Expression Of Iron Uptake Genes Research Articles

The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe

Pentatricopeptide repeat proteins are involved in mitochondrial both transcriptional and posttranscriptional regulation. Schizosaccharomyces pombe Ppr2 is a general mitochondrial translation factor that plays a critical role in the synthesis of all mitochondrial DNA-encoded oxidative phosphorylation subunits, which are essential for mitochondrial respiration. Our previous analysis showed that ppr2 deletion resulted in increased expression of iron uptake genes and caused ferroptosis-like cell death in S. pombe. In the present work, we showed that deletion of ppr2 reduced viability on glycerol- and galactose-containing media.Php4 is a transcription repressor that regulates iron homeostasis in fission yeast. We found that in the ppr2 deletion strain, Php4 was constitutively active and accumulated in the nucleus in the stationary phase. We also found that deletion of ppr2 decreased the ferroptosis-related protein Gpx1 in the mitochondria. Overexpression of Gpx1 improves the viability of Δppr2 cells. We showed that the deletion of ppr2 increased the production of ROS, downregulated heme synthesis and iron-sulfur cluster proteins, and induced stress proteins. Finally, we observed the nuclear accumulation of Pap1-GFP and Sty1-GFP, suggesting that Sty1 and Pap1 in response to cellular stress in the ppr2 deletion strain. These results suggest thatppr2 deletion may cause mitochondrial dysfunction, which is likely to lead to iron-sensing defect and iron starvation response, resulting in perturbation of iron homeostasis and increased hydroxyl radical production. The increased hydroxyl radical production triggers cellular responses in theppr2 deletion strain.

Zinc excess impairs Mycobacterium bovis growth through triggering a Zur-IdeR-iron homeostasis signal pathway.

Zinc excess is toxic to bacteria and, thus, represents an important innate defense mechanism of host cells, especially against mycobacterial infections. However, the signaling pathway triggered by zinc excess and its relationship with iron homeostasis remain poorly understood in mycobacteria. Here, we characterize a novel Zur-IdeR-iron homeostasis signaling pathway that modulates the growth of Mycobacterium bovis under zinc toxicity. We found that the regulator Zur interacts with the iron-homeostasis regulator IdeR, enhancing the DNA-binding ability of IdeR. Excess zinc disrupts this interaction and represses ideR transcription through Zur, which promotes the expression of iron uptake genes and leads to the accumulation of intracellular iron in M. bovis. The elevated iron levels lower the bacterial survival ability under excess zinc stress. Consistently, deleting zur hinders intracellular iron accumulation of M. bovis and enhances bacterial growth under stress, while silencing ideR impairs the growth of the wild-type and zur-deleted strains under the same conditions. Interestingly, both Zur and IdeR are conserved in bacteria facing zinc toxicity. Overall, our work uncovers a novel antimicrobial signal pathway whereby zinc excess disrupts iron homeostasis, which may deepen our understanding of the crosstalk mechanism between iron and zinc homeostasis in bacteria.IMPORTANCEAs a catalytic and structural cofactor of proteins, zinc is essential for almost all living organisms. However, zinc excess is toxic and represents a vital innate immunity strategy of macrophages to combat intracellular pathogens, especially against mycobacterial pathogens such as Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we first characterize an antibacterial signaling pathway of zinc excess and its relationship with iron homeostasis in M. bovis. We found that excess zinc inhibits the transcription of ideR and its DNA-binding activity through Zur, which, in turn, promotes the expression of iron uptake genes, causes intracellular iron accumulation, and finally impairs the bacterial growth. This study reveals the existence of the Zur-IdeR-iron homeostasis pathway triggered by zinc excess in M. bovis, which will shed light on the crosstalk mechanisms between zinc and iron homeostasis in bacteria and the antimicrobial mechanisms of host-mediated zinc toxicity.

The Iron Content of Human Serum Albumin Modulates the Susceptibility of Acinetobacter baumannii to Cefiderocol.

