TABLE 1.

Strains and plasmids used in this study

Strain or plasmidProperty(ies)Source or reference
Strains
    Isolate pairs
        JKD6009VSSA strain used for genome sequencing13
        JKD6008VISA strain used for genome sequencing13
        JKD6000Isolate pair 2, VSSA13
        JKD6001Isolate pair 2, VISA13
        JKD6021Isolate pair 3, VSSA13
        JKD6023Isolate pair 3, VISA13
        JKD6052Isolate pair 4, VSSA13
        JKD6051Isolate pair 4, hVISA13
        JKD6004Isolate pair 5, VSSA13
        JKD6005Isolate pair 5, hVISA13
    Others
        RN4220S. aureus strain capable of stably maintaining shuttle plasmids17
        JKD6112In vitro-derived VISA strain (vancomycin MIC of 4 μg/ml) from JKD6009 by incremental broth culturingThis study
        JKD6118In vitro-derived VISA strain (vancomycin MIC of 4 μg/ml) from JKD6009 on vancomycin gradient platesThis study
        JKD6196JKD6009 ΔgraRSThis study
        JKD6205JKD6196 with pCU1This study
        JKD6206JKD6196 with pJKD6151This study
        JKD6207JKD6196 with pJKD6148This study
        JKD6208JKD6009 with point mutation in the graS product, resulting in T136I mutationThis study
        Escherichia coliF endA1 hsdR17(rK mK) thi-1 λ recA1 gyrA96NEB
        DH5αrelA1 rhoA supE44 deoR φ80dlacZΔM15 Δ(lacZYA-argF)U169
Plasmids
    pCU1Apr Cmr, E. coli/S. aureus shuttle-cloning vector2
    pJKD6148pCU1 HindIII Ω (3-kb PCR product from JKD6008 containing the region upstream of SACOL0715 to the region downstream of SACOL0717, generated using primers insertF and insertR); contains the T136I point mutationRecombination
    pJKD6151pCU1 HindIII Ω (3-kb PCR product from JKD6009 containing the region upstream of SACOL0715 to the region downstream of SACOL0717, generated using primers insertF and insertR); does not contain a point mutationRecombination
    pKOR1E. coli/S. aureus shuttle vector for the construction of allelic-exchange mutants3
    pJKD6191pKOR1 with the graRS knockout constructThis study
    pJKD3138pKOR1 with the graRS loci from JKD6008, generated with DAP1367_attB1 and DAP1365_attB2This study