1 Bacterial Molecular Genetics Unit, Centro de Investigaciones, Universidad El Bosque, Transv 9a Bis No. 13325, Bogotá, Colombia; 2 Department of Biochemistry, University of Cambridge, Cambridge, UK
Received 16 October 2002; returned 16 November 2002; revised 16 December 2002; accepted 17 December 2002
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Abstract |
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Keywords: Enterococcus, vancomycin, racemase, serine, resistance
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Introduction |
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Pyridoxal phosphate-dependent alanine and serine racemases have been classified in three distinct groups according to amino acid sequence and structural analysis: (i) alanine racemases and the VanT serine racemase from bacteria; (ii) fungal alanine racemase; and (iii) serine racemase from mammalian brain.8 VanT differs from the other bacterial racemases firstly because it contains a transmembrane domain that is not necessary for racemase activity and also because it is much more effective in racemizing L-Ser than L-Ala; the alanine racemase activity is only 18% of that of serine racemase.9,10 The function and role in vancomycin resistance of the transmembrane domain of VanT are unknown. Amino acid sequence analysis and computer modelling indicate that the protein is likely to possess 10 membrane-spanning segments. Database searches have not yielded any primary sequence homology of the membrane domain with any protein. Interestingly, VanT and members of the glutamate transporter family share certain structural similarities: (i) the presence of 10 predicted membrane-spanning segments and (ii) a serine and threonine-rich stretch in the region located between predicted transmembrane helices 6 and 7 (SLSKT in VanT).11 We report in this paper a functional analysis of the transmembrane domain of VanT, which suggests that it plays a role in resistance to vancomycin in E. gallinarum BM4174, probably functioning in the transport of L-Ser.
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Materials and methods |
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Bacterial strains and plasmids are described in Table 1 and Figure 1. Enterococci were grown in brainheart infusion (BHI) (Difco Laboratories, Detroit, MI, USA) broth or agar, or in brainheart infusion yeast extract (BHY) broth. E. gallinarum BM4175 is a vancomycin-susceptible derivative of wild-type (vancomycin-resistant) E. gallinarum BM4174; the strain was obtained by single crossover insertional inactivation of the vanC-1 gene after electroporation of plasmid pAT217, which contains a 690 bp internal fragment of vanC-1 cloned into pAT114 (which cannot replicate in Gram-positive bacteria). Clones expected to harbour the pAT217 integration into vanC-1 were selected on erythromycin.12 Growth media for BM4175 were supplemented with erythromycin (Sigma, Steinheim, Germany) (8 mg/L). Gentamicin (Sigma), 100 mg/L, was added to the medium for pAT392-containing derivatives of BM4175 and E. faecalis JH2-2. Escherichia coli XL1-Blue was grown in LuriaBertani (LB) (Difco Laboratories) broth or agar with gentamicin (8 mg/L) when containing derivatives of pAT392.13,14 MICs were determined on MuellerHinton agar or in broth (final volume of 4 mL) with an inoculum of 104 cfu/spot and 5 x 105 cfu, respectively. MICs were determined at least three times in the presence of L- or D-Ser (50 mM) or in the absence of any growth supplement.
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E. gallinarum BM4174 total DNA was extracted as described previously.15 Cloning, digestion with restriction endonucleases (Boehringer-Mannheim, Germany), isolation of plasmid DNA (Wizard Plus SV Minipreps, Promega), ligation and transformation were carried out by standard methods.16,17 Plasmid pAT392 was used for all plasmid constructions (Table 1). Plasmid pCA10 containing the vanC-1, vanXYC and vanT genes has been described previously (Figure 1).7 Plasmid pJP1 contained the genes vanC-1, XYC and the 3' end of vanT encoding the soluble domain of VanT, i.e. the region encoding the transmembrane domain was deleted (deletion of codons 2322: vanT2322) (Figure 1). The plasmid was constructed as follows: two separate PCR products were obtained with Pwo polymerase. The first contained vanC-1 (including its ribosomal binding site, RBS) and vanXYC using primers A and B (Table 2).9,18 The second encoded the racemase domain of VanT (codons 323698) and was obtained with primers C and D (Table 2). The two PCR products were purified, mixed, denatured at 95°C and the complementary regions were allowed to anneal at 42°C for 30 min. Pwo polymerase, the corresponding buffer and deoxynucleotides were then added and a PCR assay was performed (40 cycles) with primers A and D, starting with an extension step of 3 min at 72°C. The 2.7 kb PCR product was purified, digested with SacI and XbaI, and cloned into pAT392 under the control of the P2 promoter leading to pJP1(vanC-1-XYC-T
2322). Sequencing of the insert was performed on both strands 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, USA) on a 373A automated DNA sequencer (Perkin-Elmer).19
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Extraction and analysis of peptidoglycan precursors from E. gallinarum BM4174, BM4175, E. faecalis JH2-2 and derivatives
Extraction and analysis of peptidoglycan precursors was performed as described previously.20 Briefly, E. gallinarum, E. faecalis and derivatives were grown in BHY broth supplemented with either vancomycin in BM4174 (to maximize the synthesis of the resistance proteins), erythromycin (BM4175), erythromycin plus gentamicin (BM4175/pAT392 derivatives), gentamicin only (derivatives of E. faecalis JH2-2) or in the absence of any antibiotic (E. faecalis JH2-2). E. gallinarum BM4175, E. faecalis JH2-2 and derivatives were grown in the presence of L-Ser, D-Ser (50 mM) or in the absence of any growth supplement. Ramoplanin (3 mg/L) was added to inhibit peptidoglycan synthesis, and incubation continued for 0.5 mean generation time (19 min) to allow accumulation of peptidoglycan precursors. Erythromycin selection was maintained during growth of E. gallinarum BM4175 and derivatives, since loss of the insertion was observed when experiments were performed in the absence of the antibiotic. Bacteria were harvested, cytoplasmic precursors extracted, desalted on G10 sephadex and analysed by HPLC.
