Transcriptional Analysis of the vanC Cluster from Enterococcus gallinarum Strains with Constitutive and Inducible Vancomycin Resistance

Abstract

The vanC glycopeptide resistance gene cluster encodes enzymes required for synthesis of peptidoglycan precursors ending in d-Ala-d-Ser. Enterococcus gallinarum BM4174 and SC1 are constitutively and inducibly resistant to vancomycin, respectively. Analysis of peptidoglycan precursors in both strains indicated that UDP-MurNAc-tetrapeptide and UDP-MurNAc-pentapeptide[d-Ser] were synthesized in E. gallinarum SC1 only in the presence of vancomycin (4 μg/ml), whereas the “resistance” precursors accumulated in the cytoplasm of BM4174 cells under both inducing and noninducing conditions. Northern hybridization and reverse transcription-PCR experiments revealed that all the genes from the cluster, vanC-1, vanXY_C, vanT, vanR_C, and vanS_C, were transcribed from a single promoter. In the inducible SC1 isolate, transcriptional regulation appeared to be responsible for inducible expression of resistance. Promoter mapping in E. gallinarum BM4174 revealed that the transcriptional start site was located 30 nucleotides upstream from vanC-1 and that the −10 promoter consensus sequence had high identity with that of the vanA cluster. Comparison of the deduced sequence of the vanS_C genes from isolates with constitutive and inducible resistance revealed several amino acid substitutions located in the X box (R200L) and in the region between the F and G2 boxes (D312N, D312A, and G320S) of the putative sensor kinase proteins from isolates with constitutive resistance.

Vancomycin is a glycopeptide antibiotic that binds to the d-alanyl-d-alanine (d-Ala-d-Ala) terminus of peptidoglycan precursors, inhibiting the transglycosylation and transpeptidation reactions of cell wall synthesis (28). Glycopeptide resistance in enterococci results from replacement of the last d-Ala residue of the UDP-MurNAc pentapeptide precursors by d-lactate or d-serine (6, 9) and the destruction of normal d-Ala-ending precursors (4, 25, 26). High-level resistance to glycopeptides is characteristic of the VanA, VanB, and VanD phenotypes (which synthesize d-Lac-ending peptidoglycan precursors) (6, 23). Low-level resistance to vancomycin is typical of the VanC, VanE, and VanG phenotypes (9, 15, 20, 27). The common feature of the latter types of resistance is the production of UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ser for peptidoglycan synthesis (11).

The vanC-1 gene cluster of E. gallinarum BM4174 consists of five genes: vanC-1, vanXY_C, vanT, vanR_C, and vanS_C. The genes encode proteins that are involved in the synthesis of d-Ser and its incorporation into the growing peptidoglycan chain (VanC-1 and VanT), the hydrolysis of d-Ala-ending precursors [VanXY_C], and proteins which exhibit structural homology with members of two component regulatory systems [VanR_C and vanS_C] (2, 5) that are likely to be involved in regulation of the cluster.

Expression of the vanA and vanB gene clusters is also controlled by two-component regulatory systems (5, 14, 32). The resistance and regulatory genes in these clusters are transcribed from two promoters that appear to be regulated in a coordinated fashion (3, 14, 32). Amino acid substitutions affecting VanS (3) or VanS_B enzymatic activity are usually responsible for constitutive expression of the operon (7, 8, 12).

E. gallinarum isolates are constitutively or inducibly resistant (17, 29). Sequence and transcriptional analysis of the vanE gene cluster (1) provided evidence that, unlike VanA- and VanB-type strains, the genes for resistance and regulation are transcribed from a single promoter. We have studied transcription of the vanC cluster in two isolates of E. gallinarum that express vancomycin resistance in either a constitutive (BM4174) or inducible (SC1) manner. Our results confirm that the vanC operon is transcribed from a single promoter and is tightly regulated in isolates with inducible resistance. Sequence comparison of the vanS_C sensor kinase genes between isolates with constitutive and inducible resistance revealed mutations that may affect the enzymatic activity of isolates with constitutive resistance.

