The vanC-3 vancomycin resistance gene cluster of Enterococcus flavescens CCM 439

Ireena Dutta and Peter E. Reynolds*

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK

Received 16 September 2002; returned 4 November 2002; revised 20 December 2002; accepted 20 December 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Enterococcus flavescens CCM 439 is phenotypically similar to Enterococcus casseliflavus; it possesses intrinsic low-level resistance to vancomycin and has the VanC phenotype. The complete vanC-3 vancomycin resistance gene cluster was cloned and sequenced, and found to contain five open reading frames. These encoded five proteins that displayed a high degree of amino acid identity to the proteins of the vanC-2 cluster of E. casseliflavus. The serine racemases displayed the lowest degree of identity (97%), whereas the response regulators VanRC-2 and VanRC-3 were 100% identical. Long-PCR-RFLP analysis of the vanC-3 and vanC-2 gene clusters distinguished E. flavescens CCM 439 from E. casseliflavus ATCC 25788 due to the absence of a single EcoRV restriction endonuclease site from the E. flavescens gene cluster. However, the lack of nucleotide divergence between the sequences of the vanC-2 and vanC-3 clusters casts doubt on the validity of E. flavescens and E. casseliflavus being classed as distinct species.

Keywords: Enterococcus flavescens, vanC-3, vancomycin resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The VanC vancomycin resistance phenotype is possessed by three species of enterococci, Enterococcus gallinarum, Enterococcus casseliflavus and Enterococcus flavescens. They have intrinsic, low-level resistance to the glycopeptide antibiotic vancomycin with MIC values ranging from 2 to 32 mg/L, but remain susceptible to the related antibiotic teicoplanin. Resistance to vancomycin is mediated by the production of cell wall precursors which terminate in D-alanyl-D-serine (D-Ala-D-Ser).1 In the absence of vancomycin, VanC strains that are inducible for resistance synthesize precursors that terminate in D-alanyl-D-alanine (D-Ala-D-Ala).

The vanC-2 vancomycin resistance gene cluster of E. casseliflavus consists of five genes.2 The first three genes of the cluster, which are essential for resistance, encode a D-Ala: D-Ser ligase, VanC-2, followed by VanXYC-2, a protein possessing both D,D-dipeptidase and D,D-carboxypeptidase activities, and a serine racemase, VanTC-2. Expression of the resistance genes is controlled by a two-component regulatory system, which is present downstream of vanTC-2, consisting of a response regulator, VanRC-2 and a histidine kinase, VanSC-2. The proteins encoded within the vanC-2 gene cluster all display a high degree of amino acid identity to the equivalent proteins from the vanC cluster of E. gallinarum. The highest degree of amino acid identity, 91%, was observed between the response regulators and the lowest, 65%, between the serine racemases, VanTC-2 and VanT.2

E. flavescens is a yellow-pigmented species of Enterococcus that has only been identified recently.3 Like E. casseliflavus, which also possesses yellow pigmentation, E. flavescens is motile and possesses intrinsic low-level resistance to vancomycin; it is differentiated from E. casseliflavus only through its inability to produce acid from the fermentation of ribose.3 Since its first identification, some doubt has remained over the validity of describing E. flavescens as a distinct species. Only an internal portion of the vanC-3 D-Ala:D-Ser ligase gene from the vanC-3 cluster has been sequenced previously, and this 500 bp fragment displays 98.3% nucleotide identity to the vanC-2 gene from E. casseliflavus.4

This study describes the sequencing of the complete vanC-3 gene cluster of E. flavescens and comparison with the vanC-2 cluster of E. casseliflavus by sequence analysis and restriction fragment length polymorphisms (RFLPs).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacteria

E. flavescens CCM 439 was originally derived from a clinical isolate and was obtained from the Central Public Health Laboratory, London NW9 5HT, UK.

Three strains of E. casseliflavus were used in the course of this study, ATCC 25788 (the type strain), NCTC 2321 and E93/640. E. casseliflavus E93/640 was also obtained from the Central Public Health Laboratory, and was derived from a clinical isolate. Bacteria were grown in Brain Heart Yeast extract (0.5% w/v) medium (Difco Bacto) at 37°C with gentle shaking and maintained on BHY agar.

