In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus.
Staphylococcus aureus peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t-RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP-MurNAc-pentapeptide, lipid I and lipid II and the staphylococcal t-RNA pool, as well as His-tagged Gly-tRNA-synthetase and His-tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II-Gly1 and lipid II-Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane-associated steps of S. aureus peptidoglycan biosynthesis.
Schneider
Tanja
T
Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, D-53105 Bonn, Germany.
Senn
Maria Magdalena
MM
Berger-Bächi
Brigitte
B
Tossi
Alessandro
A
Sahl
Hans-Georg
HG
Wiedemann
Imke
I
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry; D006032:Glycine-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D010453:Peptide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis; D012353:RNA, Transfer, Gly / Q000302:isolation & purification / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000737:chemistry*; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; C096686:FemA protein, Bacteria; D008565:Membrane Proteins; D010457:Peptidoglycan; D012353:RNA, Transfer, Gly; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; D006032:Glycine-tRNA Ligase; C480726:FemX UDP-MurNAc-pentapeptide l-alanine ligase; D010453:Peptide Synthases