Crystal structure of the Vibrio cholerae ferric uptake regulator (Fur) reveals insights into metal co-ordination

The ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as both a repressor and an activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, opening up the potential of Fur as a drug target for cholera. Here we present the crystal structure of V. cholerae Fur that reveals a very different orientation of the DNA-binding domains compared with that observed in Pseudomonas aeruginosa Fur . Each monomer of the dimeric Fur protein contains two metal binding sites occupied by zinc in the crystal structure. In the P. aeruginosa study these were designated as the regulatory site (Zn1) and structural site (Zn2). This V. cholerae Fur study, together with studies on Fur homologues and paralogues, suggests that in fact the Zn2 site is the regulatory iron binding site and the Zn1 site plays an auxiliary role. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including Escherichia coli Fur. An analysis of the metal binding properties shows that V. cholerae Fur can be activated by a range of divalent metals.     

Genomic insights into microbial iron oxidation and iron uptake strategies in extremely acidic environments

This minireview presents recent advances in our understanding of iron oxidation and homeostasis in acidophilic Bacteria and Archaea. These processes influence the flux of metals and nutrients in pristine and man-made acidic environments such as acid mine drainage and industrial bioleaching operations. Acidophiles are also being studied to understand life in extreme conditions and their role in the generation of biomarkers used in the search for evidence of existing or past extra-terrestrial life. Iron oxidation in acidophiles is best understood in the model organism Acidithiobacillus ferrooxidans. However, recent functional genomic analysis of acidophiles is leading to a deeper appreciation of the diversity of acidophilic iron-oxidizing pathways. Although it is too early to paint a detailed picture of the role played by lateral gene transfer in the evolution of iron oxidation, emerging evidence tends to support the view that iron oxidation arose independently more than once in evolution. Acidic environments are generally rich in soluble iron and extreme acidophiles (e.g. the Leptospirillum genus) have considerably fewer iron uptake systems compared with neutrophiles. However, some acidophiles have been shown to grow as high as pH 6 and, in the case of the Acidithiobacillus genus, to have multiple iron uptake systems. This could be an adaption allowing them to respond to different iron concentrations via the use of a multiplicity of different siderophores. Both Leptospirillum spp. and Acidithiobacillus spp. are predicted to synthesize the acid stable citrate siderophore for Fe(III) uptake. In addition, both groups have predicted receptors for siderophores produced by other microorganisms, suggesting that competition for iron occurs influencing the ecophysiology of acidic environments. Little is known about the genetic regulation of iron oxidation and iron uptake in acidophiles, especially how the use of iron as an energy source is balanced with its need to take up iron for metabolism. It is anticipated that integrated and complex regulatory networks sensing different environmental signals, such as the energy source and/or the redox state of the cell as well as the oxygen availability, are involved.     

铁摄取调节蛋白Fur功能和作用模式的研究进展

铁是大多数生物必需的微量元素,在健康和疾病,尤其是宿主-病原菌互作过程中发挥着至关重要的作用.细菌胞内铁离子浓度的高低不仅是调节自身高亲和力铁运输系统表达的信号,更是病原菌产生毒素和其他必要毒力因子的关键调控因素.而另一方面,超负荷的铁也会导致致命的细胞毒性.因此,生物体内铁稳态的维持受到严格控制,其中以铁摄取调节蛋白(ferric uptake regulator,Fur)的作用最为显著,其调控网络涵盖了细菌生命活动的各个方面.本综述将基于Fur的生物学功能,围绕其家族分类、结构特点和差异、调控网络和调控机制等方面进行总结和分析,以期为Fur和铁稳态调节等研究提供参考.     

