Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174

C. A. Arias1,2,*, J. Peña1, D. Panesso1 and P. Reynolds2

1 Bacterial Molecular Genetics Unit, Centro de Investigaciones, Universidad El Bosque, Transv 9a Bis No. 133–25, 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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enterococcus gallinarum BM4175 (a vancomycin-susceptible derivative of BM4174 obtained by insertional inactivation of vanC-1) was transformed with plasmid constructs pCA10 (containing the genes necessary for resistance, vanC-1-XYc-T), pJP1 (with a fragment lacking the DNA encoding the transmembrane region of VanT, -vanC-1-XYc-T{Delta}2–322-) and with plasmids containing fragments encoding either the transmembrane (mvanT1–322) or racemase (svanT323–698) domains of VanT under the control of a constitutive promoter. Accumulated peptidoglycan precursors were measured in all strains in the presence of L-Ser, D-Ser (50 mM) or in the absence of any growth supplement. Uptake of 0.1 mM L-[14C]serine was also determined in BM4174, BM4175 and BM4175/pCA10. Vancomycin resistance was restored in BM4175 transformed with pCA10(C-1-XYc-T), and the profile of peptidoglycan precursors was similar to wild-type E. gallinarum BM4174. Transformation of E. gallinarum BM4175 with plasmid pJP1(vanC-1-XYc-T{Delta}2–322) resulted in: (i) vancomycin MICs remaining within susceptible levels (<=4 mg/L) in the absence of any growth supplement, but increasing to 8 mg/L when either L-Ser or D-Ser was added to the medium; and (ii) the relative amounts of accumulated UDP-MurNAc-pentapeptide[D-Ser] and tetrapeptide precursors decreasing substantially compared with BM4175/pCA10 and BM4174. The effect on the appearance of tetrapeptide appeared to be host dependent, since a substantial amount was present when the same plasmid construct pJP1(vanC-1-XYc-T{Delta}2–322) was electroporated into Enterococcus faecalis JH2-2. The uptake of L-[14C]Ser at 240 s was decreased by ~40% in BM4175 compared with BM4174. Plasmid pCA10(C-1-XYC-T) restored uptake of L-[14C]Ser at 180 and 240 s in BM4175. The results suggest that the transmembrane domain of VanT is likely to be involved in the transport of L-Ser, and that in its absence the resistance phenotype is compromised.

Keywords: Enterococcus, vancomycin, racemase, serine, resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Low-level resistance to vancomycin in Enterococcus gallinarum, Enterococcus casseliflavus and Enterococcus faecalis is due to the synthesis of peptidoglycan precursors terminating in D-Ser.13 The synthesis of D-alanyl-D-Ser in E. gallinarum requires two proteins: VanT, an unusual membrane-bound serine racemase that converts L-Ser into its D-enantiomer, and VanC-1, a ligase catalysing the synthesis of D-Ala-D-Ser.4 This dipeptide is then utilized for the assembly of peptidoglycan precursors that exhibit reduced affinity for the antibiotic.5,6 The usual synthesis of D-Ala-D-Ala-ending peptidoglycan precursors is shut down by the hydrolysis of D-Ala-D-Ala dipeptides and D-Ala-terminating pentapeptides. This dual function is carried out by the enzyme (D,D-peptidase/D,D-carboxypeptidase) VanXYC.7

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, plasmids and growth conditions

Bacterial strains and plasmids are described in Table 1 and Figure 1. Enterococci were grown in brain–heart infusion (BHI) (Difco Laboratories, Detroit, MI, USA) broth or agar, or in brain–heart 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 Luria–Bertani (LB) (Difco Laboratories) broth or agar with gentamicin (8 mg/L) when containing derivatives of pAT392.13,14 MICs were determined on Mueller–Hinton 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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Table 1.  Strains and plasmids used
 


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Figure 1. Physical map of the chromosome of E. gallinarum BM4174 and plasmid constructions. White arrows represent the sense of transcription. The broken line represents a deletion (codons 2–322) of the 5' region of vanT (encoding the transmembrane domain) in pJP1. Numbers indicate codons deleted (pJP1) or present (pJP3 and pJP4) in the corresponding constructs. Plasmids were constructed by cloning PCR products obtained with oligonucleotides containing SacI and XbaI sites at the 5' and 3' end, respectively, in pAT392.13 Restriction sites are as follows: B, BamHI; E, EcoRI; Hc, HincII; H, HindIII; P, PvuI; Ps, PstI; S, SacI.

