INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

The glycopeptide antibiotic vancomycin and the structurally related antibiotic teicoplanin are considered to be the last lines of defense against a variety of serious infections caused by gram-positive organisms, such as enterococci, methicillin-resistant Staphylococcus aureus, and Clostridium difficile (11). The recent rapid emergence and spread of vancomycin-resistant enterococci (VRE) has therefore become a grave concern of both medical practitioners and their patients (22, 27).

The molecular target of glycopeptide antibiotics is the D-alanyl-D-alanine (D-Ala-D-Ala) terminus of growing peptidoglycan, the rigid polymer which protects bacterial cells from osmotic lysis. By binding to this terminal dipeptide, glycopeptide antibiotics interfere with proper cell wall formation, which results in eventual cell death (6). Glycopeptide-resistant organisms avoid such a fate by modifying the drug's peptide target, specifically, by modifying it to the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac) (40). The biosynthetic machinery required to effect this transformation in vancomycin-resistant Enterococcus faecium is found on a transposable element, Tn1546, which incorporates five genes necessary and sufficient to confer high-level inducible glycopeptide resistance (2). Two of these gene products, VanR and VanS, are required for the vancomycin-induced resistance response and are members of a two-component regulatory system directing the transcription of vanH, vanA, and vanX. These genes encode the three proteins which are necessary for D-Ala-D-Lac synthesis and are thus essential for glycopeptide resistance (1). VanA is the pivotal enzyme, producing the ester D-Ala-D-Lac instead of the usual D-Ala-D-Ala dipeptide for incorporation into peptidoglycan precursors (9). VanH is an -keto acid reductase (D-lactate dehydrogenase [D-LDH]) that supplies VanA with substrate by converting pyruvate into D-lactate (9), while VanX is a highly specific D,D-dipeptidase which hydrolyzes the D-Ala-D-Ala generated by the endogenous cell wall biosynthetic pathway but does not recognize D-Ala-D-Lac (34, 43). This elegant system is the means by which VRE can overcome current antibiotic regimens. A second variant of this mechanism and gene structure is encountered and distributed on plasmids (42), conjugative chromosomal elements (32), or transposons (e.g., Tn1547 [31]) with the D-Ala-D-Lac ligase designated VanB (18, 33).

The origin of the vanH, vanA/B, and vanX (vanHAX) resistance cassette has remained obscure. Both the intrinsically vancomycin-resistant lactic acid bacteria, such as organisms from the genera Leuconostoc, Pediococcus, and Lactobacillus, and the glycopeptide-producing organisms are potential reservoirs. Alternatively, the genes may have arisen through mutations in homologous genes within enterococci or other organisms. The vancomycin-resistant lactic acid bacteria such as Leuconostoc mesenteroides have both the requisite D-Ala-D-Lac ligase (15, 29) and D-LDH (12) enzymes, although the D-Ala-D-Lac ligases differ in their primary sequence from VanA/B, notably in the -loop region which is critical to catalysis (16). We have recently cloned a gene encoding a VanA/B homolog, designated DdlM, from the glycopeptide antibiotic-producing organism Streptomyces toyocaensis NRRL 15009 (23). The striking homology between VanA/B and DdlM (62% identity, >77% similarity) suggested that these enzymes evolved from a common ancestor. Similarly, we reported a segment of the ligase gene (ddlN) cloned from the vancomycin-producing organism Amycolatopsis orientalis C329.2 which also encoded a clear VanA/B homolog (23). Importantly, the -loop segment is conserved between VanA, VanB, DdlM, and DdlN. Thus, these results suggested that the VanA/B enzymes originated in glycopeptide-producing organisms but that the origins of the VanH and VanX accessory proteins were unknown. We now report on the cloning and sequencing of genes encoding VanH and VanX homologs from the same glycopeptide producers as well as the presence of vanHAX gene clusters in several other glycopeptide-producing organisms. The strong homology of the gene products and the arrangement of the cloned genes indicate a common origin for the vancomycin resistance genes in VRE and antibiotic-producing organisms.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

Cloning of vanX from S. toyocaensis NRRL 15009. Cloning of a vanX gene from S. toyocaensis NRRL 15009 (vanX_st) was achieved by the isolation of a specific gene probe by PCR amplification and degenerate deoxyoligonucleotide primers p1 and p2 designed to amplify an internal vanX fragment of approximately 360 bp (Table 1). PCR amplification reactions contained S. toyocaensis NRRL 15009 total DNA as template (500 ng), 1 U of Taq polymerase, each deoxynucleoside triphosphate (dNTP) at a concentration of 0.4 mM, 1 µM primers, and 2 mM MgCl₂. The 360-bp product was cloned into the pGEM-T plasmid vector (Promega), and both strands were sequenced. To isolate a clone encompassing the entire vanX_st gene, the probe fragment was randomly labeled with ³²P by using the Klenow fragment (4). Labeling reactions contained 200 ng of template, 1 µl of each PCR primer, 1× Klenow buffer (10 mM Tris-Cl [pH 7.5], 5 mM MgCl₂, 7.5 mM dithiothreitol), and 10 U of the Klenow fragment in a final volume of 55 µl. This probe was used to screen Southern hybridization blots of total S. toyocaensis NRRL 15009 DNA which had been digested to completion with several restriction enzymes in separate experiments. Hybridizing fragments (5.2 kb) of DNA from digestion with PstI were cloned into pBluescript II KS+ (Stratagene), transformed into Escherichia coli DH5, and confirmed to contain the vanX_st gene by colony hybridization. A 5.2-kb fragment was found to contain the vanX_st gene and was sequenced in its entirety.

