Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular “free” iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the “iron-bound” Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron–sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is ∼31% in the E. coli iscA/sufA mutant cells and is decreased to ∼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

Virulence mechanisms used in the pathogenesis of periodontal diseases caused by Porphyromonas gingivalis

Periodontal diseases are characterized by progressive inflammation that destroys the tooth-supporting tissues, leading to gum bleeding and tooth loss. Porphyromonas gingivalis is considered one of the main etiological agents responsible for the initiation and progression of chronic periodontitis. This gram-negative, anaerobic bacterium is a part of a multi-species oral biofilm. P. gingivalis does not have the full pathway of protoporphyrin IX synthesis, nor does it produce siderophores. Therefore, for survival and proliferation, it requires heme as a source of iron and protoporphyrin IX. In order to obtain heme, P. gingivalis uses a number of mechanisms that affect the ability of this bacterium to initiate a pathological condition. This review presents the current knowledge regarding the best-known and characterized systems involved in heme acquisition by P. gingivalis. We focused on processes occurring in the initial states of infection, where gingipain, hemagglutinins, and hemolysins play a crucial role. The mechanisms encoded by hmu, iht and hus operons, including proteins with hemophore-like properties, as well as TonB-dependent outer membrane receptors are described. We present their function and participation in the progression of the infection. In addition, we describe mechanisms produced by P. gingivalis and other periodontopathogens in synergistic processes promoting the growth and virulence of P. gingivalis. We also describe processes regulating iron and heme homeostasis, including the homolog of the Fur protein, the two-component system HaeSR, as well as the OxyR, SigH, and PgDps proteins.

Regulation of Iron Uptake by Fine-Tuning the Iron Responsiveness of the Iron Sensor Fur

ABSTRACTIron is one of most abundant environmental metal ions but is highly limited in organisms. It is an important metal ion as it facilitates various biological processes, including catalysis of metabolic enzymes and DNA biogenesis. In bacteria, the ferric uptake regulator (Fur) protein controls iron uptake by regulating genes coding for iron transporters in response to iron concentration. This iron response is ascribed to Fur’s intrinsic affinity for iron because its binding to iron dictates its regulatory function. However, we now report that the pathogenSalmonellaachieves a proper response of Fur to changes in environmental iron concentrations via EIIANtr(a nitrogen metabolic phosphotransferase system component). We establish that EIIANtrincreases expression of iron transporter-coding genes under low-iron conditions (i.e., nanomolar ranges) in a Fur-dependent manner, which promotesSalmonellagrowth under such conditions. EIIANtrdirectly hampers Fur binding to DNA, thereby inducing expression of those genes. This regulation allowsSalmonellato express Fur-regulated genes under low-iron conditions. Our findings reveal a potentially widespread control mechanism of bacterial iron uptake systems operating in response to iron availability.IMPORTANCEIron is a fundamental metal ion for living organisms as it facilitates various biological processes. The ferric uptake regulator (Fur) protein controls iron homeostasis in various bacterial species. It is believed that Fur’s iron-dependent regulatory action is sufficient for it to function as an iron sensor. However, we now establish that the bacterial pathogenSalmonellaenables Fur to properly reflect changes in surrounding iron availability by fine-tuning its responsiveness to iron. This process requires a protein that hampers Fur DNA binding at low iron concentrations. In this way,Salmonellabroadens the range of iron concentrations that Fur responds to. Our findings reveal a potentially widespread control mechanism of bacterial iron homeostasis.

Ferric Uptake Regulator Fur Is Conditionally Essential in Pseudomonas aeruginosa

ABSTRACT 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.