Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174.
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 (mvanT(1-322)) 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-[(14)C]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 (< or =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-[(14)C]Ser at 240 s was decreased by approximately 40% in BM4175 compared with BM4174. Plasmid pCA10(C-1-XY(C)-T) restored uptake of L-[(14)C]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.
Arias
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Bacterial Molecular Genetics Unit, Centro de Investigaciones, Universidad El Bosque, Transv 9a Bis No. 133-25, Bogotá, Colombia. caa22@cantab.net
Peña
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Panesso
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Reynolds
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D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D016983:Enterococcus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013293:Enterococcus faecalis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D020713:Vancomycin Resistance / Q000235:genetics; D000099300:Serine Racemase
D001426:Bacterial Proteins; D008565:Membrane Proteins; D019998:Racemases and Epimerases; D000099300:Serine Racemase