MATERIALS AND METHODS

Strains, plasmids, and growth curves.

The bacterial strains and plasmids used in this work are described in Table 1. Enterococci were grown in brain heart infusion (BHI) broth or on agar (ICN, Biomedicals, Inc, Madison, Wis.) at 37°C. Inducible and constitutive expression of vancomycin resistance was studied in 15 clinical isolates of E. gallinarum. Following overnight incubation in BHI broth (in the presence and absence of vancomycin at 4 μg/ml), 1.5 ml of each culture was inoculated into two flasks containing 60 ml of BHI broth. When the A₆₀₀ reached 0.2 to 0.3, vancomycin (4 μg/ml) was added to one of the flasks to induce expression of resistance. The A₆₀₀ in each culture was determined hourly for up to 6 h, and growth curves were compared.

TABLE 1.

Strains and plasmids

Strain or plasmid	Relevant characteristicsa	MIC of vancomycin (μg/ml)	Reference or sourceb
E. gallinarum
BM4174	Constitutive resistance	32	13
SC1	Inducible resistance	16	17
CA4176	Inducible resistance	8	CPHL (UK)
CA4177	Inducible resistance	16	CPHL (UK)
CA4178	Inducible resistance	8	CPHL (UK)
CA4179	Inducible resistance	16	CPHL (UK)
CA4180	Inducible resistance	8	CPHL (UK)
CA4181	Constitutive resistance	16	CPHL (UK)
CA4182	Constitutive resistance	16	CPHL (UK)
CA4183	Inducible resistance	16	CPHL (UK)
CA4184	Constitutive resistance	8	UAA (France)
CA4185	Constitutive resistance	32	UAA (France)
CA4186	Constitutive resistance	16	UAA (France)
CA4187	Constitutive resistance	32	UAA (France)
CA4189	Inducible resistance	16	CPHL (UK)
E. coli Top10	F− [lacIq Tn10(Tcr)] mcrA Δ (mrr-hsdRMS mcrBC) φ80lacZΔM15ΔlacX74 recA araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG		Invitrogen
Plasmids
pCR2.1	Apr Kmr; lacZα oriR from ColE1		Invitrogen
pDP1	0.8-kb PCR product containing intergenic region between orf1 and vanC-1 (PC-I) cloned into pCR2.1		This work

^aAbbreviations: Tc^r, tetracycline resistance; Str^r, streptomycin resistance; Ap^r, ampicillin resistance; Km^r, kanamycin resistance.

^bUAA, Unité des Agents Antibactériens, Institute Pasteur, France; CPHL, Central Public Health Laboratory, London, United Kingdom.

Escherichia coli Top 10 (Invitrogen, Groningen, The Netherlands) was grown in Luria-Bertani (LB) broth or on agar supplemented with ampicillin (100 μg/ml) when harboring derivatives of pCR2.1.

Analysis of peptidoglycan precursors.

Peptidoglycan precursors from enterococci were extracted and analyzed as described (21). Briefly, strains were grown in BHI medium in the presence or absence of vancomycin (4 μg/ml). When the A₆₀₀ reached 1.0, ramoplanin (3 μg/ml) was added to inhibit peptidoglycan synthesis and incubation was continued for 0.5 generation time (≈19 min) to allow cytoplasmic accumulation of cell wall precursors. Bacteria were harvested and cytoplasmic precursors were extracted, desalted on a G10 Sephadex column, and analyzed by high-pressure liquid chromatography.

DNA manipulation and sequencing.

Total DNA from E. gallinarum was extracted as described (24). Cloning, digestion with restriction endonucleases, ligation, and transformation were carried out by standard methods (30). The intergenic region between orf1 and vanC-1 (Fig. 1A) in E. gallinarum BM4174 was amplified by PCR with primers A and B (Table 2) with total DNA as the template and Taq polymerase (Amersham, Pharmacia Biotech, Buckinghamshire, England). The resulting 0.8-kb fragment was digested with EcoRI and ligated into plasmid pCR.2.1 DNA (Table 1) digested similarly and transformed into E. coli Top 10. The recombinant plasmid pDP1 was purified with a commercial kit (Wizard Plus SV Minipreps; Promega, Madison, Wis.) and used for DNA sequence comparison during primer extension analysis (see below).

