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Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator.

ABSTRACT: Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

SUBMITTER: Deng Z

PROVIDER: S-EPMC4506495 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator.

Deng Zengqin Z Wang Qing Q Liu Zhao Z Zhang Manfeng M Machado Ana Carolina Dantas AC Chiu Tsu-Pei TP Feng Chong C Zhang Qi Q Yu Lin L Qi Lei L Zheng Jiangge J Wang Xu X Huo XinMei X Qi Xiaoxuan X Li Xiaorong X Wu Wei W Rohs Remo R Li Ying Y Chen Zhongzhou Z

Nature communications 20150702

Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding ...[more]

PMID: 26134419

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Project description:Iron limitation is a universal strategy of host immunity during bacterial infection. However, the mechanisms by which pathogens antagonize host nutritional immunity have not been fully elucidated. Here, we identified a requirement for the UMPylator YdiU for this process in Salmonella. The expression of YdiU was dramatically induced by the metal starvation signal. The intracellular iron content was much lower in the ΔydiU strain than in wild-type Salmonella, and the ΔydiU strain exhibited severe growth defect under metal deficiency environments. Genome-wide expression analyses revealed significantly decreased expression of iron uptake genes in ΔydiU strain compared with the wild-type strain. Interestingly, YdiU did not affect the expression level of the major iron uptake regulator Fur but directly UMPylated Fur on its H118 residue in vivo and in vitro. UMPylation destroyed the Fur dimer, promoted Fur aggregation, and eliminated the DNA-binding activity of Fur, thus abolishing the ability of Fur to inhibit iron uptake. Restricting Fur to the deUMPylated state dramatically eliminates Salmonella iron uptake in iron deficiency environments. In parallel, YdiU facilitates Salmonella survival within host cells by regulating the iron uptake pathway. IMPORTANCE Salmonella is the major pathogen causing bacterial enteric illness in both humans and animals. Iron availability is strictly controlled upon Salmonella entry into host cells. The mechanisms by which Salmonella balances the acquisition of sufficient iron while preventing a toxic overload has not been fully understood. Here, we reveal a novel regulation process of iron acquisition mediated by the UMPylator YdiU. Fur acts as the central regulator of bacterial iron homeostasis. YdiU UMPylates Fur on H118 and prevents Fur from binding to target DNA, thus activating the expression of iron uptake genes under iron-deficient conditions. We describe the first posttranslational modification-based regulation of Fur and highlight a potential mechanism by which Salmonella can adapt to eliminate host nutritional immunity.

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

Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator.

Publications

Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator.

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