Molecular characterization of an autolytic amidase of Listeria monocytogenes EGD.
The gene encoding a 102 kDa autolysin has been cloned from an expression library of Listeria monocytogenes EGD genomic DNA, using a direct screening protocol. The encoded protein has two domains, an N-terminal enzymic domain showing a high level of homology to the amidase domain of the major autolysin (atl) of Staphylococcus aureus, and a C-terminal, putative cell-wall-binding domain containing four imperfect direct repeats. In order to examine the role of the enzyme, the autolysin-encoding gene was insertionally inactivated by site-specific integration of a temperature sensitive plasmid. The enzyme accounts for 66% of the total lytic enzyme activity when L. monocytogenes walls are used as substrate and several of the major autolytic bands are missing on renaturing gels when compared to the wild-type. The enzyme does not appear to be directly involved in cell separation but has a role in motility. Characterization of the recombinant enzyme expressed in Escherichia coli has revealed it to be an amidase and to be able to hydrolyse a range of peptidoglycan substrates.
McLaughlan
Anna M
AM
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Foster
Simon J
SJ
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
D000581:Amidohydrolases / Q000737:chemistry; D000595:Amino Acid Sequence; D001433:Bacteriolysis; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000581:Amidohydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase; C019665:amidase; D008274:Magnesium