title: Characterization of AtlL, a bifunctional autolysin of Staphylococcus lugdunensis with N-acetylglucosaminidase and N-acetylmuramoyl-l-alanine amidase activities.
abstract: The nucleotide sequence of atlL, a gene encoding a putative Staphylococcus lugdunensis peptidoglycan hydrolase, was determined using degenerate consensus PCR and genome walking. This 3837-bp gene encodes a protein, AtlL, that appears as a putative bifunctional autolysin with a 29-amino acid putative signal peptide and two enzymatic putative centres (N-acetylmuramoyl-l-alanine amidase and N-acetylglucosaminidase) interconnected with three imperfect repeated sequences displaying glycine-tryptophan motifs. In order to determine whether both lytic domains were functional, and verify their exact enzymatic activities, gene fragments harbouring both putative domains, AM (N-acetylmuramoyl-l-alanine amidase enzymatic centre plus two repeated sequences) and GL (N-acetylglucosaminidase enzymatic centre plus one repeated sequence), were isolated, subcloned, and expressed in Escherichia coli. Purified recombinant AM and GL protein truncations exhibited cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis, and S. lugdunensis. AtlL is expressed during the whole growth, with an overexpression in the early-exponential stage. Liquid chromatography-mass spectrometry analysis of muropeptides generated by digestion of B. subtilis cell walls demonstrated the hydrolytic bond specificities and confirmed both of the acetyl domains' activities as predicted by sequence homology data. AtlL is the first autolysin described in S. lugdunensis, with a bifunctional enzymatic activity involved in peptidoglycan hydrolysis.
authors.affiliation: Groupe de Recherche sur les Antimicrobiens et les Microorganismes, Rouen University Hospital, Rouen, France.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Bourgeois
authors.forename: Emilie
authors.initials: E
authors.lastname: Camiade
authors.forename: Raja
authors.initials: R
authors.lastname: Biswas
authors.forename: Pascal
authors.initials: P
authors.lastname: Courtin
authors.forename: Laure
authors.initials: L
authors.lastname: Gibert
authors.forename: Friedrich
authors.initials: F
authors.lastname: Götz
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Chapot-Chartier
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Pons
authors.forename: Martine
authors.initials: M
authors.lastname: Pestel-Caron
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001433:Bacteriolysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D000118:Acetylglucosaminidase; D009238:N-Acetylmuramoyl-L-alanine Amidase