L-Ser transport assays
E. gallinarum BM4174, BM4175 and BM4175/pCA10(C1-XYC-T)18 were grown in BHY broth with the following antibiotics: vancomycin only (4 mg/L) for BM4174, erythromycin only (8 mg/L) for BM4175 and gentamicin (100 mg/L) plus erythromycin (8 mg/L) for BM4175/pCA10(C-1-XYC-T). Bacteria were grown at 37°C until A600 was 0.8. Cells were harvested, washed twice with buffer containing 100 mM Bis-Tris Propane (pH 7.5), 5 mM MgCl2, 0.5% glucose and chloramphenicol (30 mg/L) to inhibit protein synthesis, and resuspended in the same buffer (4 mL). One millilitre of the cell suspension was incubated at 30°C for 2 min and L-[14C] Ser (Amersham, UK) was added to a final concentration of 0.1 mM. Samples (100 µL) were filtered on a glass fibre filter under vacuum using a Millipore multi-filter apparatus at 0, 30, 60, 90, 120, 180 and 240 s and washed with 4 mL of the same buffer. Radioactivity was determined by liquid scintillation counting. Each experiment was performed three times using independent bacterial preparations. The means of radioactivity levels (cpm) at each time interval for the different strains were compared to investigate statistically significant differences using an ANOVA one-way test (a confidence interval of 95%).
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Results |
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The analysis of peptidoglycan precursors in strains of E. gallinarum BM4174, BM4175 and BM4175 transformed with various combinations of the resistance genes, and fragments encoding different domains of VanT, is shown in Table 3. As expected, BM4175 was susceptible to vancomycin and unable to synthesize UDP-MurNAc-pentapeptide[D-Ser] in the absence of any growth supplement (Table 3), indicating that insertional inactivation of vanC-1 had most likely affected the expression of the resistance genes.12 Furthermore, no D,D-peptidase activity catalysed by VanXYC encoded by the gene immediately downstream from vanC-1 was present in cytoplasmic or membrane extracts of this strain (data not shown). The organization of the vanC gene cluster suggested that the resistance genes were transcribed from the same promoter located upstream of the vanC-1 gene.18 This finding has been confirmed by RTPCR, northern blot and hybridization experiments studying the expression of the vanC-1 (D. Panesso and C. A. Arias, unpublished results) and vanC-221 gene clusters. The data indicate that the resistance (vanC-1/2-XYC-1/C-2-TC-1/C-2) and regulatory genes (vanRC-1/C-2SC-1/C-2) are co-transcribed. Our findings suggest that a polar effect on the transcription of the downstream genes (as a consequence of the disruption of vanC-1) is the most likely explanation for the inability of BM4175 to synthesize the resistant precursors.
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Role of the transmembrane domain of VanT on resistance to vancomycin
As shown previously, the racemase domain of VanT is sufficient for enzymic activity.9,10 This result was confirmed when BM4175 was transformed with pJP3(svanT323698) (Table 3). The putative protein product of this construct was predicted to have three additional amino acids at the N-terminal end compared with the construct characterized previously.10 The detection of UDP-MurNAc-pentapeptide[D-Ser] when L-Ser was present at a high concentration in the growth medium indicated that cytoplasmic serine racemase activity could provide sufficient D-Ser for synthesis of peptidoglycan precursors. The difference found in the amount of accumulated pentapeptide[D-Ser] between BM4175/pJP3(svanT323698) and BM4175/pJP2(vanT) (40% versus 17%) was likely to reflect differences in the level of expression of both constructs: pJP3(svanT323698) had the strong putative RBS of the vanC cluster, which is likely to maximize translation of a cytoplasmic protein product. Nonetheless, the effect on the phenotype of both strains was minimal as vancomycin MICs remained at 4 mg/L (Table 3). The findings also indicated that in the absence of a high concentration of L-Ser in the growth medium the presence of a functional D-Ala:D-Ser ligase is likely to be crucial for the synthesis of UDP-MurNAc-pentapeptide[D-Ser] (Table 3).
