Fur Protein Research Articles

Identification of iron-acquisition proteins of Avibacterium paragallinarum

When Avibacterium paragallinarum reference strain 0083 (serovar A) was grown in an iron-restricted culture medium, the expression of the 60, 68 and 93 kDa outer membrane proteins increased as compared with normal media. Sera of chickens experimentally infected with Av. paragallinarum recognized these iron-restriction induced proteins, suggesting their expression in vivo. The three outer membrane proteins were identified as transferrin receptor and iron transport proteins by mass spectroscopy and a search in sequence databases. As these proteins have been reported to be regulated by the Fur protein in many bacteria, we investigated, through molecular methods, the presence of the fur gene in Av. paragallinarum. A candidate fur gene of Av. paragallinarum was amplified by polymerase chain reaction using complementary primers to conserved regions of fur gene sequences from members of the Pasteurellaceae family. The nucleotide sequence of the cloned gene, from ATG to TAA stop codon, was 453 base pairs in length and the deduced amino acid sequence showed 94% identity with Fur sequences of Actinobacillus pleuropneumoniae and Haemophilus ducreyi. The Av. paragallinarum deduced Fur protein (17.8 kDa) amino acid sequence contains the N-terminal helix–turn–helix DNA-binding domain and the two iron-binding sites in the C-terminal end, typical of other described Fur proteins. The study of iron-restriction-induced proteins and the mechanism regulating their expression could lead to an understanding of the responses of Av. paragallinarum to survive in an iron-restricted environment on host mucosal surfaces.

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.

130 Trend of First-Line and Second-Line Eradication Therapy for Helicobacter pylori Infection in Korea

G A A b st ra ct s to yield these reduction products that are directly toxic to the bacterium. Although mutational inactivation of the rdxA gene is strongly associated with the development of resistance to MNZ, a number of MNZ-resistant strains have also been reported that show no evident change in the rdxA gene (Aliment. Pharmacol. Ther. 24:81-87, 2006), suggesting the potential existence of other mechanisms of resistance. We assumed that these resistant strains exhibit enhanced ability to defend themselves against superoxide radicals by overexpressing bacterial superoxide dismutase (SodB). The present study was designed to examine the expression of sodB and the function of the ferric uptake regulator (Fur) that acts as a transcriptional repressor protein exhibiting iron-dependent binding to the sodB promoter. Methods. H. pylori strain ATCC 700392 was used as the MNZ-susceptible strain and the MNZ-resistant strains used for the study (KS0033, KS0048 and KS0145) were isolated from patients at Keio University Hospital. The expression of sodB under exposure to MNZ was measured by quantitative RT-PCR, and the levels in the susceptible strain and resistant strains were compared. The complete fur of resistant strains was amplified by PCR, cloned into the pCR2.1-TOPO vector, and then sequenced. The Fur proteins were aligned using Genetyx and the protein structures were modeled using Swiss-Model and DeepView-SwisspdbViewer. Results.While sodB expression was scarcely induced by MNZ in ATCC 700392, overexpression of sodB by about 1.7, 2.6 and 2.1-fold was observed in KS0033, KS0048 and KS0145, respectively, under MNZ exposure. Thus, these MNZ-resistant strains showed enhanced sodB expression, as compared with that in ATCC 700392, under exposure to MNZ. The MNZ-resistant strains had a mutant-type of Fur protein, in which Asn 118 was replaced by His (N118H). From the results of the homologymodeling, Asn 118 was predicted to be located in the 5th alpha-helix region that projected outward. It is conceivable that the N118H of Fur affected the affinity of Fur for iron or the sodB promoter region, resulting in sodB overexpression. Conclusion. Fur mutation in H. pylori, resulting in substitution of Asn 118 by His, is associated with the development of resistance to MNZ.

