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. 2021 Feb 2;12(1):e03392-20. doi: 10.1128/mBio.03392-20

FIG 2.

FIG 2

FhtR controls hrtBAEf expression. (A) Schematic representation of the fhtR gene and hrtBAEf operon. The fhtR gene (OG1RF_RS02765) encodes a TetR family transcriptional regulator. The hrtBEf (OG1RF_RS02770) and hrtAEf (OG1RF_RS02775) genes encode a permease and ATPase, respectively. (B) FhtR controls hrtBAEf expression. WT and ΔfhtR strains carrying the reporter plasmid pPhrtBA-lac and a ΔfhtR strain carrying a plasmid, pfhtR, combining both PhrtBA-lac and PfhtR-fhtR were grown to an OD600 of 0.5, and β-gal expression was quantified by luminescence as reported in the legend to Fig. 1 with the indicated concentrations of hemin. Results represent the means plus standard deviations (error bars) from three biological replicates. Statistical significance was determined by t test as follows: ns, not significant (P > 0.5); ****, P < 0.0001. (C) fhtR deletion abrogates heme toxicity. Stationary-phase cultures of WT, ΔfhtR, and ΔfhtR(pfhtR) strains were plated on solid medium. Hemin (10 μl of a 1 mM stock solution) was pipetted directly onto plates, which were incubated for 24 h. Inhibition zones appear as a circular clearing in the center of each panel. No inhibition zone was observed for the ΔfhtR strain. The results are representative of three independent experiments. (D) Visualization of the impact of FhtR on heme accumulation. WT, ΔfhtR, and ΔfhtR(pfhtR) strains were grown and incubated with 5 μM hemin as described above for panel A. The bacteria were pelleted by centrifugation and photographed. The results are representative of three independent experiments. (E) FhtR expression is constitutively induced. β-Gal expression upon hemin addition to the culture medium in WT and ΔfhtR strains transformed with the pPfhtR-lac reporter plasmid was determined by luminescence as described in the legend to Fig. 1. Results represent the means plus standard deviations from three biological replicates. (F) fhtR transcription is not mediated by hemin. The ΔfhtR strain transformed with pPfhtR-fhtR-HA or carrying an empty vector (control) was used to monitor FhtR expression by Western blotting (WB) using an antihemagglutinin (anti-HA) antibody (α-HA). Bacteria were grown to an OD600 of 0.5 and incubated with 2.5 μM hemin for 1.5 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on cell lysates (80 μg per lane). The results are representative of three independent experiments.