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Alternative glycopeptide resistance in Amycolatopsis balhimycina Hans-Jörg Frasch1, Philip Steimle1, G. Gallo2, L. Kalan3, T. Schäberle1, A.-M. Puglia2, W. Wohlleben1, G. Wright3,E. Stegmann1 1 Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany 2 Dipartimento di Biologica Cellulare e dello Sviluppo, Università di Palermo, Viale delle Scienze, edificio 16, 90128 Palermo (Sicily) 3 Health Science Receiving, MacMaster University,1200 Main STW, L8N3Z5 Hamilton (Canada) Introduction sensitive cell wall transglycosylation The actinomycete Amycolatopsis balhimycina produces the vancomycin-type glycopeptide balhimycin, which inhibits cell wall biosynthesis by binding to cell wall precursors. Glycopeptide resistance is usually achieved by the synthesis of an alternative cell wall. The endstanding D-alanine (D-Ala) in the pentapetide is replaced by a Dlactate (D-Lac), which reduces binding of the glycopeptide to its target (Fig. 1). From resistant enterococci it is known that, this alteration of cell wall precursors requires three enzymes: (I) VanH converts pyruvate to D-Lac, (II) VanA ligates D-Ala and D-Lac and (III) VanX cleaves the ubiquitous D-Ala-D-Ala dipeptide. The expression of the corresponding genes is controlled by the two component system VanRS. VanS senses the presence of glycopeptides and phosphorylates VanR, which subsequently activates the expression of the vanHAX-genes. An occassionally additional enzyme, VanY (IV), cleaves the end standing D-Ala from pentapeptide precursors, thereby increasing the resistance level (Fig 1). Resistance to glycopeptides probably originated from producer strains which are immune to their own product. Normally resistance genes are part of the biosynthetic gene cluster. Surprisingly, the balhimycin gene cluster does not contain vanHAX-genes, but vanHAXhomologues were identified somewhere else in the chromosome. To elucidate the complete resistance mechanism in A. balhimycina we have analysed the function of the vanHAXb-genes by genetic, biochemical and analytical means. Analysis of the vanHAXb-genes A transpeptidation ADP ATP Pyruvate P D-Lac D-Ala D-Lac (I) (II) promoter activation phosphorylation of VanR orf1 IRL transposase orf2 vanR resolvase response regulator (IV) vanS histidine kinase vanH vanA dehydrogenase ligase vanX vanY dipeptidase IRR carboxy peptidase Fig 1 The biosynthesis of a glycopeptide resistant cell wall in enterococci Analysis of the vanHAXb-mutant GlcNac UDP PEP Surprisingly, the vanHAXb deletion mutant produces balhimycin under certain growth conditions, although the vanHAXb-genes are missing (data not shown). Cell wall precursor analysis revealed the production of resistant cell wall precusors under production conditions (Fig 5). Homology searches in the genome of A. balhimycina delivered two genes encoding putative DAla-D-Ala-ligases, ddlAAb and ddlBAb. In vitro assays with purified protein showed that DdlAAb exclusively forms D-Ala-D-Lac didepsipeptides (Fig 6). DdlBAb showed no activity in the same assay (data not shown). A global proteomic analysis showed that DdlAAb is not upregulated in response to the loss of vanA, but is constitutively expressed, suggesting an innate lowlevel resistance to glycopeptides. GlcNac - Enolpyruvate MurB NADP+ A L-Ala D-Glu m-DAP D-Ala D-Ala (III) D-ala 1193 NADPH UDP UDP A MurNAc MurC D-lac 1194 D-ala 1193 D-Ala D-Ala P MurA Fig 2 A PCR Screeening of an A. balhimycina cosmid library using degenerated primers, a 1,3 kB fragment was amplified which corresponds to vanA- homlogues. B RT-PCR with primers overlapping vanHb and vanAb , RNA extraction after 15, 39 and 54 h hours of growth L-Ala D-Glu m-DAP D-Ala D-Lac D-Ala UDP B glycopeptide glycopeptide binding Interplay of cell wall biosynthesis pathways Homologues to vanHAXb-genes were identified in an A. balhimyina cosmid library by a PCR-screening using degenerated primers (Fig 2A). RT-PCR analysis revealed that these genes are expressed as an operon prior to antibiotic biosynthesis (Fig 2B). LC-MS analysis of extracted cell wall precursors (cwp) showed that resistant cell wall precursors ending on D-Lac are predominant under different growth conditions (Fig. 3) and deletion of the vanHAXb-operon resulted in sensitivity to 50 µg/ml balhimycin (Fig 4) showing that the vanHAXb– genes are functional. resistant cell wall L-Ala D-lac 1194 D-ala 1193 B Fig 5 Cell wall precursor pattern in LC-MS of A. balhimycina ΔvanHAX A In non production medium B In production medium Sensitive cwp ending on D-alanine elute at retention time of 10-11 min. Resistant cwp ending on D-lactate elute at retention time of 15-16 min. MurNAc D-lac 1194 B MurD Fig 3 Cell wall precursor pattern in LC-MS of A. balhimycina WT A In non production medium B In production medium Sensitive cwp ending on D-alanine elute at retention time of 10 -11 min Resistant cwp ending on D-lactate elute at retention time of 15 -16 min UDP A. balhimycina vanHAXb WT m-Dap Fig 6 Thin-layer-chromatography of DdlA product formation A) 2 mM D-Lac B) 2 mM D-Lac + 2 mM D- Ala C) 2 mM D-Ala + 1 mM D-Ala VanA forms D-Ala-D-Lac-didepsipeptide as reference DdlBEC forms D-Ala-D-Ala dipeptide as reference Wildtyp ΔvanHAXb MurNAc Fig 7 Comparison of DdlA-spots (outlined) in a 2D-DIGE analysis in A. balhimycina WT and ΔvanHAXb. In both strains DdlAAb is expressed at the same level. Fig 4 Resistance assay of A. balhimycina WT and ΔvanHAXb on YM-Agar 50 µg/ml balhimycin MurF MurF D-Ala-D-Ala Ligase VanA Pyr UDP Alanine racemase L-Ala MurNAc MurE UDP VanH D-Glu D-Lac D-Ala MurNAc UDP MurNAc D-Ala-D-Ala D-Ala-D-Lac D-Ala-D-Ala Ligase D-Ala-D-Ala VanX Canonical Resistance mechanism Alanine racemase Non-canonical Resistance mechanism D-Ala DdlAAb D-Ala-D-Lac L-Ala ? D-Lac Amycolatopsis balhimycina possesses an alternative glycopeptide resistance mechanism Stegmann et al, 2010, Glycopeptide biosynthesis in the context of basic cellular functions, Current opinion in microbiology, submitted Wohlleben et al, 2009, Chapter 18: Molecular genetic approaches to analyze glycopeptide biosynthesis, Methods Enzymol, 458: 459-489 Gallo et al., 2010, Differentiell proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina, Proteomics , [Epub head of print] Pelzer et al., 1999, Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediteranei DSM5908; Antimicrob. Agents Chemother., 43: 1565-1573. Pyr