The mntH gene encodes the major Mn2+ transporter in Bradyrhizobium japonicum and is regulated by manganese via the Fur protein

The bacterial Nramp family protein MntH is a divalent metal transporter, but mntH mutants have little or no phenotype in organisms where it has been studied. Here, we identify the mntH homologue of Bradyrhizobium japonicum, and demonstrate that it is essential for Mn(2+) transport and for maintenance of cellular manganese homeostasis. Transport activity was induced under manganese deficiency, and Fe(2+) did not compete with (54)Mn(2+) for uptake by cells. The steady-state level of mntH mRNA was negatively regulated by manganese, but was unaffected by iron. Control of mntH expression and Mn(2+) transport by manganese was lost in a fur strain, resulting in constitutively high activity. Fur protected a 35 bp region of the mntH promoter in DNase I footprinting analysis that includes three imperfect direct repeat hexamers that are needed for full occupancy. Mn(2+) increased the affinity of Fur for the mntH promoter by over 50-fold, with a K(d) value of 2.2 nM in the presence of metal. The findings identify MntH as the major Mn(2+) transporter in B. japonicum, and show that Fur is a manganese-responsive regulator in that organism. Furthermore, Fe(2+) is neither a substrate for MntH nor a regulator of mntH expression in vivo.

Alteration of growth-phase-dependent protein regulation by afurmutation inHelicobacter pylori

The ferric-uptake regulator (Fur) protein is an Fe(2+)-dependent transcriptional repressor. To clarify the global regulation of Helicobacter pylori proteins by Fur according to the growth phase, we compared the proteome profiles of H. pylori 26695 and its isogenic fur mutant, harvested during in vitro culture. Clustering analysis of the proteome profiles of the two strains revealed that the growth-phase-dependent protein regulation in the wild-type strain was largely altered in the fur mutant. Reverse transcriptase-PCR analysis of several H. pylori genes showed that a major switch in transcription occurred 12 h earlier than in the wild type, indicating that the fur mutation induced an earlier transcriptional switch from log to stationary phase. Several H. pylori proteins also showed changes in their patterns of protein post-translational modification (PTM). In particular, the HydB protein, which was detected as four spots on 2-dimensional electrophoresis gels, underwent two types of PTM, which were under different kinds of regulation. These data demonstrate that a fur mutation affects the growth-phase-dependent regulation of proteins and mRNAs, suggesting a role for Fur in controlling the global regulation of cellular processes in response to changing growth environments.

Fur‐Independent Induction of Helicobacter pylori Flavodoxin‐Encoding Gene (fldA) Under Iron Starvation

Helicobacter pylori infection is associated with a variety of diseases including gastric cancer. Flavodoxin is an electron transfer protein containing a flavin mononucleotide prosthetic group and substituted an iron-containing electron transfer protein under iron-limiting conditions. H. pylori flavodoxin has been reported but its pathogenic role is unclear. The aim of this study is to understand a pathogenic role of H. pylori flavodoxin under iron-limiting condition. The flavodoxin-encoding gene (fldA) was cloned from one of clinical H. pylori isolates (DU17) and its transcript was quantified by primer extension, Northern hybridization, and real-time polymerase chain reaction in different concentrations of an iron chelator. The fldA transcript was also quantified in H. pylori ATCC 700392, lacking a ferric uptake regulatory (fur) protein. Nucleotide sequence of the fldA from H. pylori DU17 revealed a 492-bp (164 amino acids) open reading frame with a deduced amino acid sequence having 97% identity to that from the complete genomic sequence of H. pylori 26695. The deduced promoter [-35, -10, and +1] of the fldA was 56-bp upstream from the first codon of FldA. The fldA transcript (approximately 0.55-kb) was induced up to 14-fold in both wild-type and fur-knocked-out strains under iron-limiting conditions, suggesting that the fldA induction is independent to the Fur protein. The fldA gene may play an important role in iron starvation conditions.

