A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.
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