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-TDelta2-322-) 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-[C-14]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-TDelta2-322) resulted in: (i) vancomycin MICs remaining within susceptible levels (less than or equal to4 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-TDelta2-322) was electroporated into Enterococcus faecalis JH2-2. The uptake of L-[C-14]Ser at 240 s was decreased by similar to40% in BM4175 compared with BM4174. Plasmid pCA10(C-1-XYC-T) restored uptake of L-[C-14]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.