New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress

Fur (ferric uptake regulator) is a prokaryotic transcriptional regulator that controls a large number of genes mainly related to iron metabolism. Several Fur homologues with different physiological roles are frequently found in the same organism. The genome of the filamentous cyanobacterium Anabaena (Nostoc) sp. PCC 7120 codes for three different fur genes. FurA is an essential protein involved in iron homoeostasis that also modulates dinitrogen fixation. FurA interacts with haem, impairing its DNA-binding ability. To explore functional differences between Fur homologues in Anabaena, factors affecting their regulation, as well as some biochemical characteristics, have been investigated. Although incubation of FurB with haem severely hinders its ability to interact with DNA, binding of haem to FurC could not be detected. Oxidative stress enhances the transcription of the three fur genes, especially that of furB and furC. In addition, overexpression of FurA and FurB in Escherichia coli increases survival when the cells are challenged with H(2)O(2) or Methyl Viologen (paraquat), a superoxide-anion-generating reagent. When present in saturating concentrations, FurB exhibits unspecific DNA-binding activity and protects DNA from cleavage produced by hydroxyl radicals or DNaseI. On the basis of these results, we suggest that, whereas at low concentrations FurB would act as a member of the Fur family, at saturating concentrations FurB protects DNA, showing a DNA-protection-during-starvation-like behaviour.

Domain analysis of the Edwardsiella tarda ferric uptake regulator

Recent studies have shown that the ferric uptake regulator (Fur) of Edwardsiella tarda (Fur(Et)) shares high sequence identity with the Escherichia coli Fur (Fur(Ec)) at the N-terminal DNA-binding region. In the present study, the functional importance of the C-terminal region of Fur(Et) was investigated. It was found that Fur(Et) bearing deletion of the C-terminal 12 residues still possesses most of the repressor activity, whereas Fur(Et) bearing deletions of the C-terminal 16 and more than 16 residues are severely affected in activity. Domain swapping analyses indicated that the chimeric Fur proteins (Et75Ec73 and Et75Vh74) consisting of the N-terminal 1-75 region of Fur(Et) fused to the C-terminal 76-148 region of Fur(Ec) and the C-terminal 76-149 region of the Vibrio harveyi Fur (Fur(Vh)), respectively, are fully active. C92 of Fur(Ec) and C137 of Fur(Vh), which are functionally essential in Fur(Ec) and Fur(Vh), respectively, are also essential in Et75Ec73 and Et75Vh74, respectively. Further study identified an artificial Fur protein, EtMF54, which is composed of the N-terminal 49 residues of Fur(Et) and five artificial residues. Compared to Fur(Et), EtMF54 possesses partial Fur activity that is iron-dependent. These results (i) indicate that there exist certain functional/structural compatibilities among Fur(Et), Fur(Ec), and Fur(Vh) at the C-terminal region; (ii) provide insights to the potential location of the regulatory ion-binding site of Fur(Et).

Contribution of the FeoB transporter to Streptococcus suis virulence.

The contribution of iron transporter systems encoded by feo genes to the pathogenic traits of streptococci is largely unknown, despite the fact that those systems are required for the full virulence of several gram-negative bacterial species. In this work, we show that the swine pathogen and zoonotic agent Streptococcus suis has a feoAB operon similar to that encoding an iron transporter system in Escherichia coli. Electrophoretic mobility assays and transcriptional analyses confirmed that the expression of S. suis feo genes is under the negative control of the ferric uptake regulator (Fur) protein. In vivo trials in mice using a feoB defective mutant strain were carried out to investigate the contribution of this gene to the virulence of S. suis. The results showed that the median lethal dose (LD50) of the mutant was approximately 10-fold higher than that of the wild-type parent strain. These data suggest that the Feo metal transporter plays a significant role in streptococcal infectious disease. This is in contrast to previous results reported for this same gene in other gram-positive bacterial species.

