Figure 6. Intracellular heme controls the expression of hrtBAhssRS in GBS.

(A) HrtBA expression is mediated by HssRS. Cultures of WT, ∆hssRS and ∆hssRS(pP_gbs0119-hssRS-HA) GBS strains were plated as in Fig. 1B. (B) HssS is expression is induced by hemin. GBS ∆hssRS and ∆hssRS(pP_gbs0119-hssRS-HA) strains were grown to OD_600nm = 0.5 and treated for 1 h with hemin as indicated. Western blot was performed with cell lysates using an anti-HA antibody (Supplementary methods). (C) The hrtBAhssRS operon is controlled by HssRS. β-galactosidase activity of WT and ∆hssRS GBS strains transformed with P_gbs0119-lac was performed as in (B). (D) P_gbs0119 induction depends on heme intracellular accumulation. WT, ∆hrtBA and ∆hrtBA(P₂₃-hrtBA) carrying the P_gbs0119-lac expression cassette were treated with hemin as in (B). (E) GBS internalizes heme from Hb. WT(pP_gbs0119-lac) and ∆hrtBA(pP_gbs0119-lac) GBS strains were incubated with 1 μM hemin or freshly prepared bovine Hb (Methods). β-gal expression was followed as described in (B). The Hb solution did not contain measurable amounts of free heme as verified by UV-visible spectroscopy. Two-tailed Student test was used to determine P values: WT (hemin/Hb), P = 0.0001; ΔhrtBA (hemin/Hb), P = 0.009. **P < 0.01 and ***P < 0.001. (F) GBS scavenges heme from blood. WT(pP_gbs0119-lac), ∆hrtBA(pP_gbs0119-lac), and negative control WT (pTCV-lac) were grown to OD_600nm = 0.5 and mixed with 25% BALB/c fresh blood for 3 h at 37 °C (Methods). Luminescence was determined as in (C) and CFU from blood samples were determined by serial dilutions on plates. (G) Heme dependent light emission by the heme sensing GBS strain. GBS WT(pP_gbs0119-lux) was resuspended in soft agar and overlayed on agar plates. Hemin, Hb (10 μl of a 10 mM stock solution) or fresh BALB/c mice blood (10 μl) were directly spotted on plates. Plates were incubated at 37 °C for 24 h and luminescence was visualized using an IVIS 200 luminescence imaging system (Methods).