Wildtyp ΔvanHAXb

A. balhimycina vanHAXb 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 vanHAXb-genes by genetic, biochemical and analytical means.

orf1 orf2 vanR vanS vanH vanA vanX vanY IRRIRL

ADP ATP

promoter activation

P

L-AlaD-Glum-DAPD-AlaD-Ala

L-AlaD-Glum-DAPD-AlaD-Lac

transposase resolvase response histidine dehydro- ligase dipeptidase carboxy regulator kinase genase peptidase

D-LacPyruvate

D-Ala

D-AlaD-Lac

glycopeptide binding

phosphorylation of VanR

sensitive cell wall resistant cell walltransglycosylation

transpeptidation

D-AlaD-Ala

P

(I)

(II)

(III)

(IV)

Alternative glycopeptide resistance in Amycolatopsis balhimycinaHans-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)

MurNAcUDP

GlcNacUDP

MurNAcUDP

MurNAcUDP

MurNAcUDP

MurNAcUDP

MurNAcUDP

MurC

GlcNac - EnolpyruvateUDP

MurB

MurD

MurE

MurA

D-Ala-D-AlaLigase

DdlAAb

Interplay of cell wall biosynthesis pathways

L-Ala

D-Glu

m-Dap

VanH VanA

VanX

Alanine racemase

Alanine racemase

?

Analysis of the vanHAXb-genes

PEP

D-Ala L-Ala

PyrD-Lac

D-lac

D-alaD-lac

11931194

1194

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.

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: 459-489Gallo 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 vanHb and vanAb , RNA extraction after 15, 39 and 54 h hours of growth

Fig 4 Resistance assay of A. balhimycina WT and ΔvanHAXb on YM-Agar 50 µg/ml balhimycin

Analysis of the vanHAXb-mutant

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 D-Ala-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 low-level resistance to glycopeptides.

D-Ala-D-AlaLigase

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 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 referenceDdlBEC forms D-Ala-D-Ala dipeptide as reference

1193 1194

1193

D-lacD-ala

D-ala

Fig 3 Cell wall precursor pattern in LC-MS of A. balhimycina WTA In non production mediumB In production mediumSensitive 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 mediumB In production mediumSensitive 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

A B

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