FIG 5.
FhtR controls hrtBAEf transcription via binding to two repeated 14-nt palindromic sequences. (A) Two 14-nt palindromic motifs are present upstream of hrtBAEf. The two palindromes are shown in blue (P1) and in red (P2); the ribosome binding sequence (RBS) is underlined, and the start codon is shown in green. (B) FhtR binds to the promoter region of hrtBAEf. EMSA shows binding of FhtR to PhrtBA. The hrtBAEf promoter fragment (0.25 pmol) was incubated with increasing amounts of MBP-FhtR as indicated in molar ratios. DNA shift was visualized with GelRed (Biotium) following PAGE. The two shifted DNA-protein complexes (C1 and C2) and noncomplexed DNA (NC) are indicated. The results are representative of at least three independent experiments. (C) Roles of P1 and P2 on FhtR binding to the promoter region of hrtBAEf. EMSA was performed as described for panel B with either the native PhrtBA DNA fragment (WT) (as in panel A) or mutated fragments PhrtBA P1*, P2*, PhrtBA P1*, and PhrtBA P2*. MBP-FhtR: DNA (2.5 pmol: 0.25 pmol). The results are representative of at least three independent experiments. (D) Effect of hemin on the binding of FhtR to the hrtBAEf promoter. The hrtBAEf promoter DNA (0.25 pmol) was incubated with 2.5 pmol of MBP-FhtR together with increasing amounts of hemin as indicated (molar ratios) and analyzed by EMSA as described in the legend to panel B. The results are representative of at least three independent experiments. (E) Substitution of the two palindromic nucleotide sequences, P1 and P2, in PhrtBA abrogates FhtR-mediated control of hrtBAEf transcription. The WT strain was transformed either with the reporter plasmid pPhrtBA-lac or pPhrtBA P1*P2* -lac. β-Gal activity was determined as described in the legend to Fig. 1 following incubation with 2.5 μM hemin. Results represent the means plus standard deviations from three biological replicates. Statistical significance was determined by t test with statistical significance indicated as follows: ns, not significant (P > 0.5); ****, P < 0.0001. (F) Substitution of either P1 or P2 nucleotide sequences in PhrtBA enhances its transcriptional activation by hemin. The WT strain was transformed either with the reporter plasmid pPhrtBA-lac, pPhrtBA P1* -lac, or pPhrtBA P2* -lac. β-Gal activity was determined as described in the legend to Fig. 1 following incubation with hemin. Results represent the means plus standard deviations from three biological replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test with significance indicated as follows: ns, not significant (P > 0.5); *, P = 0.0140; ****, P < 0.0001.