TABLE 1.

Plasmids and strains used in this study

Plasmid or strainRelevant characteristicsSource or reference
Plasmids
    pMutinCFPB. subtilis integrative vector for C-terminal CFP fusions; Ampr Eryr11
    pMutinGFP+B. subtilis integrative vector for C-terminal GFP fusions; Ampr Eryr11
    pMutinYFPB. subtilis integrative vector for C-terminal YFP fusions; Ampr Eryr11
    pROD17Plasmid containing the mCherry geneD. Sherratt
    pDG792Plasmid containing a kanamycin resistance gene8
    pBCB1-GES. aureus integrative vector for N- and C-terminal GFP fusions; Ampr EryrThis study
    pBCB2-YES. aureus integrative vector for N- and C-terminal YFP fusions; Ampr EryrThis study
    pBCB3-CES. aureus integrative vector for N- and C-terminal CFP fusions; Ampr EryrThis study
    pBCB4-ChES. aureus integrative vector for N- and C-terminal mCherry fusions; Ampr EryrThis study
    pBCB5-YKS. aureus integrative vector for N- and C-terminal YFP fusions; Ampr KanrThis study
    pBCB6-CKS. aureus integrative vector for N- and C-terminal CFP fusions; Ampr KanrThis study
    pBCB7-ChKS. aureus integrative vector for N- and C-terminal mCherry fusions; Ampr KanrThis study
    pBCB8-GKS. aureus integrative vector for N- and C-terminal GFP fusions; Ampr KanrThis study
    pMADE. coli-S. aureus shuttle vector with a thermosensitive origin of replication for Gram-positive bacteria; Ampr Eryr lacZ2
    pDH88Plasmid containing the Pspac promoter and the lacI gene10
    pMAD-spapMAD derivative with the up- and downstream regions of spaThis study
    pBCB13pMAD-spa derivative with Pspac-lacI region from pDH88; Ampr Eryr lacZThis study
    pGEreporterpBCB1-GE with an intergenic region between pbpB and SACOL1491 cloned downstream of the gfp gene; Ampr EryrThis study
    pPBP4t-YFPpBCB5-YK encoding a YFP fusion to the C terminus of truncated PBP4; Ampr KanrThis study
    pPBP4-mChpBCB7-ChK encoding an mCherry fusion to the C terminus of PBP4; Ampr KanrThis study
    pEzrA-CFPpBCB3-CE encoding a CFP fusion to the C terminus of EzrA; Ampr EryrThis study
    pHM232Plasmid containing the tetR-mCherry fusion14
    pBCB13-TetRmChpBCB13 derivative with tetR-mCherry; Ampr Eryr lacZThis study
Strains
    E. coli strain
        DH5αrecA endA1 gyrA96 thi-1 hsdR17 supE44 relA1 f80dlacZDM15Gibco-BRL
    S. aureus strains
        RN4220Restriction-deficient derivative of NCTC8325-4R. Novick
        NCTC8325-4Methicillin-sensitive S. aureus strainR. Novick
        COLMRSA strain7
        RNpGEreporterRN4220 with integrated pGEreporter plasmid expressing GFP in the cytoplasm, under the control of Pspac; EryrThis study
        RNpPBP4t-YFPRN4220 with integrated pPBP4t-YFP plasmid encoding C-terminal PBP4-YFP fusion; KanrThis study
        RNpPBP4-mChRN4220 with integrated pPBP4-mCh plasmid encoding C-terminal PBP4-mCherry fusion; KanrThis study
        RNpEzrA-CFPRN4220 with integrated pEzrA-CFP plasmid encoding C-terminal EzrA-CFP fusion; EryrThis study
        RNpPBP4t-YFP pEzrA-CFPRNpPBP4t-YFP with integrated pEzrA-CFP plasmid encoding C-terminal EzrA-CFP fusion; Eryr KanrThis study
        RN pBCB13-TetRmChRN4220 with integrated pBCB13-TetRmCh; Eryr lacZThis study
        NCTCΔspaTetRmChNCTC8325-4 derivative with spa gene replaced by Pspac-tetR-mCherry-lacIThis study