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International audience; In the available Staphylococcus aureus genomes, four different genes have been annotated to encode tRNA(Gly) isoacceptors. Besides their prominent role in protein synthesis, some of them also participate in the formation of pentaglycine bridges during cell wall synthesis. However, until today, it is not known how many and which of them are actually involved in this essential procedure. In the present study we identified, apart from the four annotated tRNA(Gly) genes, a putative pseudogene which encodes and expresses an unusual fifth tRNA(Gly) isoacceptor in S. aureus (as detected via RT-PCR and subsequent direct sequencing analysis). All the in vitro transcribed tRNA(Gly) molecules (including the "pseudogene-encoded" tRNA(Gly)) can be efficiently aminoacylated by the recombinant S. aureus glycyl-tRNA synthetase. Furthermore, bioinformatic analysis suggests that the "pseudo"-tRNA(Gly(UCC)) identified in the present study and two of the annotated isoacceptors bearing the same anticodon carry specific sequence elements that do not favour the strong interaction with EF-Tu that proteinogenic tRNAs would promote. This observation was verified by the differential capacity of Gly-tRNA(Gly) molecules to form ternary complexes with activated S. aureus EF-Tu.GTP. These tRNA(Gly) molecules display high sequence similarities with their S. epidermidis orthologs which also actively participate in cell wall synthesis. Both bioinformatic and biochemical data suggest that in S. aureus these three glycylated tRNA(Gly) isoacceptors that are weak EF-Tu binders, possibly escape protein synthesis and serve as glycine donors for the formation of pentaglycine bridges that are essential for stabilization of the staphylococcal cell wall.