Figure 3

The CC2193 and CC3059 operons are members of the C. crescentus Fur regulon. (A) Promoter activities of the CC2193 and CC3059 operons in response to iron and Fur. Wild type (NA1000) and fur mutant (∆fur) strains containing plasmids pLAC2193 or pLAC3059 were grown in PYE medium and treated with 100 μM FeSO₄ (Fe) or 100 μM 2,2-dipyridyl (DP) for two hours. The β-galactosidase activity generated by these lacZ fusions was determined. The experiments were performed in duplicate from three independent biological cultures. (B) Fur binds directly to the promoter of the CC3059 operon. EMSAs were performed using the purified His-Fur protein and a probe containing the promoter region of CC3059. The ³²P-labeled probe was incubated with increasing concentrations of protein (0, 50, 200, 500 and 1000 nM) (left). A competition assay using 250 nM Fur and the labeled CC3059 probe was performed, where binding of Fur was challenged with a 30-fold excess of unlabeled DNA fragments of the same region (SE) or the 16S rRNA coding region (SI) as competitors (right). Below is shown the promoter region of the CC3059 operon, indicating the previously identified transcriptional start site (+1) and conserved −35 and −10 sequences of Caulobacter σ⁷⁰ promoters (TTGAC-16 bp-G/CCTANA) [39]. The initiation codon (GTG) is underlined. The Fur binding site predicted in silico is shaded.