Role of N-Acetylglucosaminidase and N-Acetylmuramidase Activities in Enterococcus faecalis Peptidoglycan Metabolism

Abstract

Identification of the full complement of peptidoglycan hydrolases detected by zymogram in Enterococcus faecalis extracts led to the characterization of two novel hydrolases that we named AtlB and AtlC. Both enzymes have a similar modular organization comprising a central catalytic domain fused to two LysM peptidoglycan-binding modules. AtlB and AtlC displayed N-acetylmuramidase activity, as demonstrated by tandem mass spectrometry analyses of peptidoglycan fragments generated by the purified enzymes. The genes encoding AtlB and AtlC were deleted either alone or in combination with the gene encoding AtlA, a previously described N-acetylglucosaminidase. No autolytic activity was detected in the triple mutant indicating that AtlA, AtlB, and AtlC account for the major hydrolytic activities in E. faecalis. Analysis of cell size distribution by flow cytometry showed that deletion of atlA resulted in the formation of long chains. Thus, AtlA digests the septum and is required for cell separation after cell division. We found that AtlB could act as a surrogate for AtlA, although the enzyme was less efficient at septum digestion. Deletion of atlC had no impact on cell morphology. Labeling of the peptidoglycan with N-[ 14 C]acetylglucosamine revealed an unusually slow turnover as compared with model organisms, almost completely dependent upon the combined activities of AtlA and AtlB. In contrast to atlA, the atlB and atlC genes are located in putative prophages. Because AtlB and AtlC were produced in the absence of cell lysis or production of phage progeny, these enzymes may have been hijacked by E. faecalis to contribute to peptidoglycan metabolism.

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

“…Similar long-pulse-chase experiments have revealed that other ovococcus-shaped bacteria, such as Enterococcus faecalis and Streptococcus mutans, exhibit low rates of PG fragment release from sacculi (56,57). One interpretation of these combined results is that there is minimal PG turnover and PG fragment release in exponentially growing S. pneumoniae and, likely, in other Gram-positive ovococcus species compared to the PG turnover and fragment release in B. subtilis.…”

“…Thus, minimal turnover is a general property of the PG of growing S. pneumoniae cells. There may also be minimal PG turnover in other ovococcus species, based on an apparent absence of recycling enzymes and results from previous radiolabeling experiments showing low rates of PG fragment release from sacculi (13,56,57).…”

Identification of Rgg Binding Sites in the Streptococcus pyogenes Chromosome

“…Binding to three sites was not associated with changes in the expression of proximal genes, as determined by comparing the transcript levels of the wild-type strain to those of the rgg mutant strain (Table 3); however, binding to the other three sites was associated with changes in the expression of the downstream ORFs (19). These binding sites were upstream of spd-3, which is carried by prophage NZ131.3; a gene encoding an integrase/excisionase (Spy49_0746) carried by prophage NZ131.2; and a gene encoding a surface antigen (Spy49_0396), which has a motif present in lysins (Table 3) (5,6,24,38) and is carried by prophage NZ131.3. Differences in the expression of the prophage integrase between the rgg mutant and the wild-type strain were previously associated with changes in the frequency of prophage excision from the genome (19).…”

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

“…Two autolysins of Staphylococcus epidermidis, AtlE and Aae, are adhesins that contribute to bacterial attachment to polymeric surfaces and biofilm formation via release of eDNA (20,21,58). E. faecalis produces several autolysins, which were recently identified and characterized (12,24,35). The major E. faecalis autolysin, Atn (also known as AtlA), is an N-acetylglucosaminidase important for daughter cell separation during cellular division (12).…”