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

C. A. Arias; J. Peña; D. Panesso; P. Reynolds

doi:10.1093/jac/dkg128

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

C. A. Arias, J. Peña, D. Panesso, P. Reynolds

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

Abstract

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_Δ2-322-) and with plasmids containing fragments encoding either the transmembrane (mvanT_1-323) or racemase (svanT_323-698) 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_Δ2-322) 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_Δ2-322) 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-XY_c-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.

Original language	English (US)
Pages (from-to)	557-564
Number of pages	8
Journal	Journal of Antimicrobial Chemotherapy
Volume	51
Issue number	3
DOIs	https://doi.org/10.1093/jac/dkg128
State	Published - Mar 1 2003

Keywords

Enterococcus
Racemase
Resistance
Serine
Vancomycin

ASJC Scopus subject areas

Pharmacology
Microbiology (medical)
Pharmacology (medical)
Infectious Diseases

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10.1093/jac/dkg128

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@article{077e705d4106436f84a2366084d87569,

title = "Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174",

abstract = "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-TΔ2-322-) and with plasmids containing fragments encoding either the transmembrane (mvanT1-323) 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-TΔ2-322) 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-TΔ2-322) 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, Racemase, Resistance, Serine, Vancomycin",

author = "Arias, {C. A.} and J. Pe{\~n}a and D. Panesso and P. Reynolds",

note = "Funding Information: 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 Jes{\'u}s 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 Pe{\~n}a 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.",

year = "2003",

month = mar,

day = "1",

doi = "10.1093/jac/dkg128",

language = "English (US)",

volume = "51",

pages = "557--564",

journal = "Journal of Antimicrobial Chemotherapy",

issn = "0305-7453",

publisher = "Oxford University Press",

number = "3",

}

TY - JOUR

T1 - Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174

AU - Arias, C. A.

AU - Peña, J.

AU - Panesso, D.

AU - Reynolds, P.

N1 - Funding Information: 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.

PY - 2003/3/1

Y1 - 2003/3/1

N2 - 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-TΔ2-322-) and with plasmids containing fragments encoding either the transmembrane (mvanT1-323) 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-TΔ2-322) 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-TΔ2-322) 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.

AB - 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-TΔ2-322-) and with plasmids containing fragments encoding either the transmembrane (mvanT1-323) 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-TΔ2-322) 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-TΔ2-322) 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.

KW - Enterococcus

KW - Racemase

KW - Resistance

KW - Serine

KW - Vancomycin

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U2 - 10.1093/jac/dkg128

DO - 10.1093/jac/dkg128

M3 - Article

C2 - 12615855

AN - SCOPUS:0037339066

VL - 51

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JO - Journal of Antimicrobial Chemotherapy

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SN - 0305-7453

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ER -

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

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Keywords

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