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. 2011 Sep;55(9):4283-9.
doi: 10.1128/AAC.01372-10. Epub 2011 Jun 20.

Self-resistance and cell wall composition in the glycopeptide producer Amycolatopsis balhimycina

Till F Schäberle  1 , Waldemar Vollmer, Hans-Jörg Frasch, Stephan Hüttel, Andreas Kulik, Marlene Röttgen, Anna-Katharina von Thaler, Wolfgang Wohlleben, Evi Stegmann
Affiliations

Self-resistance and cell wall composition in the glycopeptide producer Amycolatopsis balhimycina

Till F Schäberle et al. Antimicrob Agents Chemother. 2011 Sep.

Abstract

The prevailing resistance mechanism against glycopeptides in Gram-positive pathogens involves reprogramming the biosynthesis of peptidoglycan precursors, resulting in d-alanyl-d-lactate depsipeptide termini. Amycolatopsis balhimycina produces the vancomycin-like glycopeptide balhimycin and therefore has to protect itself from the action of the glycopeptide. We studied the roles of the accessory resistance gene orthologs vanY(b), vnlR(b), and vnlS(b), which are part of the balhimycin biosynthetic gene cluster (represented by the subscript "b"). The VanY(b) carboxypeptidase cleaved the terminal d-Ala from peptidoglycan precursors, and its heterologous expression enhanced glycopeptide resistance in Streptomyces coelicolor. The VanRS-like two component system VnlRS(b) was not involved in glycopeptide resistance or in the expression of the vanHAX glycopeptide resistance genes. Mature A. balhimycina peptidoglycan contained mainly tri- and tetrapeptides, with only traces of the d-Ala-d-Ala-ending pentapeptides that are binding sites for the antibiotic produced. The structure of the peptidoglycan precursor is consistent with the presence of vanHAX genes, which were identified outside the balhimycin synthesis cluster. Both wild-type and non-antibiotic-producing mutant strains synthesized peptidoglycan precursors ending mainly with d-Lac, indicating constitutive synthesis of a resistant cell wall. A. balhimycina could provide a model for an ancestral glycopeptide producer with constitutively expressed resistance genes.

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Figures

Fig. 1.
Fig. 1.
Separation and proposed structures of cell wall muropeptides. (A) Separation of cell wall muropeptides by HPLC. Fraction I, disaccharide tripeptide (Tri); fraction II, disaccharide tetrapeptide (Tetra); fraction III, disaccharide pentapeptide (Penta); fraction IV, multimers. The disaccharide pentapeptide is present in S. coelicolor (left) and virtually absent in A. balhimycina (right). (B) Proposed muropeptide structures of S. coelicolor, including Tri(Gln)Gly (1), Tetra(Gln)Gly (2), and Penta(Gln)Gly (3), and of A. balhimycina, including Tri(Gln) (1a), Tri(Gln)(NH2) (1b), Tetra(Gln) (2a), and Tetra(Gln)(NH2) (2b). The amidation of Dap is highlighted in gray. The letter “r” indicates that MurNAc was reduced during sample preparation. The measured molecular masses (Da) of the reduced muropeptides (Na+ form) are given below the structures. (C) Proposed structures of precursors from S. coelicolor, including the pentapeptide precursor (5) and pentadepsipeptide precursor (6), and from A. balhimycina, including the pentapeptide precursor (7) and pentadepsipeptide precursor (8). The neutral masses calculated from the measured molecular masses of the H+ forms are given below the structures.
Fig. 2.
Fig. 2.
MS/MS fragmentation of cell wall precursors. (A) Structure of the UDP-linked cell wall precursor and its MS/MS fragmentation pattern. The neutral mass is given. (B) MS/MS analysis in negative ion mode of the precursor from A. balhimycina with a molecular mass of 1,193 Da. The fragmentation of the precursor with a mass of 1,193 Da resulted in a fragment with a mass of 403 Da corresponding to the identical UDP part of the molecule. Further fragments of 790 Da and 870 Da contained the peptide part of the molecule and confirmed the presence of the expected pentapeptide moiety.
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