The mortality rates of patients infected with Acinetobacter baumannii who were treated with cefiderocol (CFDC) were not as favorable as those receiving the best available treatment for pulmonary and bloodstream infections. Previous studies showed that the presence of human serum albumin (HSA) or HSA-containing fluids, such as human serum (HS) or human pleural fluid (HPF), in the growth medium is correlated with a decrease in the expression of genes associated with high-affinity siderophore-mediated iron uptake systems. These observations may explain the complexities of the observed clinical performance of CFDC in pulmonary and bloodstream infections, because ferric siderophore transporters enhance the penetration of CFDC into the bacterial cell. The removal of HSA from HS or HPF resulted in a reduction in the minimal inhibitory concentration (MIC) of CFDC. Concomitant with these results, an enhancement in the expression of TonB-dependent transporters known to play a crucial role in transporting iron was observed. In addition to inducing modifications in iron-uptake gene expression, the removal of HSA also decreased the expression of β-lactamases genes. Taken together, these observations suggest that environmental HSA has a role in the expression levels of select A. baumannii genes. Furthermore, the removal of iron from HSA had the same effect as the removal of HSA upon the expression of genes associated with iron uptake systems, also suggesting that at least one of the mechanisms by which HSA regulates the expression of certain genes is through acting as an iron source.

Highly Metastatic Subpopulation of TNBC Cells Has Limited Iron Metabolism and Is a Target of Iron Chelators.

Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate a possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo. Lung metastasis was further significantly reduced, leading to the hypothesis that iron metabolism between metastatic and non-metastatic cells may be different. An analysis of existing database demonstrated that the expression of iron-uptake genes was significantly suppressed in TNBC cells that metastasized to lymph nodes or lungs compared to those in primary tumors. A highly metastatic clone of the murine 4T1 TNBC cells (4T1-HM) did not proliferate well under iron-rich or iron-depleted conditions by iron chelators compared to a low-metastatic clone (4T1-LM). Bulk RNA-seq analysis of RNA from 4T1-HM and 4T1-LM cells suggested that the PI3K-AKT pathway might be responsible for this difference. Indeed, DFX suppressed the proliferation via the AKT-mTOR pathway in 4T1-HM and the human MDA-MB-231 cells, a human mesenchymal-like TNBC cell line. DFX also suppressed the growth of 4T1-HM tumors in comparison to 4T1-LM tumors, and reduced lung metastases after surgical resection of primary 4T1 tumors. These results indicated, for the first time, that highly metastatic TNBC cells have limited iron metabolism, and they can be more effectively targeted by iron chelators.

Non-mitochondrial aconitase regulates the expression of iron-uptake genes by controlling the RNA turnover process in fission yeast.

Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.

Vibrio parahaemolyticus FcrX, a Fur-controlled regulator that inhibits repression by Fur.

Iron is an essential nutrient for most organisms, but its limited availability and inherent toxicity necessitate the strict regulation of iron homeostasis. In bacteria, iron starvation affects a broad range of phenotypes including virulence, motility and biofilm formation. For Vibrio parahaemolyticus, a marine bacterium and pathogen, iron limitation is a signal modulating swarmer cell differentiation. In this work, we show the iron regulation of swarming works through the ferric uptake regulator protein Fur. We identified a new Fur-controlled regulator that is upregulated upon iron starvation. FcrX is a 144-amino acid protein containing a domain of unknown function (DUF2753) with three tetratricopeptide repeats. We found that overexpressing fcrX+ was sufficient to induce swarming, luminescence and iron uptake gene expression in multiple Vibrio species; furthermore, ectopic expression increased the transcription of a Fur-controlled gene in Escherichia coli. FcrX production increased intracellular iron. Thus, the overexpression of fcrX+ phenocopied a fur mutant and may prove a generally useful tool to ectopically derepress the Fur regulon. Both V. parahaemolyticus and E. coli Fur interacted with FcrX, and this interaction was altered by iron availability. These data support a model in which this new regulator of iron homeostasis limits the repressive action of Fur.

