Abstract
The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.
Highlights
Murein hydrolases are hydrolytic enzymes that are involved in degradation, turnover, and maturation of bacterial peptidoglycan
Construction of Atl Deletion Mutants The S. aureus AtlA murein hydrolase is a bifunctional protein that is proteolytically cleaved into two active enzymes, an amidase (AM) and a glucosaminidase (GL)
While previous studies have given insight into the function of AtlA in cell wall metabolism and biofilm formation [1,2,3,4,5,13,14,15], no studies have examined the individual contributions of AM and GL in these processes
Summary
Murein hydrolases are hydrolytic enzymes that are involved in degradation, turnover, and maturation of bacterial peptidoglycan. As such, they play an essential role in cell division by assuring proper daughter cell separation. They play an essential role in cell division by assuring proper daughter cell separation Due to their activity, they are tightly regulated to prevent accidental cell lysis, a phenomenon that has led them to be termed autolysins. AM contains an enzymatic domain and two repeat domains that are involved in localization and substrate recognition [2,3]. GL is a 53.6 kDa endo-b-N-acetylglucosaminidase that hydrolyzes the bond between N-acetyl-b-D-glucosamine and N-acetyl muramic acid and contains an enzymatic domain and a single repeat domain [1]
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