This plasmid was electroporated into S. aureus RN4220 and then transduced into the relevant strains as described previously. The complementation of isdB was carried out also using plasmid pSK5630. The isdB gene together with its promoter and terminator was amplified by PCR using primer pair RM10 and RM12 introducing SalI and BamHI sites resulting in a 2298-bp fragment. After digestion with SalI and BamHI, the PCR product was cloned into similarly cut pSK5630. A transformant in E. coli selected on Amp-containing growth media containing the correct insert was verified with SalI and BamHI digestion. This plasmid (pRM1012) was transformed into S. aureus RN4220 and then transduced into the relevant strains
as described previously. For growth experiments, overnight cultures of Newman, SH1000, AFH012, and AFH013 selleck chemicals were grown in CLR. These were used to inoculate 50 mL of prewarmed CLR supplemented with appropriate iron sources in a 250-mL flask to an OD600 nm 0.05. The cultures were incubated at 37 °C at 250 r.p.m., and the OD600 nm was measured at regular intervals. Overnight cultures (10 mL) were grown and then 1 mL mixed with 10 mL CLR top agar
(0.7% w/v) and overlaid onto 20 mL CLR agar. Filter paper disks impregnated with different concentrations of hemin, hemoglobin, and iron-loaded lactoferrin were laid on top. The plates Sorafenib clinical trial were incubated at 37 °C overnight, and the halo of growth was measured. Under these conditions, cells would only grow overnight with the addition of an iron source. Ten mice were infected with ≈ 1.5 × 107 CFU Newman or AFH013 by injection into the tail vein, using an established protocol (Clarke et al., 2007). The mice were sacrificed after 7 days, and the CFU in kidneys was calculated
by homogenizing the kidneys in 10 mL PBS and serial dilution. The mean CFU mL−1 for each pair of kidneys was calculated. The P value was calculated using the Mann–Whitney test. The weight of each mouse was recorded Epothilone B (EPO906, Patupilone) every day and the percentage weight loss calculated. The P value was calculated using the Student’s t-test. Cell wall extracts were made as described previously (Clarke et al., 2002). Proteins were separated by 11% w/v SDS-PAGE, then stained with Coomassie blue, or transferred to a polyvinylidene difluoride membrane. Membranes were probed with anti-IsdA, anti-IsdB, or anti-IsdH antibodies raised in a rabbit or mouse (Clarke et al., 2004; Clarke & Foster, 2006) at a 1 : 3000 dilution and blocked with a 5% w/v milk powder solution in PBS. Secondary antibodies were commercially available anti-mouse or anti-rabbit alkaline phosphatase conjugates used at the directed dilutions (Sigma Aldrich). Both the isdB and isdH genes were inactivated using markers that would permit the construction of a multiple mutant strain missing IsdA, IsdB, and IsdH. The three isd mutations were all transformed into the Newman and SH1000 backgrounds to prevent any strain-specific anomalies.