 
Plasmid construction and DNA sequencing

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 2–322: vanT{Delta}2–322) (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 323–698) 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{Delta}2–322). 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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Table 2.  Oligonucleotides used as primers for PCR
 
Plasmids pJP2, pJP3 and pJP4 (Figure 1) contained SacI and XbaI fragments cloned into pAT392 under the control of the P2 promoter.14 For pJP2, a fragment of 2.1 kb containing the vanT gene (including its putative RBS) was obtained by PCR amplification using total DNA from E. gallinarum BM4174 as a template with primers E and D (Table 2). For plasmids pJP3 and pJP4, 1.0 and 0.9 kb amplification products encoding the racemase (codons 323–698) (svanT323–698) and membrane domains (codons 1–322) (mvanT1322) of VanT, respectively (Figure 1), were obtained using total DNA from E. gallinarum BM4174 as template. For pJP3, primers F and D were utilized (Table 2).18 The fragment contained in pJP4 was obtained with primers G and H (Table 2). E. gallinarum BM4175 was electrotransformed with all plasmid constructs.17 Plasmids pCA10(vanC-1-XYC-T) and pJP1(vanC-1-XYC-T{Delta}2–322) were also electroporated into E. faecalis JH2-2.

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%).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peptidoglycan precursors in vancomycin-susceptible E. gallinarum BM4175

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 RT–PCR, 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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Table 3.  Analysis of peptidoglycan precursors in E. gallinarum BM4175, E. faecalis JH2-2 and derivatives and MICs of vancomycin
 
Synthesis of UDP-MurNAc-pentapeptide[D-Ser] in E. gallinarum BM4175 was restored by adding D-Ser (50 mM) to the growth medium. However, in the absence of VanXYC activity (D,D-carboxypeptidase/D,D-dipeptidase), indicated by the lack of tetrapeptide (Table 3), the effect on the MIC of vancomycin was minimal (only raised two-fold). A similar phenomenon was observed in E. faecalis JH2-2 (Table 3): this strain was able to synthesize pentapeptide[D-Ser] in the presence of a high concentration of D-Ser. The finding confirms that the D-Ala:D-Ala ligases of both organisms are able to synthesize D-Ala-D-Ser if an appropriate concentration of D-Ser is available.

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(svanT323–698) (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(svanT323–698) and BM4175/pJP2(vanT) (40% versus 17%) was likely to reflect differences in the level of expression of both constructs: pJP3(svanT323–698) 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-T{Delta}2–328) 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{Delta}2–328 was the same as in SVanT323–698. 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{Delta}2–328 in pJP1 was maintained (as in the original sequence of vanXYC and vanT from BM4174),9 and (ii) a different RBS was present in svanT323–698. 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{Delta}2–328) 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{Delta}2–328) 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{Delta}2–328), VanXYC in E. faecalis JH2-2/pJP1(C-1-XYC-T{Delta}2–328) 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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Figure 2. L-[14C]Ser uptake in E. gallinarum BM4174, BM4175 and BM4175/pCA10(C-1-XYC-T). Radioactivity measurements were taken every 30 s up to 120 s and at 180 and 240 s.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amongst the pyridoxal-phosphate (PLP)-dependent alanine and serine racemases, the presence of a transmembrane domain is a unique characteristic of VanT. Previous structural studies have indicated that the protein may contain up to 10 membrane-spanning regions.9 We have suggested that the transmembrane domain of VanT may function as an L-Ser transporter.9 Characterization of serine transporters from E. coli indicated that they were also transmembrane proteins and could use Na+ and H+ to drive amino acid transport.2224 The L-[14C]Ser transport experiments described here support this hypothesis: the only transmembrane protein likely to participate in amino acid transport within the vanC gene cluster is VanT. The uptake of L-[14C]Ser was affected in a time-dependent fashion. The results suggest that, in the absence of the transmembrane domain of VanT and of protein synthesis, the uptake of L-Ser reached a plateau after 2 min (Figure 2). This ‘plateau’ was not reached when the transmembrane domain of VanT was present: uptake of L-[14C]Ser continued to increase for at least 4 min. When the amino acid sequence of the transmembrane domain of VanT was compared with those of the E. coli serine transporters or any other amino acid transporter, no homology was evident. The serine transporter SssT has high sequence identity with YgjU from Haemophilus influenzae, which belongs to the glutamate transporter family.24 Based on structural studies of glutamate transporters, Slotboom et al.11 proposed a ‘signature’ motif for serine transporters: GXLQDSXETALNSSTD. However, this motif was not present in VanT.

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(svanT323–698) (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 SVanT323–698.

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.


    Acknowledgements
 
We thank Michel Arthur for helpful discussions, and J. Lester and C. Hill, Cambridge Centre for Molecular Recognition, for DNA sequencing and synthesis of oligonucleotides, respectively. We are grateful to Patrice Courvalin for the gift of plasmid pAT392 and for reading the manuscript prior to submission, and to Jesús Jaimes for statistical analysis. This work was funded by an International Development Award from the Wellcome Trust. Part of this work was carried out at Cambridge by Julieta Peña with financial support from the School of Dentistry, Universidad El Bosque. P.E.R. thanks the Leverhulme Trust for the award of an Emeritus Fellowship.


    Footnotes
 
* Corresponding author. Fax: +571-216-5116; E-mail: caa22{at}cantab.net Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Billot-Klein, D., Gutmann, L., Sable, S., Guittet, E. & van Heijenoort, J. (1994). Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. Journal of Bacteriology 176, 2398–405.[Abstract]

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