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. Author manuscript; available in PMC: 2010 Sep 22.
Published in final edited form as: Mol Microbiol. 2009 May;72(3):763–778. doi: 10.1111/j.1365-2958.2009.06684.x

Figure 3. B. anthracis HssRS displays elevated hemin sensing activity in vivo.

Figure 3

A. Complementation of S. aureus ΔhssRS by S. aureus and B. anthracis HssRS. Top: triplicate cultures of S. aureus wildtype (wt) harboring pOS1 (p.), ΔhssRS with pOS1, ΔhssRS with pOS1 encoding S. aureus HssR-HssSmyc (phssR-hssSmyc.Sa), and ΔhssRS with pOS1 encoding B. anthracis HssR-HssSmyc (phssR-hssSmyc.Ba) were grown in TSB + 30 µM hemin (a hemin concentration chosen due to the exceptional hemin resistance of strains containing the phssR-hssSmyc.Ba plasmid). Proliferation was tracked by growth curve analysis. Average culture density at 8 hour (white bars) and 16 hour (grey bars) time points are shown. Bottom: anti-C-Myc immunoblot showing expression of S. aureus and B. anthracis HssSmyc in S. aureus ΔhssRS. Lane 1: molecular weight ladder (L). Lanes 2–4: S. aureus ΔhssRS transformed with pOS1 (−), phssR-hssSmyc.Sa (Sa), or phssR-hssSmyc.Ba (Ba). B. Top: Complementation of B. anthracis ΔhssRS by S. aureus and B. anthracis HssRS (same plasmids described in A). Experiment was carried out as described in A, except that earlier time points (6 hour (white) and 12 hour (grey)) are shown due to sporulation and autolysis observed at late time points in B. anthracis cultures. Bottom: anti-C-Myc immunoblot showing expression of S. aureus and B. anthracis HssSmyc in B. anthracis ΔhssRS. Lanes are the same as those described in A. C. Top: Complementation of S. aureus ΔhssS by S. aureus and B. anthracis HssS. Top: S. aureus wildtype harboring plasmid pOS1, ΔhssS with pOS1, ΔhssS with pOS1 encoding S. aureus HssSmyc (phssSmyc.Sa), and ΔhssS with pOS1 encoding B. anthracis HssSmyc (phssSmyc.Ba) were analyzed as described in A, except that subcultures were performed in 20 µM hemin (chosen due to lower hemin resistance of complemented ΔhssS strains compared to strains described in A). Bottom: anti-C-Myc immunoblot showing expression of S. aureus and B. anthracis HssSmyc in S. aureus ΔhssS. Lane 1: molecular weight ladder (L). Lanes 2–4: S. aureus ΔhssS transformed with empty plasmid (−), phssSmyc.Sa (Sa), or phssSmyc.Ba (Ba). D. Top: Complementation of B. anthracis ΔhssS by S. aureus and B. anthracis HssS. Experiment was performed as described in C. Bottom: anti-C-Myc immunoblot showing expression of S. aureus and B. anthracis HssSmyc in B. anthracis ΔhssS. Lanes are the same as those described in C. All results are representative of at least three independent experiments. Error bars correspond to one standard deviation from the mean of triplicate samples within the same experiment. Asterisks denote statistically significant differences in complementation by Student’s t-test (p < 0.05).