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Research Support, Non-U.S. Gov't
- Experimental Phasing Using Zinc and Sulfur Anomalous Signals Measured at the Zinc Absorption Peak
- Sangmin Lee , Min-Kyu Kim , Chang-Jun Ji , Jin-Won Lee , Sun-Shin Cha
- J. Microbiol. 2013;51(5):639-643. Published online October 31, 2013
- DOI: https://doi.org/10.1007/s12275-013-3412-2
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Abstract
- Iron is an essential transition metal required for bacterial growth and survival. Excess free iron can lead to the generation of reactive oxygen species that can cause severe damage to cellular functions. Cells have developed iron-sensing regulators to maintain iron homeostasis at the transcription level. The ferric uptake regulator (Fur) is an iron-responsive regulator that controls the expression of genes involved in iron homeostasis, bacterial virulence, stress resistance, and redox metabolism. Here, we report the expression, purification, crystallization, and phasing of the apo-form of Bacillus subtilis Fur (BsFur) in the absence of regulatory metal ions. Crystals were obtained by microbatch crystallization method at 295 K and diffraction data at a resolution of 2.6 Å was collected at the zinc peak wavelength (λ=1.2823 Å). Experimental phasing identified the positions of one zinc atom and four sulfur atoms of cysteine residues coordinating the zinc atom, indicating that the data contained a meaningful anomalous scattering originating from the ordered zinc-coordinating sulfur atoms, in spite of the small anomalous signals of sulfur atoms at the examined wavelength.
Research Support, N.I.H., Extramural
- Helicobacter pylori apo-Fur Regulation Appears Unconserved Across Species
- Shana Miles , Beth M. Carpenter , Hanan Gancz , D. Scott Merrell
- J. Microbiol. 2010;48(3):378-386. Published online June 23, 2010
- DOI: https://doi.org/10.1007/s12275-010-0022-0
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- 25 Scopus
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Abstract
- The Ferric Uptake Regulator (Fur) is a transcriptional regulator that is conserved across a broad number of bacterial species and has been shown to regulate expression of iron uptake and storage genes. Additionally, Fur has been shown to be an important colonization factor of the gastric pathogen Helicobacter pylori. In H. pylori, Fur-dependent regulation appears to be unique in that Fur is able to act as a transcriptional repressor when bound to iron as well as in its iron free (apo) form. To date, apo-regulation has not been identified in any other bacterium. To determine whether Fur from other species has the capacity for aporegulation, we investigated the ability of Fur from Escherichia coli, Campylobacter jejuni, Desulfovibrio vulgaris Hildenborough, Pseudomonas aeruginosa, and Vibrio cholerae to complement both iron-bound and apo-Fur regulation within the context of a H. pylori fur mutant. We found that while some Fur species (E. coli, C. jejuni, and V. cholerae) complemented iron-bound regulation, apo-regulation was unable to be complemented by any of the examined species. These data suggest that despite the conservation among bacterial Fur proteins, H. pylori Fur contains unique structure/function features that make it novel in comparison to Fur from other species.
Research Support, Non-U.S. Gov'ts
- Experimental and Computational Characterization of the Ferric Uptake Regulator from Aliivibrio salmonicida (Vibrio salmonicida)
- Hege Lynum Pedersen , Rafi Ahmad , Ellen Kristin Riise , Hanna-Kirsti Schrøder Leiros , Stefan Hauglid , Sigrun Espelid , Bjørn Olav Brandsdal , Ingar Leiros , Nils-Peder Willassen , Peik Haugen
- J. Microbiol. 2010;48(2):174-183. Published online May 1, 2010
- DOI: https://doi.org/10.1007/s12275-010-9199-5
- 35 View
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- 5 Scopus
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Abstract
- The Ferric uptake regulator (Fur) is a global transcription factor that affects expression of bacterial genes in an iron-dependent fashion. Although the Fur protein and its iron-responsive regulon are well studied, there are still important questions that remain to be answered. For example, the consensus Fur binding site also known as the “Fur box” is under debate, and it is still unclear which Fur residues directly interact with the DNA. Our long-term goal is to dissect the biological roles of Fur in the development of the disease cold-water vibriosis, which is caused by the psychrophilic bacteria Aliivibrio salmonicida (also known as Vibrio salmonicida). Here, we have used experimental and computational methods to characterise the Fur protein from A. salmonicida (AS-Fur). Electrophoretic mobility shift assays show that AS-Fur binds to the recently proposed vibrio Fur box consensus in addition to nine promoter regions that contain Fur boxes. Binding appears to be dependent on the number of Fur boxes, and the predicted “strength” of Fur boxes. Finally, structure modeling and molecular dynamics simulations provide new insights into potential AS-Fur–DNA interactions.
- Cys-92, Cys-95, and the C-Terminal 12 Residues of the Vibrio harveyi Ferric Uptake Regulator (Fur) are Functionally Inessential
- Kun Sun , Shuang Cheng , Min Zhang , Fang Wang , Li Sun
- J. Microbiol. 2008;46(6):670-680. Published online December 24, 2008
- DOI: https://doi.org/10.1007/s12275-008-0113-3
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- 10 Scopus
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Abstract
- Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (FurVh) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. FurVh shares 77% overall sequence identity with the Escherichia coli Fur (FurEc) and could complement a mutant of FurEc. Like FurEc, FurVh possesses two cysteine residues at positions 92 and 95, yet unlike FurEc, in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of FurVh proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of FurVh are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of FurVh are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of FurVh is possibly different from that of FurEc; and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of FurVh; and (iii) provided insights into the potential function of the local structure involving C137 and K138.
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