It is likely that clinically isolated heme-auxotrophic SCVs are able to obtain heme from the host via heme transport systems, which may contribute to the pathogenesis and persistence of these strains. Characterization of a heme-auxotrophic, heme transport–defective mutant in appropriate in vivo infection models would enable the contribution of heme transport in these SCVs to be assessed. With this in mind, we set out to construct a ΔhemBΔhtsAΔisdE S. aureus strain to investigate the role of heme acquisition via these transport systems in a heme-auxotrophic SCV. Characterization of this strain in vitro demonstrates that S. aureus is still able to acquire heme

added to the growth medium in the form of either hemin or hemoglobin in the absence of both htsA and isdE. This learn more lends support to the hypothesis that the Hts system is responsible only for the transport of staphyloferrin A and contradicts the argument that IsdE AZD4547 may transfer heme to the HtsBC permease (Hammer & Skaar, 2011). Furthermore, these data strongly suggest that additional, as yet uncharacterized, heme transport system components operate in S. aureus. This may take the form of an additional lipoprotein that is able to transport heme in conjunction with

HtsBC or IsdDF, or possibly another transport system altogether. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was grown on Luria–Bertani (LB) agar or in LB broth, supplemented with 100 μg mL−1 ampicillin and 10 μg mL−1 chloramphenicol where appropriate, at 37 °C under aerobic conditions. Staphylococcus aureus was cultured on tryptone soy agar (TSA) or in tryptone soy broth (TSB), supplemented with 10 μg mL−1 chloramphenicol where required, at 37 °C under aerobic conditions. Gene deletion mutants were constructed in S. aureus LS-1 according to the method of Bae and Schneewind (Bae & Schneewind,

2006). DNA fragments flanking the gene of interest of S. aureus LS-1 were amplified by PCR using primers listed in Table 2 and cloned into the vector pKOR1 in E. coli DH5α. Staphylococcus aureus RN4220 was used to passage plasmids prior Branched chain aminotransferase to transformation of target S. aureus strains. Double- and triple-deletion mutant strains were constructed by sequential allelic replacement using the plasmid constructs listed in Table 1. Gene deletions were confirmed by PCR amplification and DNA sequencing using the primers listed in Table 2, which flank the manipulated regions. The hemB gene was amplified by PCR from S. aureus LS-1 genomic DNA using primers JAW418 and JAW419 (Table 2) to yield a product of 996 bp, then purified, and digested with BamHI and XbaI. Plasmid pSK236 was digested with SacI and XbaI, and pHCMC05 was digested with BamHI and SacI to excise the Pspac promoter.