Four IVa bHLH Transcription Factors Are Novel Interactors of FIT and Mediate JA Inhibition of Iron Uptake in Arabidopsis

Plants have evolved sophisticated genetic networks to regulate iron (Fe) homeostasis for their survival. Several classes of plant hormones including jasmonic acid (JA) have been shown to be involved in regulating the expression of iron uptake and/or deficiency-responsive genes in plants. However, the molecular mechanisms by which JA regulates iron uptake remain unclear. In this study, we found that JA negatively modulates iron uptake by downregulating the expression of FIT (bHLH29), bHLH38, bHLH39, bHLH100, and bHLH101 and promoting the degradation of FIT protein, a key regulator of iron uptake in Arabidopsis. We further demonstrated that the subgroup IVa bHLH proteins, bHLH18, bHLH19, bHLH20, and bHLH25, are novel interactors of FIT, which promote JA-induced FIT protein degradation. These four IVa bHLHs function redundantly to antagonize the activity of the Ib bHLHs (such as bHLH38) in regulating FIT protein stability under iron deficiency. The four IVa bHLH genes are primarily expressed in roots, and are inducible by JA treatment. Moreover, we found that MYC2 and JAR1, two critical components of the JA signaling pathway, play critical roles in mediating JA suppression of the expression of FIT and Ib bHLH genes, whereas they differentially modulate the expression of bHLH18, bHLH19, bHLH20, and bHLH25 to regulate FIT accumulation under iron deficiency. Taken together, these results indicate that by transcriptionally regulating the expression of different sets of bHLH genes JA signaling promotes FIT degradation, resulting in reduced expression of iron-uptake genes, IRT1 and FRO2, and increased sensitivity to iron deficiency. Our data suggest that there is a multilayered inhibition of iron-deficiency response in the presence JA in Arabidopsis.

Distinct iron homeostasis in Th1- and Th2-biased mouse strains

Abstract Iron is a proinflammatory transition metal ion, yet limited information is documented on the differences in iron homeostasis under Th1 and Th2 biased conditions. We used C57BL/6 (BL6) and Balb/C (BB) strains, which exhibit prototypical Th1 and Th2 immune responses, to investigate the extent to which Th1 and Th2 biased responses dictate iron homeostasis during healthy and inflammatory conditions. Systemic and tissue iron levels were assessed in age and gender matched BL6 and BB mice. Gene expression for iron absorption, transport, and storage proteins were quantified in duodenum and liver. Intracellular labile iron pool (LIP) was assessed in bone marrow-derived macrophages (BMDM) and neutrophils (BMDN). Hypoferremia of inflammation was assessed by measuring serum iron before and after LPS treatment. BB mice displayed significantly higher levels of systemic and storage iron than BL6 mice. Although the expression of hepcidin and ferroportin were comparable between the two strains, hepcidin-independent iron absorption is more prominent in BB mice, as indicated by higher expression of genes involved in iron absorption and transport: DcytB, DMT1, SFT, and hephaestin. Similarly, hepatic TfR1, TfR2, transferrin, and DMT1 expression was higher in BB mice. Moreover, the basal levels of LIP in BB BMDM and BMDN were 3.4 and 2.6 folds higher than BL6, respectively. The LPS-induced hypoferremia of inflammation is more pronounced in BL6 than BB mice. Taken together, iron homeostasis is distinct between these two strains during both healthy and inflammatory conditions. BB mice showed more iron load than BL6 mice, which could be explained, in part, by the higher expression of iron uptake genes and also reduced Th1 responses in BB mice.

Tissue-Specific Regulation of Gibberellin Signaling Fine-Tunes Arabidopsis Iron-Deficiency Responses

Iron is an essential element for most living organisms. Plants acquire iron from the rhizosphere and have evolved different biochemical and developmental responses to adapt to a low-iron environment. In Arabidopsis, FIT encodes a basic helix-loop-helix transcription factor that activates the expression of iron-uptake genes in root epidermis upon iron deficiency. Here, we report that the gibberellin (GA)-signaling DELLA repressors contribute substantially in the adaptive responses to iron-deficient conditions. When iron availability decreases, DELLAs accumulate in the root meristem, thereby restraining root growth, while being progressively excluded from epidermal cells in the root differentiation zone. Such DELLA exclusion from the site of iron acquisition relieves FIT from DELLA-dependent inhibition and therefore promotes iron uptake. Consistent with this mechanism, expression of a non-GA-degradable DELLA mutant protein in root epidermis interferes with iron acquisition. Hence, spatial distribution of DELLAs in roots is essential to fine-tune the adaptive responses to iron availability.

