Abstract
Ferric uptake regulators (Fur) are a family of transcription factors coupling gene regulatory events to metal concentration. Recent evidence has expanded the mechanistic repertoires employed by Fur to activate or repress gene expression in the presence or absence of regulatory metals. However, the mechanistic basis underlying this extended repertoire has remained largely unexplored. In this study, we used an extensive set of mutations to demonstrate that Campylobacter jejuni Fur (CjFur) employs the same surface to positively and negatively control gene expression regardless of the presence or absence of metals. Moreover, the crystal structure determination of a CjFur devoid of any regulatory metals shows that subtle reorientation of the transcription factor DNA binding domain negatively impacts DNA binding, gene expression and gut colonization in chickens. Overall, these results highlight the versatility of the CjFur DNA binding domain in mediating all gene regulatory events controlled by the metalloregulator and that the full metalation of CjFur is critical to the Campylobacter jejuni life cycle in vivo.
Highlights
Transition metals are crucial components of several metabolic pathways and are critical for DNA, RNA and protein synthesis[1,2]
Our results show that the substitutions of R20, K25, K28 and R69 by glutamic acid residues abolish the apo-Campylobacter jejuni Fur (CjFur) binding to the cj1345c promoter region
After confirming that each mutant was expressed at a level similar to the wild-type (Fig. S1), we evaluated the expression of cj1345c and cj0948c, two genes known to be activated and repressed by CjFur, respectively, in the absence of iron (Fig. 1E)
Summary
Transition metals are crucial components of several metabolic pathways and are critical for DNA, RNA and protein synthesis[1,2]. Previous structural studies of members of the Fur family of proteins[16,17,18,19,20,21,22,23,24] revealed that these transcription factors fold into two distinct domains, including a DNA binding domain (DBD) and a dimerization domain (DD) localized on the N- and C-terminus of the protein, respectively. The metal ion binds to Fur (referred to as the holo-form) to induce a structural reorganization of the DBD, leading to DNA binding and gene repression. To better understand the mechanistic basis underlying the extended repertoire of DNA binding by the Fur family of metalloregulators, we undertook structural and biochemical studies of CjFur. In the current study, we show that similar sets of residues are involved in the positive and negative regulation of holoand apo-regulated genes. We demonstrate that metal coordination in the S3 site is important for CjFur DNA binding activity and contributes to processes related to chick ceca colonization by Campylobacter jejuni
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