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Iron Binding Site in a Global Regulator in Bacteria - Ferric Uptake Regulator (Fur) Protein: Structure, Mossbauer Properties, and Functional Implication.

ABSTRACT: Fur protein plays key roles in regulating numerous genes in bacteria and is essential for intracellular iron concentration regulation. However, atomic level pictures of the iron binding site and its functional mechanism remain to be established. Here we present results of the first quantum chemical investigation of various first- and second-shell models and experimental Mössbauer data of E. Coli Fur, including 1) the first robust evidence that site 2 is the Fe binding site with a 3His/2Glu ligand set, being the first case in non-heme proteins, with computed Mössbauer data in excellent accord with experiment; 2) the first discovery of a conservative hydrogen bonding interaction in the iron binding site based on X-ray and homology structures; 3) the first atomic level hypothesis of active site reorganization upon iron concentration increase, triggering the conformational change needed for its function. These results shall facilitate structural and functional studies of Fur family proteins.

SUBMITTER: Katigbak J

PROVIDER: S-EPMC3507992 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Iron Binding Site in a Global Regulator in Bacteria - Ferric Uptake Regulator (Fur) Protein: Structure, Mössbauer Properties, and Functional Implication.

Katigbak Joseph J Zhang Yong Y

The journal of physical chemistry letters 20121101 3

Fur protein plays key roles in regulating numerous genes in bacteria and is essential for intracellular iron concentration regulation. However, atomic level pictures of the iron binding site and its functional mechanism remain to be established. Here we present results of the first quantum chemical investigation of various first- and second-shell models and experimental Mössbauer data of E. Coli Fur, including 1) the first robust evidence that site 2 is the Fe binding site with a 3His/2Glu ligan ...[more]

PMID: 23205186

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Project description:UnlabelledClostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea. Although iron acquisition in the host is a key to survival of bacterial pathogens, high levels of intracellular iron can increase oxidative damage. Therefore, expression of iron acquisition mechanisms is tightly controlled by transcriptional regulators. We identified a C. difficile homologue of the master bacterial iron regulator Fur. Using targetron mutagenesis, we generated a fur insertion mutant of C. difficile. To identify the genes regulated by Fur in C. difficile, we used microarray analysis to compare transcriptional differences between the fur mutant and the wild type when grown in high-iron medium. The fur mutant had increased expression of greater than 70 transcriptional units. Using quantitative reverse transcriptase PCR (qRT-PCR), we analyzed several of the Fur-regulated genes identified by the microarray and verified that they are both iron and Fur regulated in C. difficile. Among those Fur- and iron-repressed genes were C. difficile genes encoding 7 putative cation transport systems of different classes. We found that Fur was able to bind the DNA upstream of three Fur-repressed genes in electrophoretic mobility shift assays. We also demonstrate that expression of Fur-regulated putative iron acquisition systems was increased during C. difficile infection using the hamster model. Our data suggest that C. difficile expresses multiple iron transport mechanisms in response iron depletion in vitro and in vivo.ImportanceClostridium difficile is the most common cause of hospital-acquired infectious diarrhea and has been recently classified as an "urgent" antibiotic resistance threat by the CDC. To survive and cause disease, most bacterial pathogens must acquire the essential enzymatic cofactor iron. While import of adequate iron is essential for most bacterial growth, excess intracellular iron can lead to extensive oxidative damage. Thus, bacteria must regulate iron import to maintain iron homeostasis. We demonstrate here that C. difficile regulates expression of several putative iron acquisition systems using the transcriptional regulator Fur. These import mechanisms are induced under iron-limiting conditions in vitro and during C. difficile infection of the host. This suggests that during a C. difficile infection, iron availability is limited in vivo.

Project description:In Pseudomonas aeruginosa, the ferric uptake regulator (Fur) protein controls both metabolism and virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion mutants of P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this bacterium. By investigating a P. aeruginosa conditional fur mutant, we demonstrate that Fur is not essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are severely impaired in colony formation. Transposon-mediated random mutagenesis experiments identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the conditional fur mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do not accumulate higher levels of reactive oxygen species (ROS) and are not rescued by antioxidant agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that pyochelin production has no inhibitory effects on a fur deletion mutant of Pseudomonas syringae pv. tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves a P. aeruginosa-specific pathway(s).IMPORTANCE Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors that control iron uptake and storage in response to iron availability, thereby playing a crucial role in the maintenance of iron homeostasis. While fur null mutants of many bacteria have been obtained, Fur appears to be essential in Pseudomonas aeruginosa for still unknown reasons. We obtained Fur-depleted P. aeruginosa cells by conditional mutagenesis and showed that Fur is dispensable for planktonic growth, while it is required for colony formation. This is because Fur protects P. aeruginosa colonies from toxicity exerted by the pyochelin siderophore. This work provides a functional basis to the essentiality of Fur in P. aeruginosa and highlights unique properties of the Fur regulon in this species.

Dataset Information

Iron Binding Site in a Global Regulator in Bacteria - Ferric Uptake Regulator (Fur) Protein: Structure, Mossbauer Properties, and Functional Implication.

Publications

Iron Binding Site in a Global Regulator in Bacteria - Ferric Uptake Regulator (Fur) Protein: Structure, Mössbauer Properties, and Functional Implication.

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