SlbHLH068 interacts with FER to regulate the iron-deficiency response in tomato.

Iron is an essential micronutrient for all organisms and its uptake, translocation, distribution and utilization are regulated in a complex manner in plants. FER, isolated from tomato (Solanum lycopersicum), was the first transcription factor involved in the iron homeostasis of higher plants to be identified. A FER defect in the T3238fer mutant drastically downregulates the expression of iron uptake genes, such as ferric-chelate reductase 1 (LeFRO1) and iron-regulated transporter 1 (LeIRT1); however, the molecular mechanism by which FER regulates genes downstream remains unknown. The aim of this work was therefore to identify the gene that interacts with FER to regulate the iron-deficiency response in tomato. The homologue of the Arabidopsis Ib subgroup of the basic helix-loop-helix (bHLH) proteins, SlbHLH068, was identified by using the program BLASTP against the AtbHLH39 amino acid sequence in the tomato genome. The interaction between SlbHLH068 and FER was detected using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. In addition, virus-induced gene silencing (VIGS) was used to generate tomato plants in which SlbHLH068 expression was downregulated. The expression of genes was analysed using northern blot hybridization and multiple RT-PCR analysis. Seedlings of wild-type and mutant plants were grown under conditions of different nutrient deficiency. SlbHLH068 is highly upregulated in roots, leaves and stems in response to iron deficiency. An interaction between SlbHLH068 and FER was demonstrated using yeast two-hybrid and BiFC assays. The heterodimer formed by FER with SlbHLH068 directly bound to the promoter of LeFRO1 and activated the expression of its reporter gene in the yeast assay. The downregulation of SlbHLH068 expression by VIGS resulted in a reduction of LeFRO1 and LeIRT1 expression and iron accumulation in leaves and roots. The results indicate that SlbHLH068, as a putative transcription factor, is involved in iron homeostasis in tomato via an interaction with FER.

Mediator subunit 16 functions in the regulation of iron uptake gene expression in Arabidopsis.

Iron is an essential nutrient for plant growth and development, and its absorption is tightly controlled. Under iron limitation, FIT dimerizes with the four Ib bHLH proteins and activates the expression of iron uptake genes. However, how the dimerized complex activates downstream genes remains unclear. Using forward genetics, a low-iron-sensitive mutant was screened. The corresponding gene (MED16) was isolated, and its biological functions in iron homeostasis were characterized using approaches such as gene expression, protein subcellular localization, protein-protein interaction and chromatin immunoprecipitation assay. Lesion of MED16 significantly reduced FRO2 and IRT1 expression in Arabidopsis roots. The MED16 mutants showed a low shoot iron concentration and severe leaf chlorosis under iron limitation, whereas it grew normally as wild-type under iron sufficiency. Furthermore, we showed that MED16 interacted with FIT and improved the binding of the FIT/Ib bHLH complex to FRO2 and IRT1 promoters under iron-deficient conditions. Additionally, we found that many iron-deficient response genes, which are regulated by FIT, were also controlled by MED16. In conclusion, MED16 is involved in the iron deficiency response, and modulates the iron uptake gene expression under iron limitation. Our results increase the understanding of the molecular regulation mechanisms underlying iron uptake and homeostasis in plants.

