OVERPRODUCTION OF A 37-KILODALTON CYTOPLASMIC PROTEIN HOMOLOGOUS TO NAD(-LINKED D-LACTATE DEHYDROGENASE ASSOCIATED WITH VANCOMYCIN RESISTANCE IA STAPHYLOCOCCUS-AUREUS())

We previously reported the isolation of a laboratory-derived Staphyloc occus aureus mutant, 523k, that has constitutive low-level resistance to vancomycin (MIC = 5 mu g/ml) and teicoplanin (MIG = 5 mu g/ml) and elaborates a ca. 39-kDa cytoplasmic protein that was not detected in t he parent strain 523 (MIG = 1 mu g/ml). We have now detected the prote in in strain 523 by immunoblotting with antiserum raised against the p rotein, Consistent with our initial observations, densitometric analys is of the immunoblots revealed an increased production of the protein in 523k compared with that of the susceptible parent 523, The 5' regio n of the gene encoding the protein of interest was identified by nucle otide sequencing a PCR product amplified from the genome of 523k with degenerate primers designed to encode the amino acid sequence of prote olytic peptides obtained from the protein. The remainder of the gene w as identified by library screening, PCR, and nucleotide sequencing, Th e gene encodes a 36.7-kDa protein with homolog to a family of bacteria l NAD(+)-dependent, D-specific 2-hydroxyacid dehydrogenases which incl udes both D-lactate dehydrogenase and the enterococcal vancomycin resi stance protein VanH and is therefore designated ddh. Increased product ion of the product of ddh, Ddh, was associated with increased D-lactat e dehydrogenase activity in 523k, a finding which suggested that Ddh i s 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, a nd VanH suggest that overproduction of Ddh might play a role in vancom ycin resistance in this strain.