The use of a Mercury-Penicillin V Derivative to Localize Penicillin-Binding Proteins in Escherichia coli
Precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in a wild-type strain and two PBP overproducing mutants of Escherichia coli JM101 was investigated by high resolution electron microscopy (EM). A novel 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 grown to exponential growth-phase. Results obtained from susceptibility tests, lysis experiments and PBP affinity assays revealed few differences between strains and between the antimicrobial activities of Hg-pen V and the parent compound, penicillin V. When cells were grown in the presence of Hg-pen V, Hg-pen V-PBP complexes could readily be visualized by EM of unstained whole mounts as distinct randomly situated electron dense particles. Thin sections of Hg-pen V-treated bacteria revealed similar electron dense particles located predominantly on the plasma membrane and seldom in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space thereby demonstrating the high resolution of the Hg-pen V probe. In addition, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron dense particles. The presence of electron dense particles in cells of the wild-type strain, demonstrated that our probe could identify PBPs in a naturally-occuring strain without inducing PBP overproduction.
KeywordsMinimum Inhibitory Concentration Luria Broth Penicillin Binding Protein Penicillin Versus Strain 1M101
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