Cloning of van genes from A. orientalis C329.2. Cloning of the A. orientalis C329.2 vanHAX cluster was achieved essentially as described for the vanX gene of S. toyocaensis NRRL 15009. A van gene probe was generated from A. orientalis C329.2 genomic DNA by degenerate PCR with primers p2 and p3 complementary to conserved sequences in vanA and vanX (Table 1). Each amplification reaction mixture consisted of 500 ng of total DNA from A. orientalis C329.2, 1× Taq buffer, 5% dimethyl sulfoxide, 2 mM MgCl₂, each dNTP at a concentration of 0.4 mM, 1 U of Taq polymerase, and 1 µM primers. The 1.3-kb product was cloned into pGEM-T (Promega), and this clone was used as a template to generate a ³²P-radiolabeled oligonucleotide probe with the Klenow fragment. This probe was used to screen Southern blots of restriction endonuclease-digested A. orientalis C329.2 genomic DNA to identify a 3.5-kb BamHI product containing van genes. This fragment was cloned into pGEM-7Z and was isolated by standard methods.

DNA sequencing. The nucleotide sequence of the S. toyocaensis NRRL 15009 gene cluster was determined with a series of nested deletions constructed by ExoIII-S1 nuclease digestion and through the synthesis of specific oligonucleotide primers. The gene cluster for A. orientalis C329.2 was sequenced by primer walking. DNA cycle sequencing was performed by B. Allore on an Applied Biosystems automated DNA sequencer at the MOBIX central facility at McMaster University.

Analysis of gene clusters in glycopeptide producing organisms. Total DNA was isolated from glycopeptide-producing organisms, and 10 to 20 µg was separated on a 1% TAE (Tris-acetate-EDTA)-agarose gel after digestion to completion with BamHI. The DNA was blotted onto nitrocellulose by standard methods. A vanHAX cluster oligonucleotide probe radiolabeled with ³²P was generated as described above by using the cloned 3.5-kb BamHI fragment from A. orientalis C329.2 as template and primers p3 and p4 (Table 1). This probe was hybridized to genomic DNA restriction products, and the blot was washed under stringent conditions prior to exposure.

PCR amplification of vanHAX from glycopeptide-producing organisms. The presence of the vanHAX cluster in glycopeptide producers was also assessed by amplification of the cluster by PCR. Degenerate oligonucleotide primers complementary to conserved regions in VanH (primer p5) and VanX (primer p2) were designed to amplify nearly the entire cluster (Table 1). The PCR mixtures (100 µl) contained 500 ng of total DNA, 1 U of Taq polymerase, 1× Taq buffer, 5% dimethyl sulfoxide, 4 mM MgCl₂, each dNTP at a concentration of 0.4 mM, and 1 µM primers. Forty cycles of 94, 55, and 72°C (1, 1, and 2 min, respectively) were required to produce a visible band of approximately 2.6 kb. This product was isolated, applied to a 1% TAE agarose gel, and blotted onto nitrocellulose. A ³²P-labeled vanHAX probe was generated from A. orientalis C329.2 total DNA and primers p3 and p4 (Table 1).

Analysis of sequence data. Contiguous sequences were constructed by using the MegAlign software from DNA Star (Madison, Wis.). Open reading frames (ORFs) were screened in all six frames by using the program FRAME (8) to identify probable streptomycete ORFs. Protein sequence alignments were performed by using CLUSTAL W software (39), version 1.7, with the Blosum matrix with a gap penalty of 10 and a gap extension of 0.05.

Nucleotide sequence accession numbers. The nucleotide sequences of vanH_st and ddlM (accession no. U82965), vanX_st (accession no. AF039028), and the gene cluster from A. orientalis C329.2 (accession no. AF060799) have been deposited in GenBank.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Cloning of van genes from S. toyocaensis NRRL 15009 and A. orientalis C329.2. S. toyocaensis NRRL 15009 and A. orientalis C329.2 are gram-positive microbes which produce the "aglyco" glycopeptide antibiotic A47934, a structural homolog of teicoplanin (44), and vancomycin, respectively. We cloned and sequenced an expanse of S. toyocaensis NRRL 15009 DNA spanning 2,650 bp and a 3,470-bp fragment of DNA from A. orientalis C329.2. Each of these contains three ORFs whose products bear striking homology to VanH, VanA (23), and VanX of VRE (Fig. 1). These genes have been designated vanH_st, ddlM (23), and vanX_st, respectively, in S. toyocaensis and vanH_aov, ddlN, and vanH_aov, respectively, in A. orientalis C329.2. (This rather ponderous terminology is necessitated by the fact that various A. orientalis strains produce different glycopeptide antibiotics; thus, the subscript ao designates A. orientalis and the subscript v indicates that it is vancomycin producer.)

