The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D 2 C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.Staphylococcus aureus is a gram-positive pathogen often involved in nosocomial infections and food-borne diseases, and there is now growing concern about the spread of multidrugresistant strains of methicillin-resistant S. aureus (4,25,26,31,39,45). The staphylococcal cell wall plays an important role for this organism's success, as it withstands tremendous turgor pressures throughout the cell cycle (ca. 20 to 30 atm), strongly interacts with the cell's external environment, particularly in infection processes, and is intimately involved in cell division (1-3, 15, 21, 22, 33). Over the past two decades much knowledge has been accumulated on the primary structure of staphylococcal cell wall components, i.e., peptidoglycan (3, 15, 45), (lipo)teichoic acids (34,47,48), and surface proteins (33, 43), but there is still a lack of structural information at high resolution on the spatial organization of this complex cell wall (4, 9, 17, 46, 49).As with other gram-positive bacteria, the envelope of S. aureus is commonly seen in conventional thin sections by electron microscopy as composed of a plasma membrane tightly bound by a thick and undifferentiated wall (Fig. 1). This simple architect...

Peptidoglycan architecture can specify division planes in Staphylococcus aureus

“…Th e similarity between the diameter of the piecrust as measured by AFM and the diameter of whole cells as measured by diff erential interference contrast microscopy shows that these features are not the leading edge of the septum, a marked diff erence from B. subtilis , the septum of which has somewhat raised leading and lagging edges 18 . Such features may also be associated with ' bridge ' regions of cell wall previously observed by cryo-transmission electron microscopy of pure S. aureus peptidoglycan 29 .…”

“…Ribs may either be recognized by a ligand -receptortype interaction between the mostly uncharacterized divisome and the distinct peptidoglycan architecture in the ribs, in the rib junctions, or by recognition of the distinct curvature that is imprinted on the cytoplasmic membrane by ribs and inter-rib junctions. Th e plane containing the quarter rib has the longest circumference, as there is the minimum protrusion of peptidoglycan impinging on the cytoplasmic membrane (by direct physical contact or by the periplasm 29 ), and thus the largest average radius of curvature. Th is may single it out for recognition.…”

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

“…Lipoproteins of Gram-positive bacteria have, thus, been proposed to be functional equivalents of periplasmic proteins in Gram-negative bacteria, a comparison that is most directly sustained by the fact that, in Gram-positive bacteria, substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters are typically lipoproteins [2,3]. Moreover, cell fractionation experiments and recent advances in electron microscopy have lent some credibility to the controversial concept of a Gram-positive 'periplasm' [4][5][6]. Cryoelectron microscopy has also provided evidence supporting the presence of an outer membrane permeability barrier in the mycolic acid-containing actinomycete bacteria Mycobacterium smegmatis, Mycobacterium bovis and Corynebacterium glutamicum [7,8].…”

Localization and expression of the Bacillus subtilis dl-endopeptidase LytF are influenced by mutations in LTA synthases and glycolipid anchor synthetic enzymes

“…Cryo-electron microscopy of frozen-hydrated sections has indicated that Gram-positive bacteria such as B. subtilis and S. aureus also have a periplasmic space, termed the inner wall zone, between the plasma membrane and thick CW (Matias & Beveridge, 2005;Matias & Beveridge, 2006;Zuber et al, 2006) and that LTA is a major component of the B. subtilis periplasm (Matias & Beveridge, 2008). Moreover, subcellular localization analysis of UgtP fused to GFP showed that the fusion protein was predominantly localized at the septa and that proper localization was abolished in the pgcA and gtaB mutants (Weart et al, 2007).…”