1. WildtypΔvanHAX b A. balhimycina  vanHAX b WT Introduction 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 D- lactate (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 vanHAX- homologues were identified somewhere else in the chromosome. To elucidate the complete resistance mechanism in A. balhimycina we have analysed the function of the vanHAX b -genes by genetic, biochemical and analytical means. orf1 orf2 vanR vanS vanH vanA vanX vanY IR R IR L ADP ATP promoter activation P L-Ala D-Glu m-DAP D-Ala L-Ala D-Glu m-DAP D-Ala D-Lac transposase resolvase response histidine dehydro- ligase dipeptidase carboxy regulator kinase genase peptidase D-Lac Pyruvate D-Ala D-Lac glycopeptide binding phosphorylation of VanR sensitive cell wallresistant cell wall transglycosylation transpeptidation D-Ala P (I) (II) (III) (IV) Alternative glycopeptide resistance in Amycolatopsis balhimycina Hans-Jörg Frasch 1, Philip Steimle 1, G. Gallo 2, L. Kalan 3, T. Schäberle 1, A.-M. Puglia 2, W. Wohlleben 1, G. Wright 3,E. Stegmann 1 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) MurNAc UDP GlcNac UDP MurNAc UDP MurNAc UDP MurNAc UDP MurNAc UDP MurNAc UDP MurC GlcNac - Enolpyruvate UDP MurB MurD MurE MurA D-Ala-D-Ala Ligase DdlA Ab Interplay of cell wall biosynthesis pathways L-Ala D-Glu m-Dap VanH VanA VanX Alanine racemase Alanine racemase ? Analysis of the vanHAX b -genes PEP D-Ala L-Ala Pyr D-Lac D-lac D-ala D-lac 1193 1194 Homologues to vanHAX b -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 vanHAX b -operon resulted in sensitivity to 50 µg/ml balhimycin (Fig 4) showing that the vanHAX b – genes are functional. Pyr D-Lac L-Ala D-Ala D-Ala-D-Ala D-Ala-D-Lac D-Ala-D-Ala D-Ala-D-Lac MurF 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. 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 vanH b and vanA b, RNA extraction after 15, 39 and 54 h hours of growth Fig 4 Resistance assay of A. balhimycina WT and ΔvanHAX b on YM-Agar 50 µg/ml balhimycin Analysis of the  vanHAX b -mutant Surprisingly, the vanHAX b deletion mutant produces balhimycin under certain growth conditions, although the vanHAX b -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 D- Ala-D-Ala-ligases, ddlA Ab and ddlB Ab. In vitro assays with purified protein showed that DdlA Ab exclusively forms D-Ala-D-Lac didepsipeptides (Fig 6). DdlB Ab showed no activity in the same assay (data not shown). A global proteomic analysis showed that DdlA Ab is not upregulated in response to the loss of vanA, but is constitutively expressed, suggesting an innate low- level resistance to glycopeptides. D-Ala-D-Ala Ligase Fig 7 Comparison of DdlA-spots (outlined) in a 2D-DIGE analysis in A. balhimycina WT and ΔvanHAX b. In both strains DdlA Ab is expressed at the same level. 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 DdlB EC forms D-Ala-D-Ala dipeptide as reference 11931194 1193 D-lac D-ala 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 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. A B A B AB Amycolatopsis balhimycina possesses an alternative glycopeptide resistance mechanism glycopeptide NADPH NADP + MurF Canonical Resistance mechanism Non-canonical Resistance mechanism Fig 1 The biosynthesis of a glycopeptide resistant cell wall in enterococci