muramidase and globomycin

The major outer membrane lipoprotein (Lpp) of Escherichia coli possesses serine at position 2, which is thought to function as the outer membrane sorting signal, and lysine at the C terminus, through which Lpp covalently associates with peptidoglycan. Arginine (R) is present before the C-terminal lysine in the wild-type Lpp (LppSK). By replacing serine (S) at position 2 with aspartate (D), the putative inner membrane sorting signal, and by deleting lysine (K) at the C terminus, Lpp mutants with a different residue at either position 2 (LppDK) or the C terminus (LppSR) or both (LppDR) were constructed. Expression of LppSR and LppDR little affected the growth of E. coli. In contrast, the number of viable cells immediately decreased when LppDK was expressed. Prolonged expression of LppDK inhibited separation of the inner and outer membranes by sucrose density gradient centrifugation, whereas short-term expression did not. Pulse-labeled LppDK and LppDR were localized in the inner membrane, indicating that the amino acid residue at position 2 functions as a sorting signal for the membrane localization of Lpp. LppDK accumulated in the inner membrane covalently associated with the peptidoglycan and thus prevented the separation of the two membranes. Globomycin, an inhibitor of lipoprotein-specific signal peptidase II, was lethal for E. coli only when Lpp possessed the C-terminal lysine. Taken together, these results indicate that the inner membrane accumulation of Lpp per se is not lethal for E. coli. Instead, a covalent linkage between the inner membrane Lpp having the C-terminal lysine and the peptidoglycan is lethal for E. coli, presumably due to the disruption of the cell surface integrity.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Binding Sites; Carrier Proteins; Cell Membrane; Escherichia coli; Escherichia coli Proteins; Kinetics; Lipoproteins; Lysine; Muramidase; Mutagenesis, Site-Directed; Peptides; Peptidoglycan; Point Mutation; Protease Inhibitors; Recombinant Proteins; Serine; Signal Transduction

Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored beta-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; beta-Lactamases; Cell Wall; Endopeptidases; Enterotoxins; Lipoproteins; Lysostaphin; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Muramidase; Peptides; Peptidoglycan; Protease Inhibitors; Protein Precursors; Protein Processing, Post-Translational; Protein Sorting Signals; Recombinant Fusion Proteins; Serine Endopeptidases; Staphylococcal Protein A; Staphylococcus aureus