FIG 4.
Mcc1229 transcription is iron-regulated. (A) A region upstream of the mctA ORF containing a putative Fur binding site was ligated into pRS551 and successfully promoted the transcription of lacZ. Promoter regions upstream of the mctB, mctC, and mctE ORFs were also tested in this manner. The transcriptional activity of the mctA promoter decreased when the medium was supplemented with 200 μM FeCl3. Significance was determined by two-way ANOVA with Sidak’s multiple-comparison test. (B) RNA was extracted from 16-h-grown LB cultures of 0.1229 and 0.1229Δfur::cat, converted to cDNA, and probed by qPCR for the mctA, mctB, mctC, and mctD ORFs. Gene expression was determined by the ΔCT method using the ribosomal gene rrsH as an internal control (80). Taking the differences of ΔCT,WT and ΔCT,Δfur gives ΔΔCT values of 10.14, 6.04, 8.60, and 8.85 for mctA, mctB, mctC, and mctD, respectively. All mct genes examined were consistently upregulated in the Δfur::cat strain. Statistical significance was determined by Student’s t test on the ΔCT values for each gene (*, P < 0.05; **, P < 0.01).