Activity of the major staphylococcal autolysin Atl.
The major autolysin of Staphylococcus aureus (AtlA) and of Staphylococcus epidermidis (AtlE) are well-studied enzymes. Here we created an atlA deletion mutant in S. aureus that formed large cell clusters and was biofilm-negative. In electron micrographs, the mutant cells were distinguished by rough outer cell surface. The mutant could be complemented using the atlE gene from S. epidermidis. To study the role of the repetitive sequences of atlE, we expressed in Escherichia coli the amidase domain encoded by the gene, carrying no repeat regions (amiE) or two repeat regions (amiE-R1,2), or the three repeat regions alone (R1,2,3) as N-terminal His-tag fusion proteins. Only slight differences in the cell wall lytic activity between AmiE and AmiE-R1,2 were observed. The repetitive sequences exhibit a good binding affinity to isolated peptidoglycan and might contribute to the targeting of the amidase to the substrate. AmiE and AmiE-R1,2 have a broad substrate specificity as shown by similar activities with peptidoglycan lacking wall teichoic acid, O-acetylation, or both. As the amidase activity of AtlA and AtlE has not been proved biochemically, we used purified AmiE-R1,2 to determine the exact peptidoglycan cleavage site. We provide the first evidence that the amidase indeed cleaves the amide bond between N-acetyl muramic acid and L-alanine.
Biswas
Raja
R
Microbial Genetics, Universität Tübingen, Tübingen, Germany.
Voggu
Lalitha
L
Simon
Uwe Karsten
UK
Hentschel
Petra
P
Thumm
Günther
G
Götz
Friedrich
F
D001422:Bacterial Adhesion; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D016296:Mutagenesis; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D013212:Staphylococcus epidermidis / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014774:Virulence
D010457:Peptidoglycan; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase