JAC Advance Access published online on February 11, 2003
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkg128
© 2003 by The British Society for Antimicrobial Chemotherapy
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Original article
1 Bacterial Molecular Genetics
Unit, Centro de Investigaciones, Universidad El Bosque, Transv 9a
Bis No. 133-25, Bogotá, Colombia; Department of Biochemistry, University
of Cambridge, Cambridge, UK
* Corresponding author. E-mail: caa22{at}cantab.net.
Received 16 October 2002
; revised 16 November 2002
; accepted 17 December 2002
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
Keywords: Enterococcus, vancomycin, racemase,
serine, resistance
Role of the transmembrane domain of the VanT serine
racemase in resistance to vancomycin in Enterococcus
gallinarum BM4174
2 Bacterial Molecular Genetics
Unit, Centro de Investigaciones, Universidad El Bosque, Transv 9a
Bis No. 133-25, Bogotá, Colombia
3 Department of Biochemistry, University
of Cambridge, Cambridge, UK
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-T2-322) 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-T2-322) 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.
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