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J. Biol. Chem., Vol. 283, Issue 28, 19845-19853, July 11, 2008

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

Stéphane Mesnage^¶1, Françoise Chau^||, Lionel Dubost^**, and Michel Arthur^¶

From the Centre de Recherche des Cordeliers, LRMA, Equipe 12, Université Pierre et Marie Curie, UMR-S 872, Paris F-75006, Université Paris Descartes, UMR-S 872, Paris F-75006, ^¶INSERM, U872, Paris F-75006, ^||EA3964, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris F-75018, ^**Muséum National d'Histoire Naturelle, USM0502, Paris F-75005, and CNRS, UMR8041, Plateforme de Spectrométrie de Masse et de Protéomique du Muséum, Département de Recherche Développement et Diversité Moléculaire, Paris F-75005, France

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-[¹⁴C]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.

Received for publication, March 25, 2008 , and in revised form, May 5, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Experimental Procedures, Table S1, and additional references.

¹ To whom correspondence should be addressed: INSERM, U872, LRMA, Equipe 12, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie et Université Paris Descartes, 15 Rue de l'Ecole de Médecine 75270 Paris Cedex 06, France. Tel.: 33-1-42-34-68-65; Fax: 33-1-43-25-68-12; E-mail: stephane.mesnage{at}crc.jussieu.fr.

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