Abstract
Full length recombinant iron regulatory protein, Fur, has been isolated and characterized from the algal-associated marine bacterium Marinobacter algicola DG893. Under nondenaturing conditions the Fur protein behaves on size exclusion chromatography as a dimer while it is monomeric under SDS PAGE conditions. ICP-MS and fluorescence quenching experiments show that Mb-Fur binds a single metal ion (Zn, Mn, or Co) per monomer. Electrophoretic mobility shift assays were used to probe the interaction of Mb-Fur with the purported Fur box in the promoter region upstream of the vibrioferrin biosynthetic operon. Interaction of Mb-Fur with a 100 bp DNA fragment containing the Fur box in the presence of 10 µM Mn, Co or Zn(II) resulted in decreased migration of DNA on a 7.5 % polyacrylamide gel. In the absence of the Fur protein or the metal, no interaction is seen. The presence of EDTA in the binding, loading or running buffers also abolished all activity demonstrating the importance of the metal in formation of the promoter-repressor complex. Based on a high degree of similarity between Mb-Fur and its homolog from Pseudomonas aeruginosa (PA) whose X-ray structure is known we developed a structural model for the former which suggested that only one of the several metal binding sites found in other Fur’s would be functional. This is consistent with the single metal binding stoichiometry we observed. Since the purported metal binding site was one that has been described as “structural” rather than “functional” in PA and yet the monometallic Mb-Fur retains DNA Fur box binding ability it reopens the question of which site is which, or if different species have adapted the sites for different purposes.
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Acknowledgments
This work was funded in part by NSF Grant CHE-0924313 as well as Grants from the CSU Council on Ocean Affairs, Science & Technology (COAST) and the SDSU University Grants Program. J.T. was supported by the National Institutes of Health SDSU MBRS Grant #2R25GM058906.
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Barker, R.A., Tisnado, J., Lambert, L.A. et al. Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893. Biometals 28, 197–206 (2015). https://doi.org/10.1007/s10534-014-9815-7
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DOI: https://doi.org/10.1007/s10534-014-9815-7