Snapshot of iron response in Shewanella oneidensis by gene network reconstruction

BackgroundIron homeostasis of Shewanella oneidensis, a γ-proteobacterium possessing high iron content, is regulated by a global transcription factor Fur. However, knowledge is incomplete about other biological pathways that respond to changes in iron concentration, as well as details of the responses. In this work, we integrate physiological, transcriptomics and genetic approaches to delineate the iron response of S. oneidensis.ResultsWe show that the iron response in S. oneidensis is a rapid process. Temporal gene expression profiles were examined for iron depletion and repletion, and a gene co-expression network was reconstructed. Modules of iron acquisition systems, anaerobic energy metabolism and protein degradation were the most noteworthy in the gene network. Bioinformatics analyses suggested that genes in each of the modules might be regulated by DNA-binding proteins Fur, CRP and RpoH, respectively. Closer inspection of these modules revealed a transcriptional regulator (SO2426) involved in iron acquisition and ten transcriptional factors involved in anaerobic energy metabolism. Selected genes in the network were analyzed by genetic studies. Disruption of genes encoding a putative alcaligin biosynthesis protein (SO3032) and a gene previously implicated in protein degradation (SO2017) led to severe growth deficiency under iron depletion conditions. Disruption of a novel transcriptional factor (SO1415) caused deficiency in both anaerobic iron reduction and growth with thiosulfate or TMAO as an electronic acceptor, suggesting that SO1415 is required for specific branches of anaerobic energy metabolism pathways.ConclusionUsing a reconstructed gene network, we identified major biological pathways that were differentially expressed during iron depletion and repletion. Genetic studies not only demonstrated the importance of iron acquisition and protein degradation for iron depletion, but also characterized a novel transcriptional factor (SO1415) with a role in anaerobic energy metabolism.

Salmonella entericaSerovar Typhimurium Strains with Regulated Delayed Attenuation In Vivo

Recombinant bacterial vaccines must be fully attenuated for animal or human hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Unfortunately, many well-studied means for attenuating Salmonella render strains more susceptible to host defense stresses encountered following oral vaccination than wild-type virulent strains and/or impair their ability to effectively colonize the gut-associated and internal lymphoid tissues. This thus impairs the ability of recombinant vaccines to serve as factories to produce recombinant antigens to induce the desired protective immunity. To address these problems, we designed strains that display features of wild-type virulent strains of Salmonella at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. We recently described one means to achieve this based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen. We report here a second means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC P(BAD) cassette for the promoters of the fur, crp, phoPQ, and rpoS genes such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur, Crp, PhoPQ, and/or RpoS proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Means for achieving regulated delayed attenuation can be combined with other mutations, which together may yield safe efficacious recombinant attenuated Salmonella vaccines.

Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

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.

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.

RstA-Promoted Expression of the Ferrous Iron Transporter FeoB under Iron-Replete Conditions Enhances Fur Activity in Salmonella enterica

The Fur protein is a primary regulator that monitors and controls cytoplasmic iron levels. We now report the identification of a regulatory pathway mediated by the Salmonella response regulator RstA that promotes Fur activity. Genome-wide expression experiments revealed that under iron-replete conditions, expression of the RstA protein from a plasmid lowered transcription levels of various genes involved in iron acquisition. The RstA protein controlled iron-responsive genes through the Fur-Fe(II) protein because deletion of the fur gene or iron depletion abrogated RstA-mediated repression of these genes. The RstA protein maintained wild-type levels of the Fur protein but exceptionally activated transcription of the feoAB operon encoding the ferrous iron transporter FeoB by binding directly to the feoA promoter. This FeoB induction resulted in increased ferrous iron uptake, which associates with the Fur protein because lack of RstA-dependent transcriptional activation of the feoA promoter and feoB-deletion abolished repression of the Fur target genes by the RstA protein. Under iron-replete conditions, RstA expression retarded Salmonella growth but enabled the Fur protein to repress the target genes beyond the levels which were simply accomplished by iron.