嗜酸异养菌对自养菌Acidithiobacillus ferrooxidans金属离子抗性和生物浸出的影响

【目的】了解嗜酸异养菌在诸如酸性矿坑水(AMD)和生物浸出体系等极端酸性环境中对浸矿微生物产生的影响。【方法】研究由嗜酸异养菌Acidiphilium acidophilum和自养菌Acidithiobacillus ferrooxidans经长期驯化后形成的共培养体系分别在Cd2+、Cu2+、Ni2+和Mg2+胁迫下的稳定性;并将此共培养体系应用于黄铁矿和低品位黄铜矿的生物浸出实验。【结果】在上述4种金属离子分别存在的条件下,异养菌Aph.acidophilum均能促进At.ferrooxidans对亚铁的氧化,提高其对能源利用的效率。共培养体系中的异养菌Aph.acidophilum使At.ferrooxidans对Cu2+的最大耐受浓度(MTC)由2.0 g/L提高到5.0 g/L,而且共培养的细胞数量与2.0 g/L Cu2+条件下生长的At.ferrooxidans纯培养相似。另外,共培养中的At.ferrooxidans对Mg2+的MTC也由12.0 g/L提高到17.0 g/L。生物浸出实验中嗜酸异养菌Aph.acidophilum促进了At.ferrooxidans对黄铁矿样品的浸出,浸出率较其纯培养提高了22.7%;但在含铁量较低的低品位黄铜矿浸出体系中共培养和At.ferrooxidans纯培养的浸出率均低于33%。在加入2.0 g/L Fe2+的低品位黄铜矿浸出体系中,共培养和At.ferrooxidans纯培养的浸出率均得到提高,分别达到52.22%和41.27%。【结论】以上结果表明,Aph.acidophilum与At.ferrooxidans共培养在一定的环境胁迫下仍能保持其稳定性并完成各自的生态功能,并且嗜酸异养菌Aph.acidophilum适合在含铁量较高的浸出体系中与铁氧化细菌共同作用来提高生物浸出的效率。     

Ferric uptake regulator protein: binding free energy calculations and per-residue free energy decomposition

Iron homeostasis is, in many bacterial species, mediated by the ferric uptake regulator (Fur). A regulatory site able to bind iron to activate Fur for DNA binding has been described, and a structural zinc site essential for the dimerization has also been proposed. They have been localized and named site 1 and site 2, respectively, from the crystal structure of a zinc-substituted Pseudomonas aeruginosa Fur (PA-Fur). Notwithstanding the studies on Fur proteins from various species, both the precise site of iron binding and the effect on DNA binding affinity are still controversial. These issues were investigated here by molecular dynamics simulations and free energy calculations. Simulations were performed for eight molecular systems represented by the three forms of Fur, that is, apo Fur, metal-substituted Fur, and Fur complexed with DNA. Because of the lack of a Fur-DNA complex crystal structure, the recently published model based on mass spectrometry experiments on Escherichia coli Fur (EC-Fur), and the crystal structure of PA-Fur, was used, after adjustment to adopt a symmetric conformation. The simulation results suggest that the formerly proposed site 2 is, in fact, the regulatory iron-sensing site. The calculations also predict that Fe(2+) at site 2 is hexacoordinated having an octahedral environment with only nitrogen and oxygen atoms, which is in accordance with previous spectroscopic characterizations. Energy decomposition pinpoints H87 as an additional amino acid that defines the regulatory metal site. Finally, free energy decomposition analysis reveals a number of amino acids potentially important in dimerization and in DNA binding.     

Effect of Salmonella typhimurium ferric uptake regulator (fur) mutations on iron- and pH-regulated protein synthesis.

Fur is an important regulatory protein known to function in the presence of iron as a repressor of iron-controlled genes. It was recently discovered that Fur is also essential to Salmonella typhimurium for mounting an adaptive acid tolerance response (J. W. Foster, J. Bacteriol 173:6896-6902, 1991). Because little is known about the effect of Fur on the physiology of this enteric pathogen, a systematic two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis was conducted to identify proteins whose synthesis is linked to iron levels. Mutations in the fur locus were identified and used to classify which proteins are controlled by Fur. Thirty-six proteins were overtly affected by iron availability, most of which were clearly under the control of Fur. Although most of the Fur-dependent proteins were under negative control, a significant portion (15 of 34) appeared to be under a form of positive control. Nine of the positively controlled proteins required Fur and iron for expression. However, Fur lacking iron was also required for the induction of six gene products. Surprisingly, not all iron-regulated proteins were controlled by Fur and not all Fur-dependent proteins were obviously regulated by iron status. Because fur mutants fail to mount an effective acid tolerance response, we made a comparative two-dimensional PAGE analysis of 100 total acid- and iron-regulated gene products. Production of most of these proteins was regulated by only one of the two stresses, yet a clear subset of seven genes were influenced by both acid and iron and were also controlled by fur. These proteins were also members of the acid tolerance response modulon. Consistent with the fur effect on pH-regulated protein synthesis, fur mutants lacked the inducible pH homeostasis system associated with the acid tolerance response. The results provide further evidence that Fur has an extensive impact on gene expression and cellular physiology and suggest an explanation for the acid-sensitive nature of fur mutants.     