Plasmid construction and cloning of the vanC-3 gene cluster by PCR

A PCR approach was used to clone and sequence the genes of the vanC-3 vancomycin resistance gene cluster. Primers were designed using sequence information from the vanC-2 gene cluster of E. casseliflavus.

A 1.6 kb PCR product containing the vanC-3 and vanXYC-3 genes was obtained with primers C1 (5'-CTAAGAGCTCTCGGAAAAGCGGAAGGAAG) and X2 (5'-GTAATCTAGATCATGCGAACTGCCTCGC) and E. flavescens CCM 439 chromosomal DNA as template. Similarly, a 2.0 kb PCR product containing vanTC-3 was obtained using primers T1 (5'-CTAAGAGCTCCTTGAACAAACTGCGAGG) and T2 (5'-GTAATCTAGACTACTTTGAACTAGAGGT). The PCR products were digested with XbaI (Roche Molecular Biochemicals, Mannheim, Germany) and SacI (Roche), purified and ligated with pUC18 digested with the same enzymes to give plasmids pUCF1 and pUCF2, respectively. Plasmid pUCF3, containing vanRC-3 and vanSC-3, was constructed by cloning the 1.7 kb PCR product obtained using primers R1 (5'-CTCAGAGCTCGATCGTAGATAGTTGGAG) and S2 (5'-GTAAGGATCCTTAAGCGGTTGGTTCAGA) into pUC18 after both the product and vector had been digested with SacI and BamHI (Roche). Plasmids were transformed into Escherichia coli XL-1 Blue.

The intergenic region between vanTC-3 and vanRC-3 was amplified by PCR using Pwo polymerase (Roche) and primers T3 (5'-TTGCACCTGATCTTGAGG) and R4 (5'-GTAATCTAGAAGGCCAAAGCGCTCGTTCCA), located at the 3' and 5' ends of vanTC-3 and vanRC-3, respectively, and was sequenced directly. The sequences of vanC-3 and vanXYC-3, of vanTC-3 and of vanRC-3 and vanSC-2 were obtained by sequencing the inserts of plasmids pUCF1, pUCF2 and pUCF3, respectively. Sequencing was carried out by the dideoxy chain terminator method using fluorescent cycle sequencing with dye-labelled terminators (ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit; Perkin-Elmer) on a 373A automated DNA sequencer. Sequence analysis was carried out using the programs of the Genetics Computer Group (GCG) v.9 (Madison, WI, USA).

The complete nucleotide sequence of the vanC-3 gene cluster has been deposited in GenBank under accession number AY033764.

RFLP analysis of long-PCR (L-PCR) amplicons

Genomic DNA was extracted from E. flavescens and E. casseliflavus and 100 ng amounts were used as templates for L-PCR.5 The complete vanC-3 and vanC-2 gene clusters were amplified using primers C1 and S2 and an Expand Long Template PCR System (Roche). Amplification was carried out using the following protocol: (i) 94°C for 2 min; (ii) 10 cycles of 94°C for 20 s, 65°C for 30 s and 68°C for 10 min; (iii) 20 cycles of 94°C for 20 s, 65°C for 30 s and 68°C for 10 min (with the elongation time increased by 20 s/cycle); and (iv) 68°C for 10 min. L-PCR products were digested with 10 U EcoRV and the resulting fragments were separated by electrophoresis through a 0.7% agarose gel containing 0.5 mg/L ethidium bromide. The gel was run in TBE electrophoresis buffer (0.09 mM Tris–HCl, pH 8.0, 0.09 M boric acid, 2 mM EDTA).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Analysis of the vanC-3 vancomycin resistance gene cluster of E. flavescens CCM 439

The vanC-3 vancomycin resistance gene cluster of E. flavescens contained five open reading frames. The genes encoding the vancomycin resistance proteins in the vanC-3, vanC and vanC-2 clusters had the same organization. The proteins encoded by the vanC-3 gene cluster displayed extensive amino acid identity to those encoded by the vanC-2 cluster (Table 1). The lowest degree of amino acid identity, 97%, was observed between VanTC-3 and VanTC-2, and the highest, 100%, between VanRC-3 and VanRC-2. The 99 bp intergenic region between vanTC-3 and vanRC-3 (98 bp in E. casseliflavus) displayed 98.0% nucleotide identity to the intergenic region between vanTC-2 and vanRC-2.


