Activity of the major staphylococcal autolysin Atl

Abstract

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.

The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus

“…In Staphylococcus epidermidis and S. aureus, disruption of the atl gene, encoding the primary murein hydrolase of these bacteria, causes a dramatic decrease in their abilities to form biofilm (65,66). Inactivation of a gene encoding an Atl homolog in S. mutans also reduced its biofilm-forming ability (67,68).…”

Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis

“…The three-Atlrepeat domain, R 1-3 (17,18), was expressed with a N-terminal His 6 tag in Escherichia coli M15 using the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible plasmid pEQ30Atl R [1][2][3] (19,20). Cells were cultivated in 2ϫ yeast extract-tryptone broth (YT) to an OD of 0.5 at 37°C and then induced with 0.5 mM IPTG for 4 h at 20°C.…”

“…It was also shown that mutation of tarO results in delocalization of amidase (21). Atl possesses three repeat sequences, each about 150 amino acids long (R 1-3 ), which bind to peptidoglycan (19,35). We examined the localization of Atl using a fluorescently labeled R 1-3 repeat domain (Cy3-R 1-3 ) (21).…”

A novel role for SarX in Staphylococcus epidermidis biofilm regulation

“…Production of the icaADBC operon-encoded polysaccharide intercellular adhesin (PIA) or polymeric N-acetylglucosamine (PNAG) remains the best-understood mechanism of biofilm accumulation in staphylococci (Mack et al, 1996;Maira-Litrán et al, 2002;O'Gara, 2007). The major cell wall-anchored autolysins AtlA and AtlE, in S. aureus and S. epidermidis, respectively, have been implicated in the primary attachment stage of biofilm production (Biswas et al, 2006;Heilmann et al, 1997). Atl contains amidase (Ami) and glucosaminidase (GL) domains separated by three direct repeats, R 1 , R 2 and R 3 .…”