LOCALIZATION OF PENICILLIN-BINDING PROTEINS TO THE SPLITTING SYSTEM OF STAPHYLOCOCCUS-AUREUS SEPTA BY USING A MERCURY-PENICILLIN-V DERIVATIVE

Precise localization of penicillin-binding protein (PBP)-antibiotic co mplexes in a methicillin-sensitive Staphylococcus aureus strain (BB255 ), its isogenic heterogeneous methicillin-resistant transductant (BB27 0), and a homogeneous methicillin-resistant strain (Col) was investiga ted by high-resolution electron microscopy. A mercury-penicillin V (Hg -pen V) derivative was used as a heavy metal-labeled, electron-dense p robe for accurately localizing PBPs in situ in single bacterial cells during growth. The most striking feature of thin sections was the pres ence of an abnormally large (17 to 24 nm in width) splitting system wi thin the thick cross walls or septa of Hg-pen V-treated bacteria of al l strains. Untreated control cells possessed a thin, condensed splitti ng system, 7 to 9 nm in width. A thick splitting system was also disti nguishable in unstained thin sections, thereby confirming that the ele ctron contrast of this structure was not attributed to binding of bulk y heavy metal stains usually used for electron microscopy. Biochemical analyses demonstrated that Hg-pen V bound to isolated plasma membrane s 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 s plitting system in penicillin V-treated bacteria was rarely visible af ter 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 particula r, in the splitting system. Inhibition of one or more high-M(r) PBPs b y beta-lactam antibiotics modified the splitting system and cross-wall structure, therefore supporting a role for these PBPs in the synthesi s and architectural design of these structures in S. aureus.