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H₂O₂ and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.     

Functional specialization within the Fur family of metalloregulators

The ferric uptake regulator (Fur) protein, as originally described in Escherichia coli, is an iron-sensing repressor that controls the expression of genes for siderophore biosynthesis and iron transport. Although Fur is commonly thought of as a metal-dependent repressor, Fur also activates the expression of many genes by either indirect or direct mechanisms. In the best studied model systems, Fur functions as a global regulator of iron homeostasis controlling both the induction of iron uptake functions (under iron limitation) and the expression of iron storage proteins and iron-utilizing enzymes (under iron sufficiency). We now appreciate that there is a tremendous diversity in metal selectivity and biological function within the Fur family which includes sensors of iron (Fur), zinc (Zur), manganese (Mur), and nickel (Nur). Despite numerous studies, the mechanism of metal ion sensing by Fur family proteins is still controversial. Other family members use metal catalyzed oxidation reactions to sense peroxide-stress (PerR) or the availability of heme (Irr).     

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species.     

Identification and characterization of four strains of Acidithiobacillus ferrooxidans isolated from different sites in China

Four strains of Acidithiobacillus ferrooxidans (A. ferrooxidans), AF1, AF2, AF3 and AFc, were isolated from samples with different geological sources using a 9K medium. These four isolates were identified as A. ferrooxidans by phenotypic and 16S rDNA sequence analyses. All four isolates were able to use ferrous ion (Fe(2+)), elemental sulfur (S0) or pyrite as a sole energy source, but they showed differences in pH optima and range of activity, optimum temperature of activity, resistance to chloride (KCl) and heavy metal ions, and oxidation rates of Fe(2+), S0 and pyrite. AF3 was the most active strain when using Fe(2+) as the energy source, while AFc grew best using pyrite as the energy source. AF2 appeared to differ from the other three strains in substrate utilization, as it oxidizes S0 and pyrite more effectively than Fe(2+). RAPD analysis of genomic DNA from these isolates showed that banding profiles of their genomic DNA exhibited some differences, and the genomic banding profile of AF2 was significantly different from that of others. To obtain an insight into the molecular biology of the process of the energy production of these strains, several genes involved in the iron respiratory chain were cloned and sequenced, including Fe(2+) oxidase (iro), rusticyanin (rus) and subunit III of aa3-type cytochrome oxidase (cox C) genes. The results revealed that the iro gene can be cloned from all of the four strains and the nucleotide sequences were shown to be completely identical in each. However, rus and coxC genes could be amplified only from AF1, AF3 and AFc, not from AF2. These results suggested that the phenotypic differences of the four strains of A. ferrooxidans from different sites correlated with their genetic polymorphism, which may result from the different environments in which they lived, and that the strain AF2 was phenotypically and genetically significantly different from the other three strains.     

Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120

Heme is an iron-containing cofactor that aside from serving as the active group of essential proteins is a key element in the control of many molecular and cellular processes. In prokaryotes, the family of Fur (ferric uptake regulator) proteins governs processes essential for the survival of microorganims such as the iron homeostasis. We show that purified recombinant FurA from Anabaena sp. PCC 7120 interacts strongly with heme in the micromolar range and this interaction affects the in vitro ability of FurA to bind DNA, inhibiting that process in a concentration-dependent fashion. Our results provide the first evidence of the possible involvement of heme in the regulatory function of cyanobacterial Fur.