Allelic replacement was used to study the role of the two-component regulator (TCR), graRS, in clinical isolates of vancomycin-intermediate Staphylococcus aureus (VISA). Genomic studies in our laboratory, utilising a pair of clinical isolates derived from a patient with S. aureus endocarditis before and after failed vancomycin monotherapy, found a point mutation in graS (T136I) in the VISA daughter strain that was not present in the vancomycin sensitive (VSSA) progenitor. It was hypothesised that this mutation may be responsible for increased tolerance to vancomycin in the VISA daughter strain. Two mutants were generated from the clinical VSSA isolate using the Escherichia coli - S. aureus shuttle vector, pKOR1. The first was a graRS knock-out, the second, an allele swap replacing the wild type graS sequence of the parent strain with the single base substitution generating the T136I mutation from the VISA isolate. Macro-method Etest and vancomycin population analysis profiling were performed to assess differences in vancomycin susceptibility. The results for both methods revealed an increased resistance to vancomycin in the graS T136I mutant, but increased susceptibility in the graRS knock-out. Efficiency of double-crossover allelic exchange using pKOR1 was high (40%, n=20). This study has confirmed a role for the graRS TCR in VISA and demonstrates the utility of pKOR1 for the rapid and efficient construction of unmarked mutations in clinical S. aureus.