FIG. 1.

Schematic representation of the vanC-1 gene cluster from BM4174. Map of 5.5 kb containing the vanC-1, vanYX_C, vanT, vanR_C, and vanS_C genes. Open arrows represent coding sequences and indicate the direction of transcription. (A) Fragment cloned into pDP1 containing the intergenic region between orf1 and vanC-1. (B) Probes used in RT-PCR and Northern hybridization experiments. (C) Oligonucleotide PE1 used in primer extensions. (D) DNA sequenced in the clinical isolates of E. gallinarum.

TABLE 2.

Primers used

Primer	Sequence	Positiona
A	5′-CTCCATGACTGTGATGGC	813
B	5′-CTAGCCAAGTATCATTGC	1600
PE1	5′-CTAGCCAAGTATCATTGCGAACATTCGCGAGG	1600
D1	5′-GGAGGTATGGAAATGTG	5140
E1	5′-TCAGTTGTTGGCAGGTT	6963
F1	5′-GATCGGTTATGGGCAAAC	4789
G1	5′-GGCTGCACACTAAATCC	5284
C1	5′-AATTCTCCAGAATACTCAG	1441
LG	5′-CGTTTGTCCTCCTCTGCCAG	2438
X1	5′-CTTAGTGCTAGCTCCTTTTAG	2501
CP	5′-CAGTGATGACTTGGCTATGG	2946
T1	5′-TATTTCCGCTGATCTTGCGC	3774
SR	5′-CTGCCGAACCAAGATGAG	4496
R1	5′-AAATTGCGGACTTAGTCACG	5230
RR	5′-TGTGCCATGACAGTATTATTG	5794
S2	5′-GAATTCTTCGAGATCACA	6457
RB1b	5′-GGAGGAAGGTGGGGATGACG	1173
RB2b	5′-ATGGTGTGACGGGCGGTGTG	1394

^aNumber corresponds to the position of the 5′ end of the priming oligonucleotides in the sequence of E. gallinarum BM4174 (GenBank accession number AF162694).

^bCorresponds to GenBank accession number J01859.

The vanS_C genes from 15 clinical isolates of E. gallinarum (Table 1) were amplified with total DNA from each strain as a template. PCR with Pwo polymerase (Boehringer, Mannheim, Germany) was performed with primers D1 and E1 (Table 2) (Fig. 1D). The 2.1-kb amplification products were sequenced on both strands by the dideoxynucleotide chain termination method (31) with fluorescent cycle sequencing with dye-labeled terminators (ABI Prism Dye terminator cycle sequencing ready reaction kit; Perkin-Elmer) on a 373A automated DNA sequencer (Perkin-Elmer). Sequence alignment was performed with ClustalW (33).

RNA manipulation and Northern hybridization.

E. gallinarum BM4174 and SC1 were grown in the absence or presence of vancomycin (4 μg/ml) to an A₆₀₀ of 0.8, and RNA was extracted with standard methods (16). Total RNA was digested with RQ1 RNase-free DNase I (5 U/μg of RNA) (Promega), and reverse transcription (RT)-PCR was performed with the Access RT-PCR kit (Promega). Briefly, synthesis of cDNA and amplification were carried out in a single mixture with 2 μg of purified total RNA in a final volume of 50 μl containing avian myeloblastosis virus reverse transcriptase and Tfl polymerase, the corresponding buffers, and 1 μM each of the primers. The fragments corresponded to cDNA fragments internal to vanC-1, vanXY_C, vanT, vanR_C, and, vanS_C (Fig. 1B). An additional RT-PCR was performed with primers F1 and G1 (Table 2) directed at detecting cDNA spanning the 3′ end of vanT and the 5′ end of vanR_C (Fig. 1B). A primer pair which directed synthesis of a 222-bp conserved region of 16S rRNA genes (RB1 and RB2) (Table 2) was used as an internal control for RT-PCR with RNA as a template and extracted from E. gallinarum SC1 grown in the absence of vancomycin.

