Localization of penicillin-binding proteins to the splitting system of Staphylococcus aureus septa by using a mercury-penicillin V derivative

Abstract

Precise localization of penicillin-binding protein (PBP)-antibiotic complexes in a methicillin-sensitive Staphylococcus aureus strain (BB255), its isogenic heterogeneous methicillin-resistant transductant (BB270), and a homogeneous methicillin-resistant strain (Col) was investigated by high-resolution electron microscopy. A mercury-penicillin V (Hg-pen V) derivative was used as a heavy metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells during growth. The most striking feature of thin sections was the presence of an abnormally large (17 to 24 nm in width) splitting system within the thick cross walls or septa of Hg-pen V-treated bacteria of all strains. Untreated control cells possessed a thin, condensed splitting system, 7 to 9 nm in width. A thick splitting system was also distinguishable in unstained thin sections, thereby confirming that the electron contrast of this structure was not attributed to binding of bulky heavy metal stains usually used for electron microscopy. Biochemical analyses demonstrated that Hg-pen V bound to isolated plasma membranes as well as sodium dodecyl sulfate-treated cell walls and that two or more PBPs in each strain bound to this antibiotic. In contrast, the splitting system in penicillin V-treated bacteria was rarely visible after 30 min in the presence of antibiotic. These findings suggest that while most PBPs were associated with the plasma membrane, a proportion of PBPs were located within the fabric of the cell wall, in particular, in the splitting system. Inhibition of one or more high-M r PBPs by ␤-lactam antibiotics modified the splitting system and cross-wall structure, therefore supporting a role for these PBPs in the synthesis and architectural design of these structures in S. aureus.Penicillin-binding proteins (PBPs) catalyze the final stages of peptidoglycan synthesis, which is necessary for maintenance of bacterial cell morphology and growth (37). In earlier work (27), we demonstrated that PBPs of Escherichia coli could be accurately localized in situ in individual bacterial cells by using a heavy metal-labeled derivative of penicillin, mercury-penicillin V (Hg-pen V), and high-resolution electron microscopy (EM). The key to visualization was the covalent attachment of a mercury atom to the secondary ring of the ␤-lactam nucleus in order to enhance its electron density and facilitate visualization at the EM level. Since ␤-lactam antibiotics are covalently bound to PBPs, forming a stable, acylated complex, this stable complex was able to survive the often harsh fixation and embedding methods used during sample preparation, and it revealed the sites of PBP-Hg-pen V complexes as electrondense particles positioned on the plasma membranes and within the periplasm of thin-sectioned E. coli cells.We now explore the utility of Hg-pen V for the identification and localization of PBPs in a gram-positive organism. Staphylococcus aureus was selected as our gram-positive model because this organism exhibits distinct morphological ...

Two alternative mechanisms of cell separation in staphylococci: one lytic and one mechanical

“…Since this splitting system disappears during cell separation ( Fig. 1 D), for more than 25 years it was thought to be responsible for lytic cell separation (Giesbrecht and Wecke 1971;Paul et al 1995). According to our data obtained during induced cell separation, however, the splitting system is not involved in those muralytic processes that result in partition (Fig.…”

“…It has been concluded, therefore, that the splitting system is lytically degraded and released into the culture medium during cell separation (Giesbrecht et al 1976(Giesbrecht et al , 1985(Giesbrecht et al , 1992Paul et al 1995). Since subinhibitory concentrations of certain β-lactams are known to inhibit the formation of a detectable splitting system without substantially interfering with cell growth, cell division, and cell separation (Beise et al 1988), the question arose whether staphylococci would have any alternative cell-separation mechanism not involving the splitting system.…”

Granular Layer in the Periplasmic Space of Gram-Positive Bacteria and Fine Structures ofEnterococcus gallinarumandStreptococcus gordoniiSepta Revealed by Cryo-Electron Microscopy of Vitreous Sections

“…6B). Interestingly, electron microscopy experiments using labeled penicillin in S. aureus cells indicated that a fraction of the PBPs may not be bound to the plasma membrane (30). Since vitreous sections cannot be immunolabeled, targeted gene deletions or selective The layered structure of the gram-positive envelope raises questions.…”

Atomic Force Microscopy of Cell Growth and Division in Staphylococcus aureus

“…This strongly suggests to us that the ring pattern is an integral part of the fabric of the new cell wall and that it most likely does not only represent structural features of the splitting system. Since new peptidoglycan is inserted at the inner face of the wall via penicillin-binding proteins (33) and since the splitting system must involve autolysins and the breakdown of peptidoglycan as the daughter cells separate, it is logical to assume that the rings are structural features or remnants of the putting in and taking out of the peptidoglycan during the division of these grampositive bacteria. For example, if the peptidoglycan strands are oriented perpendicular to the cross wall (see next paragraph), then some circular regions may have the peptidoglycan ends protrude slightly above the plane of the cross-wall while other circular regions might have the ends slightly below this plane, thus generating a landscape of circular grooves and valleys.…”