Transformation of E. gallinarum BM4175 with pJP1-(vanC1-XYC-T2328) lacking the transmembrane domain of VanT was unable to restore vancomycin resistance in the absence of any growth supplement [unlike transformation with pCA10(C-1-XYC-T), which contains the complete resistance gene cluster]. The predicted protein sequence of VanT
2328 was the same as in SVanT323698. The cloning strategy produced two important differences between the two constructs: (i) the overlap between the stop codon of vanXYC and the start codon of vanT
2328 in pJP1 was maintained (as in the original sequence of vanXYC and vanT from BM4174),9 and (ii) a different RBS was present in svanT323698. Deletion of the transmembrane domain affected the resistance phenotype. The most obvious effect was on the expression and/or synthesis of VanXYC: the proportion of accumulated tetrapeptide in BM4175/pCA10(C-1-XYC-T) compared with BM4175/pJP1(C-1-XYC-T
2328) decreased substantially (from 83% to 9%) (Table 3). To investigate whether this alteration was dependent on the host or the cloning strategy, pJP1(C-1-XYC-T
2328) and pCA10(C-1-XYC-T) were electroporated into E. faecalis JH2-2, a vancomycin-susceptible isolate. Unlike in BM4175/pJP1(C-1-XYC-T
2328), VanXYC in E. faecalis JH2-2/pJP1(C-1-XYC-T
2328) appeared to be active, as reflected by the large amount of accumulated tetrapeptide in the absence of any growth supplement, or in the presence of L-Ser (89% and 84%, respectively) (Table 3). However, the lack of detection of UDP-MurNAc-pentapeptide[D-Ser] (Table 3) indicated that synthesis of D-Ser or D-Ala-D-Ser in this strain was very poor. This finding suggests that the deletion of a DNA fragment encoding the transmembrane domain of VanT might affect the expression of the resistance genes of the cluster in a host-dependent manner. Other factors, which appear to be specific for E. gallinarum BM4174, may interact with the transmembrane domain of VanT, or the DNA encoding it, to regulate the expression of the resistance phenotype.
Uptake of L-[14C]Ser by E. gallinarum BM4174, BM4175 and derivatives
The time course of uptake of L-[14C]Ser in E. gallinarum BM4174, BM4175 and BM4175 transformed with pCA10(C-1-XYC-T) is shown in Figure 2. Insertional inactivation of the vanC-1 gene in BM4175 produced a significant decrease in serine uptake as measured from radioactivity levels at 120, 180 and 240 s in three independent experiments (P = 0.005, 0.003 and 0.008, respectively). The average decrease in radioactivity at these time intervals was 40%. When E. gallinarum BM4175 was transformed with pCA10(C-1-XYC-T), serine uptake was restored to the levels of wild-type BM4174 (Figure 2). Statistically significant differences in mean radioactive measurements between BM4175 and BM4175/pCA10(C-1-XYC-T) were obtained at 180 and 240 s (P = 0.0001 and 0.0001, respectively). At 240 s the mean radioactivity level (cpm) of E. gallinarum BM4175/pCA10(C-1-XYC-T) was almost doubled when compared with BM4175. Substantial differences in the early stages of serine uptake (the first 120 s) were also observed (Figure 2): the mean decrease in radioactivity levels at 60 and 90 s in E. gallinarum BM4175 compared with wild-type BM4174 were 40% and 42%, respectively. Differences in radioactivity levels between E. gallinarum BM4175 and BM4175/pCA10(C-1-XYC-T) at similar time points (60 and 90 s) were 29% and 46%, respectively (Figure 2).
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Discussion |
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An alternative interpretation for the plateau reached in L-[14C]Ser uptake is that transport of L-Ser ceases unless D-Ser is incorporated into the cell wall. Nonetheless, important differences between BM4175, BM4174 and BM4175/pCA10(C-1-XYC-T) were also observed in the early stages of L-Ser cell uptake. Since transport of the amino acid is likely to be proceeding in all three strains during the first 90 s, the increased radioactivity levels in early stages observed in BM4174 and BM4175/pCA10(C-1-XYC-T) may reflect the fact that additional serine transporters may be present (namely the transmembrane domain of VanT). The uptake of L-[14C]Ser continues for longer than in BM4175 because serine can be racemized to its D-enantiomer and utilized for wall synthesis.
The addition of a high concentration of D-Ser (50 mM) to the growth medium stimulated the synthesis of D-Ser-ending precursors. The accumulation of UDP-MurNAc-pentapeptide[D-Ser] differed between BM4175/pJP2(vanT) and BM4175/pJP3(svanT323698) (57% versus 30%). This difference could be due to the fact that transport of D-Ser into the cell might also be facilitated by the presence of the transmembrane domain of VanT. Alternatively, accumulation of high concentrations of D-Ser within the cytoplasm could have an inhibitory effect on the racemase activity of SVanT323698.
In summary, we conclude that the transmembrane domain of VanT plays a crucial role in resistance to vancomycin in E. gallinarum BM4174 and suggest that the protein is probably involved in the uptake of L-Ser from the external medium.
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Acknowledgements |
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Footnotes |
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References |
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