JAC Advance Access originally published online on February 11, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 557-564
© 2003 The British Society for Antimicrobial Chemotherapy
Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174
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
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
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.
Keywords: Enterococcus, vancomycin, racemase, serine, resistance
* Corresponding author. Fax: +571-216-5116; E-mail: caa22{at}cantab.net
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