Overproduction of a 37-kilodalton cytoplasmic protein homologous to NAD super(+)-linked D-lactate dehydrogenase associated with vancomycin resistance in Staphylococcus aureus

We previously reported the isolation of a laboratory-derived Staphylococcus aureus mutant, 523k, that has constitutive low-level resistance to vancomycin (MIC = 5 mu g/ml) and teicoplanin (MIC = 5 mu g/ml) and elaborates a ca. 39-kDa cytoplasmic protein that was not detected in the parent strain 523 (MIC = 1 mu g/ml). We have now detected the protein in strain 523 by immunoblotting with antiserum raised against the protein. Consistent with our initial observations, densitometric analysis of the immunoblots revealed an increased production of the protein in 523k compared with that of the susceptible parent 523. The 5' region of the gene encoding the protein of interest was identified by nucleotide sequencing a PCR product amplified from the genome of 523k with degenerate primers designed to encode the amino acid sequence of proteolytic peptides obtained from the protein. The remainder of the gene was identified by library screening, PCR, and nucleotide sequencing. The gene encodes a 36.7-kDa protein with homology to a family of bacterial NAD super(+)-dependent, D-specific 2-hydroxyacid dehydrogenases which includes both D-lactate dehydrogenase and the enterococcal vancomycin resistance protein VanH and is therefore designated ddh. Increased production of the product of ddh, Ddh, was associated with increased D-lactate dehydrogenase activity in 523k, a finding which suggested that Ddh is likely to be the D-lactate dehydrogenase previously identified in S. aureus. The increased D-lactate dehydrogenase activity in strain 523k and the structural similarities among Ddh, D-lactate dehydrogenase, and VanH suggest that overproduction of Ddh might play a role in vancomycin resistance in this strain.