Pleiotropic roles of iron-responsive transcriptional regulator Fur in Burkholderia multivorans

The fur (ferric uptake regulator) gene of Burkholderia multivorans ATCC 17616 was identified by transposon mutagenesis analysis. The fur deletion mutant of strain ATCC 17616 (i) constitutively produced siderophores, (ii) was more sensitive to reactive oxygen species (ROS) than the wild-type strain, (iii) showed lower superoxide dismutase and catalase activities than the wild-type strain, (iv) was unable to grow on M9 minimal agar plates containing several substrates that can be used as sole carbon sources by the wild-type strain, and (v) was hypersensitive to nitrite and nitric oxide under microaerobic and aerobic conditions, respectively. These results clearly indicate that the Fur protein in strain ATCC 17616 plays pleiotropic roles in iron homeostasis, removal and/or resistance to ROS and nitrosative stress, and energy metabolism. Furthermore, employment of an in vivo Fur titration assay system led to the isolation from the ATCC 17616 genome of 13 Fur-binding DNA regions, and a subsequent electrophoretic mobility-shift assay confirmed the direct binding of Fur protein to all of these DNA regions. Transcriptional analysis of the genes located just downstream of the Fur-binding sites demonstrated that Fur acts as a repressor for these genes. Nine of the 13 regions were presumed to be involved in the acquisition and utilization of iron.

The Influence of Iron on Pseudomonas aeruginosa Physiology: A REGULATORY LINK BETWEEN IRON AND QUORUM SENSING

In iron-replete environments, the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein represses expression of two small regulatory RNAs encoded by prrF1 and prrF2. Here we describe the effects of iron and PrrF regulation on P. aeruginosa physiology. We show that PrrF represses genes encoding enzymes for the degradation of anthranilate (i.e. antABC), a precursor of the Pseudomonas quinolone signal (PQS). Under iron-limiting conditions, PQS production was greatly decreased in a DeltaprrF1,2 mutant as compared with wild type. The addition of anthranilate to the growth medium restored PQS production to the DeltaprrF1,2 mutant, indicating that its defect in PQS production is a consequence of anthranilate degradation. PA2511 was shown to encode an anthranilate-dependent activator of the ant genes and was subsequently renamed antR. AntR was not required for regulation of antA by PrrF but was required for optimal iron activation of antA. Furthermore, iron was capable of activating both antA and antR in a DeltaprrF1,2 mutant, indicating the presence of two distinct yet overlapping pathways for iron activation of antA (AntR-dependent and PrrF-dependent). Additionally, several quorum-sensing regulators, including PqsR, influenced antA expression, demonstrating that regulation of anthranilate metabolism is intimately woven into the quorum-sensing network of P. aeruginosa. Overall, our data illustrate the extensive control that both iron regulation and quorum sensing exercise in basic cellular physiology, underlining how intermediary metabolism can affect the regulation of virulence factors in P. aeruginosa.

Biosynthetic and regulatory elements involved in the production of the siderophore vanchrobactin in Vibrio anguillarum

Some Vibrio anguillarum strains produce a catechol-type siderophore named vanchrobactin, whose biosynthetic pathway has not been completely elucidated. In addition to the previously described genes vabA, vabC, vabB, vabE, vabF, vabS and vabH, in the present study we have identified the genes encoding a DAHP (3-deoxy-d-arabino-heptulosonate-7-phosphate) synthetase (vabG), a phosphopantheteinyl transferase (vabD), a LysR-family transcriptional regulator (vabR) and a putative siderophore receptor (fvtA). A deletion affecting vabG or vabD greatly reduced growth under iron-limiting conditions, whereas deletion of vabR did not have significant effects. Vanchrobactin production was abolished in the vabD mutant, whereas the vabG mutant retained a residual vanchrobactin production ability. Reverse transcriptase-mediated PCR indicated that this 11-gene cluster is organized into six iron-regulated transcriptional units. Transcriptional lacZ fusions demonstrated that the ferric uptake regulator (Fur) protein is the main iron-responsive regulator of these genes. Interestingly, the vabG gene was strongly iron-repressed, but Fur was not essential for this repression. In addition, the maximal expression from the vabG promoter was achieved only in the presence of an intact copy of vabR. Analysis of the beta-galactosidase activities of a fvtA : : lacZ fusion in a vabB mutant and in the presence of added vanchrobactin suggested that a ferric-vanchrobactin-dependent activator plays a positive regulatory role in transcription of the fvtA-vabD operon. This possibility is reinforced by the presence of a predicted AraC box upstream of fvtA. We propose that vanchrobactin biosynthesis is subjected to a complex regulatory circuitry aimed at adjusting vanchrobactin production for the maintenance of iron homeostasis in V. anguillarum.

