title: Anchor structure of cell wall surface proteins in Listeria monocytogenes.
abstract: Many surface proteins of Gram-positive bacteria are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. In Staphylococcus aureus, surface proteins are cleaved between the threonine and the glycine of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino group of the pentaglycine cell wall crossbridge. Here we investigated the anchor structure of surface proteins in Listeria monocytogenes. A methionine and six histidines (MH(6)) were inserted upstream of the LPXTG motif of internalin A (InlA), a cell-wall-anchored surface protein of L. monocytogenes. The engineered protein InlA-MH(6)-Cws was found anchored in the bacterial cell wall. After peptidoglycan digestion with phage endolysin, InlA-MH(6)-Cws was purified by affinity chromatography. COOH-terminal peptides of InlA-MH(6)-Cws were obtained by cyanogen bromide cleavage followed by purification on a nickel-nitriloacetic acid column. Analysis of COOH-terminal peptides with Edman degradation and mass spectrometry revealed an amide linkage between the threonine of the cleaved LPXTG motif and the amino group of the m-diaminopimelic acid crossbridge within the listerial peptidoglycan. These results reveal that the cell wall anchoring of surface proteins in Gram-positive bacteria such as S. aureus and L. monocytogenes occurs by a universal mechanism.
authors.affiliation: Departments of Microbiology and Immunology and of Psychiatry and Biobehavioral Sciences, University of California Los Angeles School of Medicine, Los Angeles, California 90095, USA.
authors.forename: G
authors.initials: G
authors.lastname: Dhar
authors.forename: K F
authors.initials: KF
authors.lastname: Faull
authors.forename: O
authors.initials: O
authors.lastname: Schneewind
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D006639:Histidine / Q000235:genetics; D008089:Listeria monocytogenes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000235:genetics; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C069761:internalin protein, Bacteria; D006639:Histidine; D008715:Methionine