FIGURE 3.

Hemin alleviates HrtR repression of the hrtRBA operon. A, in vivo induction of hrtRBA operon by hemin. The strain carrying P_hrthrtR-lac was grown in the presence of the indicated concentrations of hemin to A₆₀₀ = 1. β-Gal activity was quantified by luminescence (see “Experimental Procedures”). Results represent the mean ± S.D. from the triplicate sample. B, effect of hemin on binding of HrtR to the hrtRBA promoter by EMSA. Thirty pmol of hrtRBA promoter fragment were incubated in the presence of 200 pmol of MBP-HrtR together with increasing amounts of hemin. C, in vivo induction of P_hrt with PPIX and metal porphyrins. β-Gal activity of the pP_hrthrtR-lac plasmid, established in WT L. lactis was evaluated on bacteria incubated without or with 5 μm of the indicated PPIX or metal-conjugated porphyrins. D, EMSA was used to test ligand specificity of HrtR. Thirty pmol of hrtRBA promoter fragment were incubated in the presence of 200 pmol of MBP-HrtR and 200 pmol of the indicated metalloporphyrin. β-Gal activity was quantified by luminescence (see “Experimental Procedures”). Results represent the mean ± S.D. from triplicate experiments. RLU, relative light units.