MUTATIONS LEADING TO INCREASED LEVELS OF RESISTANCE TO GLYCOPEPTIDE ANTIBIOTICS IN VANB-TYPE ENTEROCOCCI

The vanB gene cluster mediates glycopeptide resistance by production o f peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lacta te (D-Ala-D-Lac) instead of D-Ala-D-Ala found in susceptible enterococ ci. Synthesis of D-Ala-D-Lac and hydrolysis of D-Ala-D-Ala is controll ed by the VanR(B)S(B) two-component regulatory system that activates t ranscription of the resistance genes in response to vancomycin but not to teicoplanin. Two substitutions (A30-->G or D168-->Y) in the VanS(B ) sensor kinase resulted in induction by teicoplanin, indicating that the N-terminal domain of the protein was involved in glycopeptide sens ing. A substitution (T237-->K) located in the vicinity of the putative autophosphorylation site of VanS(B) (H233) was associated with a cons titutive phenotype and affected a conserved residue known to be critic al for the phosphatase activity of related kinases. A mutant producing an impaired host D-Ala:D-Ala ligase required vancomycin for growth, s ince D-Ala-D-Lac was only produced under inducing conditions. The ddl and vanS(B) mutations, alone or in combination, resulted in various re sistance phenotypes that were determined by the amount of D-Ala-D-Ala and D-Aka-D-Lac incorporated into peptidoglycan precursors under diffe rent inducing conditions.