Identification of Differentially Regulated Francisella tularensis Genes by Use of a Newly Developed Tn 5 -Based Transposon Delivery System

Francisella tularensis is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn5-based transposon delivery system that is capable of generating chromosomal reporter fusions with lacZ or luxCDABE, enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn5 transposase, whose expression is driven by the Francisella groES promoter. When high-temperature selection (42 degrees C) is applied to a bacterial culture carrying the transposon delivery plasmid, approximately 0.1% of the population is recovered with Tn5 insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the F. tularensis live vaccine strain as well as two different virulent F. tularensis strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the fsl operon, reported to be involved in iron acquisition, as well as genes in the igl gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of fsl but not igl reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in F. tularensis strains.

Expression of the Gonococcal Global Regulatory Protein Fur and Genes Encompassing the Fur and Iron Regulon during In Vitro and In Vivo Infection in Women

The ferric uptake regulatory protein, Fur, functions as a global regulatory protein of gene transcription in the mucosal pathogen Neisseria gonorrhoeae. We have shown previously that several N. gonorrhoeae Fur-repressed genes are expressed in vivo during mucosal gonococcal infection in men, which suggests that this organism infects in an iron-limited environment and that Fur is expressed under these conditions. In this study we have demonstrated expression of the gonococcal fur gene in vitro, in human cervical epithelial cells, and in specimens from female subjects with uncomplicated gonococcal infection. In vitro studies confirmed that the expression of the gonococcal fur gene was repressed during growth under iron-replete growth conditions but that a basal level of the protein was maintained. Using GFP transcriptional fusions constructed from specific Fur binding sequences within the fur promoter/operator region, we determined that this operator region was functional during N. gonorrhoeae infection of cervical epithelial cells. Furthermore, reverse transcription-PCR analysis, as well as microarray analysis, using a custom Neisseria Fur and iron regulon microarray revealed that several Fur- and iron-regulated genes were expressed during N. gonorrhoeae infection of cervical epithelial cells. Microarray analysis of specimens obtained from female subjects with uncomplicated gonococcal infection corroborated our in vitro findings and point toward a key role of gonococcal Fur- and iron-regulated genes in gonococcal disease.

The Iron-Responsive Fur Regulon in Yersinia pestis

The ferric uptake regulator (Fur) is a predominant bacterial regulator controlling the iron assimilation functions in response to iron availability. Our previous microarray analysis on Yersinia pestis defined the iron-Fur modulon. In the present work, we reannotated the iron assimilation genes in Y. pestis, and the resulting genes in complementation with those disclosed by microarray constituted a total of 34 genome loci (putative operons) that represent the potential iron-responsive targets of Fur. The subsequent real-time reverse transcription-PCR (RT-PCR) in conjunction with the primer extension analysis showed that 32 of them were regulated by Fur in response to iron starvation. A previously predicted Fur box sequence was then used to search against the promoter regions of the 34 operons; the homologue of the above box could be predicted in each promoter tested. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified His(6) tag-fused Fur protein was able to bind in vitro to each of these promoter regions. Therefore, Fur is a global regulator, both an activator and a repressor, and directly controls not only almost all of the iron assimilation functions but also a variety of genes involved in various non-iron functions for governing a complex regulatory cascade in Y. pestis. In addition, real-time RT-PCR, primer extension, EMSA, and DNase I footprinting assay were used to elucidate the Fur regulation of the ybt locus encoding a virulence-required iron uptake system. By combining the published data on the YbtA regulation of ybt, we constructed a concise Fur/YbtA regulatory network with a map of the Fur-promoter DNA interactions within the ybt locus. The data presented here give us an overview of the iron-responsive Fur regulon in Y. pestis.