View this table:
[in this window]
[in a new window]
 
Table 1.  Percentage identity between the deduced amino acid sequences of the proteins of the vanC-2 and vanC-3 clusters of E. casseliflavus and E. flavescens, respectively
 
Resistance to vancomycin in E. flavescens CCM 449 was inducible, but, in common with E. casseliflavus, it required several hours of incubation in growth medium containing a low concentration of vancomycin (2 mg/L) before growth resumed (data not shown), indicating that the operon was tightly regulated. The vanRC-3 and vanSC-3 genes were exactly the same length as vanRC-2 and vanSC-2 from E. casseliflavus and were separated by a region of 62 nucleotides, which displayed 100% nucleotide identity over 62 nucleotides to the same region in vanRC-2vanSC-2, although the region from E. casseliflavus contained an additional six nucleotides. The high degree of nucleotide and amino acid identity between the vanC-3 and vanC-2 gene clusters and the proteins they encode demonstrates that there has been little sequence divergence. This lack of nucleotide divergence is not limited to the vancomycin resistance genes. Sequencing of internal portions of the D-Ala:D-Ala ligase and sodA superoxide dismutase genes from E. casseliflavus and E. flavescens revealed that they were 98.5% and 99.5% identical, respectively.4,6 The minimal degree of inter-species sequence divergence was much lower than expected for housekeeping genes from different species and also relative to estimates of sequence diversity between the genomes based on DNA–DNA hybridization experiments.3

The genes of the vanC-3 cluster were considered different from those of the vanC-2 cluster because the host organism was distinct from E. casseliflavus. However, the highest degree of sequence divergence observed between the two gene clusters was only 3.6%, between vanXYC-3 and vanXYC-2. Alleles of other vancomycin resistance genes display greater sequence divergence than the vanC-3 and vanC-2 clusters; vanB3 displays 5% nucleotide divergence from vanB, and vanD4 exhibits 17% sequence divergence from vanD.7,8

L-PCR-RFLP analysis of the vanC-2 and vanC-3 gene clusters of E. casseliflavus and E. flavescens

To evaluate the closeness of the relationship between E. casseliflavus and E. flavescens, the vanC-2 gene clusters of three strains of E. casseliflavus and the vanC-3 gene cluster were amplified by L-PCR to produce fragments of 5.8 kb in each instance. With EcoRV, the three strains of E. casseliflavus all gave rise to the same RFLP (Figure 1). The sizes of the restriction fragments corresponded to the three target sites within the cluster predicted by sequence analysis. The RFLP profile of the vanC-3 L-PCR product of E. flavescens contained only two bands, indicating the absence of a single EcoRV site within the cluster (Figure 1). This led to the production of a restriction fragment of 4 kb instead of one of 1.7 kb and another of 2.3 kb as seen with E. casseliflavus strains.



View larger version (63K):
[in this window]
[in a new window]
 
Figure 1. Separation of DNA fragments after restriction of L-PCR products with EcoRV. Lane 2, MW marker. Sizes of relevant fragments (kb) of the marker are shown on the right-hand side. Digested L-PCR products of E. casseliflavus E93/640, lane 1; E. casseliflavus ATCC 25788, lane 3; E. flavescens CCM 439, lane 4; E. casseliflavus NCTC 2321, lane 5. Sizes of the restriction fragments (kb) are shown to the left of the photograph. The restriction fragment from the E. flavescens strain, not present in the E. casseliflavus strains, is 4 kb in size and is indicated on the right of the photograph. The two bands missing from the E. flavescens profile are indicated by the dashed lines (to the left).

 
Previously, it was shown that DNA from strains of E. flavescens, digested with restriction endonucleases, hybridized to probes based on E. casseliflavus genes, although their hybridization profile was distinct from those of E. casseliflavus isolates.4 These results support the L-PCR-RFLP analysis of the vanC-2 and vanC-3 gene clusters. Other techniques, such as PCR, PFGE and 16S rRNA sequence analysis, were unable to differentiate between E. casseliflavus and E. flavescens.911 RFLP analysis can detect single nucleotide changes that affect the presence or absence of restriction sites, and, as shown here, can differentiate between some strains of the two species. However, further investigation is required to determine whether this difference is found consistently in additional strains of both E. flavescens and E. casseliflavus.

