The RM5 and RM6 primers both contained a KpnI site allowing KpnI cut fragments (1282 and

1248 bp, respectively) to be ligated together. The ligation product was used as a PCR template using RM4 and RM7 primers. The resulting 2.5-kbp fragment was cut with XhoI and BamHI and cloned into XhoI and BamHI cut pMUTIN4 (Vagner et al., 1998) resulting in pMUT-LR. Finally, a tetracycline resistance cassette (from pLS1 M. Masalha and S.J. Foster, unpublished) was cloned into the KpnI site in the isdB gene of pMUT-LR creating pMUT-IsdB. Purified plasmid from Escherichia coli was transformed into S. aureus RN4220. selleck chemical Selection was made using Brain Heart Infusion (BHI) medium containing erythromycin and lincomycin, and integration was confirmed by PCR using primer pair RM4 and RM7. Phage lysates were prepared as described previously and transduced into SH1000. Tetracycline-resistant, selleck compound erythromycin/lincomycin-sensitive colonies were selected, and the mutation was confirmed

by PCR and Southern blot (results not shown). The isdH mutant was constructed using the primers shown in Table 1. Briefly, 1-kb fragments of chromosomal DNA flanking the isdH gene were amplified with the primer pairs MOL1313/MOL1314 and MOL1315/MOL1316 with SH1000 genomic DNA used as the template. The MOL1314 and MOL1315 primers both contained a KpnI site allowing KpnI cut fragments to be ligated together. The ligation product was used as a PCR template using MOL1313 and MOL1316 primers. The resulting 2-kbp fragment was cut with SalI and EcoRI and cloned into SalI and EcoRI cut pMAD (Arnaud et al., 2004). Finally, not a kanamycin resistance cassette (from pMAL7, Horsburgh et al., 2001a, b) was cloned into the KpnI site in the isdH gene creating pALX4. Purified plasmid from E. coli was transformed into S. aureus RN4220.

Selection was made using BHI containing kanamycin, and integration was confirmed by PCR using primer pair MOL1313 and MOL1316. Phage lysates were prepared as described previously and transduced into SH1000 and Newman. Kanamycin-resistant erythromycin/lincomycin-sensitive colonies were selected, and the mutation was confirmed by PCR (results not shown). The single marked isdB, and isdH mutations were then transduced into the Newman and SH1000 strain backgrounds alone and in combination with isdA (Clarke et al., 2004) using phage phi 11 as described previously (Novick, 1967). All strains are shown in Table 1. Complementation of isdA was carried out using the plasmid pSRC001 (Clarke et al., 2004). The complementation plasmid for the isdH mutant strain was created using the primers MOL1313 and MOL1316. Briefly, the whole isdH gene including its promoter and terminator was amplified by PCR and ligated into the plasmid pSK5630 using the restriction sites SalI and EcoRI. A transformant in E.