Total RNA was quantified by spectrophotometric analysis at A₂₆₀. Total RNA (20 μg) extracted from E. gallinarum grown under inducing and noninducing conditions was subjected to electrophoresis and transferred to a nylon membrane (Hybond N+, Amersham Pharmacia Biotech) with standard methodology (30). DNA probes internal to every gene of the vanC cluster, obtained by PCR and labeled (Megaprime DNA labeling system, Amersham Pharmacia Biotech) with [α-³²P]dCTP (Amersham Pharmacia Biotech), were used in hybridization experiments under stringent conditions as described (30). The size of the transcripts was determined with RNA molecular size markers (Boehringer).

Primer extension.

The 5′ end of the PE1 oligonucleotide (Fig. 1C; Table 2) was labeled with [γ-³²P]ATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (Amersham Pharmacia, Biotech). After phenol-chloroform extraction, the labeled PE1 oligonucleotide was precipitated with ethanol and resuspended in sterile water to a final concentration of 1 pmol/μl. Labeled PE1 was annealed to 50 μg of total RNA at 65°C for 3 min, and extension was performed with 40 U of Moloney murine leukemia virus modified reverse transcriptase (Superscript II; Gibco Life Technologies, Rockville, Md.) in a final volume of 20 μl for 45 min at 50°C. The reverse transcriptase products were analyzed by electrophoresis in 6% polyacrylamide-urea sequencing gels. A sequencing reaction (dideoxynucleotide chain terminator method) (31) was performed with the same primer with plasmid pDP1 DNA (Fig. 1A, Table 1) as a template. The samples were run in parallel to determine the endpoints of the extension products.

RESULTS AND DISCUSSION

Expression of vancomycin resistance in clinical isolates of E. gallinarum.

Expression of resistance was induced by the presence of vancomycin in eight clinical isolates of E. gallinarum: the average time for growth to recover after addition of subinhibitory concentrations of vancomycin was about 4 h (data not shown). The remaining seven isolates expressed resistance constitutively, since addition of vancomycin did not affect growth (Table 1).

Peptidoglycan precursors from E. gallinarum BM4174 and SC1.

Analysis of the peptidoglycan precursors of E. gallinarum SC1 grown in the absence of vancomycin revealed that only UDP-MurNAc-pentapeptide[d-Ala] was present. The profile of precursors changed after exposure of SC1 to subinhibitory concentrations of vancomycin (4 μg/ml) (Table 3): UDP-MurNAc-tetrapeptide represented the main precursor accumulated (75%). UDP-MurNAc-pentapeptide[d-Ser] accounted for the remaining 25% of the precursors, and UDP-MurNAc-pentapeptide[d-Ala] was not detected. These results confirm that expression of the resistance genes in E. gallinarum SC1 is inducible, as reported previously (17). In contrast, the profile of peptidoglycan precursors in E. gallinarum BM4174 grown both in the presence and in the absence of vancomycin was similar, 78 to 74% UDP-MurNAc-tetrapeptide and 22 to 26% UDP-MurNAc pentapeptide[d-Ser], respectively (Table 3). No pentapeptide[d-Ala] was found. This indicates that expression of the vanC gene cluster in E. gallinarum BM4174 was constitutive.

TABLE 3.

Peptidoglycan precursors of E. gallinarum BM4174 and SC1

Strain	Vancomycin in medium	Peptidoglycan precursors (%)a
		UDP-MurNAc-tetrapeptide	UDP-MurNAc-pentapeptide[Ser]	UDP-MurNAc-pentapeptide[Ala]
E. gallinarum BM4174	Yes	78	22	0
	No	74	26	0
E. gallinarum SC1	Yes	75	25	0
	No	0	0	100

^aDetermined by HPLC from integrated peaks areas.

Transcriptional analysis of the vanC gene cluster.