It therefore appears that the allocation of E. flavescens as a species distinct from E. casseliflavus is tenuous. Although a single nucleotide change leading to the absence or presence of a restriction site will affect the number of fragments obtained by L-PCR-RFLP analysis, it seems unlikely that this would be sufficient to differentiate E. flavescens from the many strains of E. casseliflavus that appear to show variation from the type strain. E. flavescens is differentiated from E. casseliflavus on its inability to acidify ribose, but this technique is not always sufficient to identify isolates of the two species correctly.7 It may therefore be more appropriate to describe E. flavescens as a phenotypic variant or subspecies of E. casseliflavus.


    Acknowledgements
 
We thank the Medical Research Council for the award of a research studentship to I.D., and J. Lester and C. Hill, Cambridge Centre for Molecular Recognition, for DNA sequencing and synthesis of oligonucleotides, respectively. P.E.R. thanks the Leverhulme Trust for the award of an Emeritus Fellowship.


    Footnotes
 
* Corresponding author. Tel: +44-1223-333644; Fax: +44-1223-333345; E-mail: per{at}mole.bio.cam.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Reynolds, P. E., Snaith, H. A., Maguire, A. J., Dutka-Malen, S. & Courvalin, P. (1994). Analysis of peptidoglycan precursors in vancomycin-resistant Enterococcus gallinarum BM4174. Biochemical Journal 301, 5–8.[ISI][Medline]

2 . Dutta, I. & Reynolds, P. E. (2002). Biochemical and genetic characterization of the vanC-2 vancomycin resistance gene cluster of Enterococcus casseliflavus ATCC 25788. Antimicrobial Agents and Chemotherapy 46, 3125–32.[Abstract/Free Full Text]

3 . Pompei, R., Berlutti, F., Thaller, M. C., Ingianni, A., Cortis, G. & Dainelli, B. (1992). Enterococcus flavescens sp. nov., a new species of enterococci of clinical origin. International Journal of Systematic Bacteriology 42, 365–9.[Abstract]

4 . Navarro, F. & Courvalin, P. (1994). Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens. Antimicrobial Agents and Chemotherapy 38, 1788–93.[Abstract]

5 . Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidinium thiocyanate. Applied Microbiology Letters 8, 151–6.

6 . Poyart, C., Quesnes, G. & Trieu-Cuot, P. (2000). Sequencing of the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci. Journal of Clinical Microbiology 38, 415–8.[Abstract/Free Full Text]

7 . Patel, R., Uhl, J. R., Kohner, P., Hopkins, M. K., Stenckelberg, J. M., Kline, B. et al. (1998). DNA sequence variation within vanA, vanB, vanC-1 and vanC-2/3 genes of clinical enterococcus isolates. Antimicrobial Agents and Chemotherapy 42, 202–5.[Abstract/Free Full Text]

8 . Dalla Costa, L. M., Reynolds, P. E., Souza, H. A. P. H. M., Souza, D. C., Palepou, M. F. I. & Woodford, N. (2000). Characterisation of a divergent vanD-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil. Antimicrobial Agents and Chemotherapy 44, 3444–6.[Abstract/Free Full Text]

9 . Clark, N. C., Teixeira, L. M., Facklam, R. R. & Tenover, F. C. (1998). Detection and differentiation of vanC-1, vanC-2 and vanC-3 glycopeptide resistance genes in enterococci. Journal of Clinical Microbiology 36, 2294–7.[Abstract/Free Full Text]

10 . Patel, R., Piper, K. E., Rouse, M. S., Steckelberg, J. M., Uhl, J. R., Kohner, P. et al. (1998). Determination of 16S rRNA sequences of enterococci and applications to species identification of non-motile Enterococcus gallinarum isolates. Journal of Clinical Microbiology 36, 3399–407.[Abstract/Free Full Text]

11 . Dutka-Malen, S., Evers, S. & Courvalin, P. (1995). Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. Journal of Clinical Microbiology 33, 24–7.[Abstract]