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Originally published In Press as doi:10.1074/jbc.M004675200 on June 26, 2000

J. Biol. Chem., Vol. 275, Issue 36, 27768-27774, September 8, 2000
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Characterization of the murMN Operon Involved in the Synthesis of Branched Peptidoglycan Peptides in Streptococcus pneumoniae*

Sergio R. FilipeDagger §, Mariana G. PinhoDagger §||, and Alexander TomaszDagger **

From the Dagger  Laboratory of Microbiology, The Rockefeller University, New York, New York 10021 and the § Molecular Genetics Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780 Oeiras, Portugal

The murMN operon, recently identified in the genome of Streptococcus pneumoniae, encodes for enzymes involved in the synthesis of branched structured muropeptides in the pneumococcal peptidoglycan; inactivation of murMN causes production of a peptidoglycan composed exclusively of linear muropeptides and a virtually complete loss of resistance in penicillin-resistant strains (Filipe, S. R., and Tomasz, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4891-4896). The experiments described in this paper follow up these observations. Primer extension analysis was used to identify the putative promoter region of the murMN operon in penicillin-susceptible and -resistant strains. Selective inactivation of the murN gene in the penicillin-resistant strain Pen6 caused production of an unusual peptidoglycan that contained only single amino acid residues in the muropeptide branches, indicating that the product of murN was involved with the addition of the second amino acid and the product of murM was involved with the addition of the first amino acid (alanine or serine) to the peptidoglycan cross-bridge. Allelic replacement of the mosaic murM gene of strain Pen6 with murM of the penicillin-susceptible laboratory strain caused enrichment of the peptidoglycan in linear muropeptides. The findings suggest that the genetic determinant primarily controlling the synthesis of branched muropeptides in the pneumococcal peptidoglycan is murM.

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

These studies were supported, in part, by National Institutes of Health Grant RO1 AI37275 and by the Irene Diamond Foundation.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ250766 (Pen6) and AJ250764 (R36A).

Supported by Fundação para a Ciência e Tecnologia PRAXIS XXI Grant BD/9071/96.

|| Supported by Fundação para a Ciência e Tecnologia PRAXIS XXI Grant BD/9079/96.

** To whom correspondence should be addressed: Laboratory of Microbiology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8278; Fax: 212-327-8688; E-mail: tomasz@rockvax.rockefeller.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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