Molecular basis for the evolution of species-specific hemoglobin capture by pathogenic Staphylococcus

Jacob E. Choby; Hanna B. Buechi; Allison J. Farrand; Eric P. Skaar; Matthew F. Barber

S. aureus strains were streaked on tryptic soy agar (containing 10 µg/ml chloramphenicol for 395 strains carrying plasmids) and grown at 37°C for 24 h. Single colonies were used to inoculate 3 ml of 396 RPMI containing 1%cas-amino acids and 0.5 mM 2,2'-dipyridyl (Acros/Fisher) to induce expression of 397 chromosomal isdB) or 10 µg/ml chloramphenicol (for strains carrying plasmids with constitutive isdB 398 expression). After 14-16 h of growth at 37°C with shaking, 2 OD 600 units were collected by centrifugation 399 in a 1.5 ml Eppendorf tube. The cell pellet was resuspended with 1 ml PBS or PBS containing 400 recombinant hemoglobin. 10 µg/ml (chromosomal IsdB) or 2.5 µg/ml (plasmid-borne IsdB) of hemoglobin 401 was used. The cells were incubated with hemoglobin or PBS for 30 min at 37°C with shaking, then cells 402 were collected by centrifugation at 4°C. Cells were washed thrice with 1 ml ice-cold PBS, centrifuging at 403 4°C. After the final wash, the cells were resuspended in 30 µl 0.5 M Tris pH 8.0 (Fisher) containing 4% 404 SDS (Fisher) and heated at 90°C for 5 min to remove surface bound proteins. Cells were collected by 405 centrifugation, and eluate was added to 6X loading buffer and heated at 90°C for 5 min. Samples were 406 subjected to 12 or 17.5% SDS-PAGE and silver stained (GE, Boston, MA). Quantification was performed 407 All rights reserved. No reuse allowed without permission.