FIGURE 3: hutA1 and hutA2 are expressed in iron- and heme-dependent manner. (A) Strategy for genomic plasmid insertion to generate AFS-I-PhutA1 and AFS-I-PhutA2. The white downward triangles indicate the predicted promoter start sites. (B) Anabaena wild-type, AFS-I-PhutA1 (left) and AFS-I-PhutA2 (right) strains were grown in YBG11 medium without iron for seven days (first bar). After seven days the culture was transferred into a new medium for growth for three days in YBG11-Fe (second bar), YBG11 medium without iron but with 10 µM protoporphyrin IX (third bar), 10 µM (forth bar) or 30 µM heme (fifth bar for AFS-I-PhutA2) or in YBG11, which contains iron (last bar). The ratio of the GFP fluorescence of the mutant strain and the background in wild-type Anabaena is shown. The results represent the average of at least three independent experiments. (C) AFS-I-PhutA1 was grown in YBG11-Fe medium cultures (OD_{750 nm} = 0.02) supplemented with 10 mM NaHCO₃ and bubbled with CO₂-enriched air (1% CO₂ v/v). Growth was monitored on day 0 (top), day 2 (middle) and day 4 (bottom). Images are representative for multiple replicas and autofluorescence (left) and GFP fluorescence (right) are shown. (D) Images of Anabaena wild-type and AFS-I-PhutA1 strains were taken from (C) and the GFP fluorescence was quantified using a cross section of representative cells. The results represent the average of five independent cultures. (E) Anabaena pre-starved for 21 days was transferred to YBG11 medium, YBG11 medium without iron or YBG11 medium without iron but with 30 µM heme. After three days of incubation, RNA was extracted and qRT-PCR was performed. Ct values for rnpB and for the indicated gene in cultures grown in YBG11 medium were used to calculate the presented values. The average and standard deviation of n>3 independent biological replicas are shown. The statistical analysis in (B), (D) and (E) was performed using ANOVA with ranks indicated with p<0.05.