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Comparison of glycopeptide resistance gene clusters from VRE and glycopeptide antibiotic-producing organisms.

170

42

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Overlap of vanH and vanA homolog genes.

Sequence analysis of vanH and vanX from glycopeptide producing organisms. The predicted amino acid sequence of VanH_st and VanH_aov clearly identify them as D-LDHs. Amino acid residues which are thought to participate in catalysis based on site-directed mutagenesis studies (21, 37, 38) of D-LDHs, as well as the three-dimensional structure of the Lactobacillus pentosus D-LDH (35), are conserved in VanH_st and VanH_aov. For example, a conserved Arg (Arg237 in VanH_st and Arg255 in VanH_aov) is predicted to bind to the carboxylate and polarize the ketone carbonyl for reduction, and an invariant His (His298 in VanH_st and His316 in VanH_aov) is predicted to act as an active-site general acid donating a proton during pyruvate reduction. Protein sequence alignment of VanH_st and VanH_aov with the VanH enzymes from VRE yielded overall amino acid identities of 51 to 55%, while alignment with other D-LDHs resulted in identities ranging from 26 to 31%. Phylogenetic analysis clearly places all the VanH enzymes in a distinct branch of the D-LDH family (Fig. 3), an observation which parallels the clustering of DdlM and DdlN with VanA and VanB within the D-Ala-D-Ala ligase family (Fig. 4) (23).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Phylogenetic relationship among VanH homologs as calculated by the CLUSTAL W method (39). The tree was generated with the PHYLIP program Drawtree (19). Lact., Lactobacillus.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Phylogenetic relationship among D-Ala-D-Ala ligase homologs as calculated by the CLUSTAL W method (39). The tree was generated with the PHYLIP program Drawtree (19). The four major branches of the tree are labeled A, B, C, and D.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Overlap of the Zn2+ binding regions of VanX proteins. The alignment was as calculated by the CLUSTAL W method (39). The Zn2+ ligands are boxed, and the putative active site base is indicated with an arrow.

Identification of vanHAX gene clusters in other glycopeptide antibiotic-producing organisms. Several other glycopeptide-producing organisms were screened for the presence of the vanHAX genes by two methods. First, a part of the gene cluster from A. orientalis C329.2 was amplified and used to probe total DNA that had been isolated from several glycopeptide producers and digested to completion with BamHI. A single hybridizing band was observed in producers of chloro-eremomycin, ristocetin, vancomycin, and teicoplanin-avoparcin (Fig. 6A), indicating that these organisms harbor similar genes. Second, a PCR screen was developed with degenerate primers complementary to conserved sequences in VanH and VanX. The presence of a vanHAX gene cluster should give rise to an amplified product of approximately 2.7 kb. By using total DNA from several glycopeptide-producing organisms, a band of the expected size was observed in all organisms tested, and the sequence represented by this band was confirmed to encode the vanHAX cluster by Southern hybridization with the A. orientalis C329.2 van cluster probe (Fig. 6B). Thus, this gene cluster appears to be ubiquitously present in glycopeptide-producing bacteria.

View larger version (41K): [in this window] [in a new window]   FIG. 6.   The presence of the vanHAX gene cluster in glycopeptide-producing organisms. (A) Southern blot of complete BamHI digests of A. orientalis 18098 (chloro-eremomycin producer) (lane 1), A. orientalis subsp. lurida (ristocetin producer) (lane 2), A. coloradensis subsp. labeda (teicoplanin and avoparcin producer) (lane 3), and A. orientalis C329.2 (vancomycin producer) (lane 4). The blot was probed with a portion of the ddlN gene amplified from A. orientalis C329.2. (B) Results of PCR amplification of the vanHAX cluster from glycopeptide-producing organism. The lane assignments and the blot probe are the same as described for panel A. Numbers to the left of the blots are in kilobases.

Implications of the van gene cluster in glycopeptide-producing organisms. In addition to the considerable similarity at the amino acid level noted above, the orientation and alignment of the vanH_st, ddlM, and vanX_st genes and the vanH_aov, ddlN, and vanX_aov genes in the glycopeptide producers is identical to the orientation of the homologous genes in VRE (Fig. 1). There are several implications of these findings. First, the fact that glycopeptide producers possess this gene cluster implies that they use a glycopeptide resistance mechanism which is similar to that found in VRE. Second, the presence of the vanX D,D-dipeptidase gene suggests that at some point during growth, the organisms switch from producing the conventional D-Ala-D-Ala peptidoglycan terminus to producing D-Ala-D-Lac. This is supported by our recent isolation of a dedicated D-Ala-D-Ala ligase which is incapable of D-Ala-D-Lac synthesis from S. toyocaensis NRRL 15009 cultures that are not producing the A47934 glycopeptide antibiotic (24).