Total RNA from E. gallinarum BM4174 and SC1 was extracted from induced and uninduced cultures in the exponential phase of growth. For transcriptional analysis, Northern hybridization and RT-PCR experiments were performed with probes and primers complementary to specific regions of the five genes of the cluster (Fig. 1B, Table 2). For E. gallinarum BM4174, a band of approximately 5,500 bases was observed after hybridization with the five probes, vanC-1, vanXY_C, vanT, vanR_C, and vanS_C, when RNA was extracted after growth in the presence or absence of vancomycin (Fig. 2). The size of the transcript was consistent with synthesis of a single mRNA encoding the five proteins of the cluster. These results were confirmed by RT-PCR with primers directed to specific regions of each member of the cluster (Fig. 3A). In addition, when a region between vanT (a resistance gene) and vanR_C (a regulatory gene located downstream from vanT) was amplified by RT-PCR, the expected band was obtained under inducing and noninducing conditions (Fig. 3A, line 8).

FIG. 2.

Transcription analysis of the vanC-1 gene cluster by Northern hybridization. Total RNA extracted from E. gallinarum BM4174 and SC1 grown in the presence (4 μg/ml) or absence of vancomycin was hybridized with probes corresponding to the five genes of the cluster: A, vanC-1; B, vanXY_C; C, vanT; D, vanR_C; and E, vanS_C. Lanes 1 and 2, E. gallinarum BM4174 with and without vancomycin, respectively; lanes 3 and 4, E. gallinarum SC1 with and without vancomycin, respectively. The size of the transcripts was determined according to RNA molecular size markers (Boehringer) (not shown).

FIG. 3.

Transcription analysis of the vanC cluster by RT-PCR. (A) Agarose gel electrophoresis of RT-PCR products with RNA from E. gallinarum BM4174 (grown in the absence of vancomycin) as the template. Lane 1, molecular size markers; lane 2, control without avian myeloblastosis virus reverse transcriptase; lane 3, vanC-1; lane 4, vanXY_C; lane 5, vanT; lane 6, vanR_C; lane 7, vanS_C; lane 8, fragment between the 3′ end of vanT and the 5′ end of vanR_C. (B) Same as A but with RNA from E. gallinarum SC1 grown in the absence of vancomycin as the template and adding primers directed to an internal fragment (222 bp) of the 16S rRNA gene (rrs). Lane 1, molecular size markers; lane 2, control without avian myeloblastosis virus reverse transcriptase; lane 3, vanC-1; lane 4, vanXY_C; lane 5, vanT; lane 6, vanR_C; lane 7; vanS_C; lane 8, vanT-R_C.

Since our experiments indicated that the resistance and regulatory genes were cotranscribed, primer extension analysis was carried out on DNA located upstream from vanC-1 (designated P_C-1) in E. gallinarum BM4174 (Fig. 1A). The results suggested that the transcriptional start site was located 30 nucleotides upstream from the start codon of vanC-1 (Fig. 4). The putative −35 (CCGCAA) and −10 (TACACT) sequences displayed limited similarities with σ⁷⁰ consensus promoter sequences of E. coli (18).

FIG. 4.

Identification of the transcriptional start site for the vanC-1 gene in E. gallinarum BM4174 by primer extension analysis in the absence of induction. Left panel: lane 1, primer elongation product obtained with oligonucleotide PE1 and 50 μg of total RNA from BM4174 (arrowhead). Lanes T, G, C, and A are the results of sequencing reactions performed with the same primer. Right panel: +1 transcriptional start site for the vanC cluster; the −35 and −10 promoter sequences upstream from the start site are in boldface. The ATG start codon of vanC-1 is indicated by a bent arrow, and the ribosome-binding site (RBS) is in boldface and underlined.

Transcriptional analysis of operons with a structural organization identical to that of the vanC cluster (e.g., vanE) (1) yielded similar results: the resistance and regulatory genes are cotranscribed from a single promoter. These findings confirm that transcriptional regulation differs substantially between vancomycin resistance operons. Gene clusters from isolates expressing high-level resistance to vancomycin and synthesizing d-Lac-ending precursors have the regulatory genes located upstream from the resistance genes and are transcribed from two independent promoters that act in a coordinated fashion to express resistance (5, 10, 14). By contrast, VanC-1, VanC-2, and VanE strains synthesize peptidoglycan precursors ending in d-Ser and express low-level resistance to vancomycin (9, 15, 20). In these isolates the regulatory genes are located downstream from the resistance genes. On the other hand, the vanG operon includes genes recruited from various van operons. In these isolates the regulatory genes are located at the 5′ end of the cluster and are transcribed from a constitutive promoter. The resistance genes are at the 3′ end, and their transcription is regulated by an inducible promoter (11).

In Northern hybridization experiments, additional bands corresponding to 2,900 bases and 1,500 bases were also noted in both E. gallinarum BM4174 and SC1 under inducing and noninducing conditions (Fig. 2). These transcripts were not identified when a probe directed to the 16S rRNA was used as the control. In E. gallinarum SC1, which expresses inducible vancomycin resistance, Northern hybridization yielded results similar to those for BM4174 when RNA was extracted after growth in the presence of vancomycin (Fig. 2). RT-PCR performed under inducing conditions confirmed expression of the genes (data not shown). However, no transcripts were identified when RNA was extracted after growth in the absence of vancomycin (Fig. 2). Moreover, RT-PCR yielded no amplification products under noninducing conditions (Fig. 3B). These results confirm that regulation of synthesis of the resistance proteins occurs at the transcriptional level.

Our data support the notion that although transcriptional regulation of the different vancomycin resistance clusters may vary considerably, the actual activation mechanism could be similar at the molecular level. In fact, the transcriptional regulator VanR_C shares almost 50% identity with VanR (2).

Amino acid sequence comparison of VanS_C from clinical isolates of E. gallinarum.

Several differences in the amino acid sequence of the sensor histidine kinase protein from two-component regulatory systems that control the expression of vancomycin resistance in VanA- and VanB-type strains have been associated with the conversion of an inducible to a constitutive phenotype. This family of proteins has specific amino acid motifs that are conserved among all members (19, 22). The H, N, X, G1, F, and G2 boxes are associated with crucial enzymatic activities (19). In particular, the N, G1, F, and G2 boxes border a unique ATP binding pocket located towards the C terminus of the protein (19).

To determine if amino acid substitutions in the putative VanS_C proteins could be associated with the expression of the phenotype, the corresponding genes in eight inducible and seven constitutive clinical isolates of E. gallinarum were sequenced in both strains. Several substitutions in VanS_C were found: four of them (Arg-200→Leu, Asp-312→Asn or Ala, and Gly-320→Ser) were present exclusively in isolates with constitutive resistance (Fig. 5). They were also the only amino acid differences found when the predicted amino acid sequences were compared to that of the putative VanS_C proteins from the majority of isolates with inducible resistance. They were located in the catalytic (receiver) domain of VanS_C. The Arg-200→Leu change was found in three isolates (CA4185, CA4186, and CA4187) and was located between the H and G1 domains (the X box) (Fig. 5).

FIG. 5.

Schematic representation of vanS_C and localization of amino acid substitutions in E. gallinarum isolates with constitutive resistance. TM1 and TM2 represent the putative membrane-associated sensor domains containing two stretches of hydrophobic amino acids. The putative catalytic ATP binding domain contains amino acid motifs (H, X, N G1, F, and G2) that are conserved in the histidine kinases are shown. The putative linker is located between the second transmembrane segment and the catalytic ATP binding domain. Numbers correspond to the sequence of E. gallinarum BM4174 (2).

Mutational studies of EnvZ indicate that mutations in this region disrupt phosphatase activity (19). The substitutions Asp-312→Asn and Asp-312→Ala were immediately adjacent to the F box (Fig. 5), which is important for kinase activity (19). The Gly-320→Ser substitution flanked the G2 box. Amino acid changes or deletions in the G2 box have been shown to affect autokinase, kinase, and phosphatase activities, demonstrating the importance of this region for all three activities (12, 19). Further work to elucidate the role of these mutations in regulation of gene expression is in progress.