Dynamics in the rhizosphere and iron-uptake gene expression in peanut induced by intercropping with maize: Role in improving iron nutrition in peanut

The intercropping of maize with peanuts is an effective cropping practice. Indeed, peanut/maize intercropping reportedly improves the iron nutrition of peanuts in calcareous soils. The limited evidence available suggests that the improved Fe nutrition in intercropping is largely attributable to a rhizosphere effect of maize. In this study, the effects of peanut/maize intercropping on the Fe nutritional status of peanut associated with the dynamics of the rhizosphere processes and Fe uptake gene expression induced by the interaction of the two species at various growth days were investigated. The results suggest that an interspecific rhizosphere effect improves Fe nutrition in peanut, as shown by changes in the rhizosphere available Fe concentration, pH, and Olsen-P concentration, based on time-course changes in peanut–maize interaction. The increase in available Fe in the rhizosphere of peanut ranged from 0.2 to 2.64 mg kg−1. The transition from the vegetative to reproductive stage was a key turning point in the time-course of changes in the rhizosphere processes in intercropping. There was more consistently positive effect of intercropping on peanut Fe nutrition after 53 days. Moreover, the expression of AhFRO1 and AhYSL1 was expressed at significantly higher level in intercropped peanuts compared to monocropped peanut at the vegetative stage, indicating a role for these genes in Fe improvement in intercropped peanuts. We conclude that the enhanced time-course changes in the rhizosphere processes and iron uptake gene expression with a consistent positive interspecific effect appear to be one of the mechanisms underlying the improved Fe nutrition in intercropped peanut plants.

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.

FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis

Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase (FRO2) and ferrous transporter (IRT1) promoters. Overexpression of FIT with either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.

Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots

Long-distance signals generated in shoots are thought to be associated with the regulation of iron uptake from roots; however, the signaling mechanism is still unknown. To elucidate whether the signal regulates iron uptake genes in roots positively or negatively, we analyzed the expressions of two representative iron uptake genes: NtIRT1 and NtFRO1 in tobacco (Nicotiana tabacum L.) roots, after shoots were manipulated in vitro. When iron-deficient leaves were treated with Fe(II)-EDTA, the expressions of both genes were significantly reduced; nevertheless iron concentration in the roots maintained a similar level to that in roots grown under iron-deficient conditions. Next, all leaves from tobacco plants grown under the iron-deficient condition were excised. The expression of two genes were quickly reduced below half within 2 h after the leaf excision and gradually disappeared by the end of a 24-h period. The NtIRT1 expression was compared among the plants whose leaves were cut off in various patterns. The expression increased in proportion to the dry weight of iron-deficient leaves, although no relation was observed between the gene expression and the position of excised leaves. Interestingly, the NtIRT1 expression in hairy roots increased under the iron-deficient condition, suggesting that roots also have the signaling mechanism of iron status as well as shoots. Taken together, these results indicate that the long-distance signal generated in iron-deficient tissues including roots is a major factor in positive regulation of the expression of NtIRT1 and NtFRO1 in roots, and that the strength of the signal depends on the size of plants.

Cobalt Stress in Escherichia coli: THE EFFECT ON THE IRON-SULFUR PROTEINS

Cobalt is toxic for cells, but mechanisms of this toxicity are largely unknown. The biochemical and genetic experiments reported here demonstrate that iron-sulfur proteins are greatly affected in cobalt-treated Escherichia coli cells. Exposure of a wild-type strain to intracellular cobalt results in the inactivation of three selected iron-sulfur enzymes, the tRNA methylthio-transferase, aconitase, and ferrichrome reductase. Consistently, mutant strains lacking the [Fe-S] cluster assembly SUF machinery are hypersensitive to cobalt. Last, expression of iron uptake genes is increased in cells treated with cobalt. In vitro studies demonstrated that cobalt does not react directly with fully assembled [Fe-S] clusters. In contrast, it reacts with labile ones present in scaffold proteins (IscU, SufA) involved in iron-sulfur cluster biosynthesis. We propose a model wherein cobalt competes out iron during synthesis of [Fe-S] clusters in metabolically essential proteins.

Iron homeostasis in the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe has acquisition processes for iron, an essential nutrient. One pathway consists to produce, excrete, and capture siderophore-iron complexes. A second pathway requires enzymatic reduction of ferric iron at the cell surface prior to uptake by a permease-oxidase complex. Genes encoding proteins involved in iron assimilation are transcriptionally regulated as a function of iron availability. Under high iron conditions, the GATA-type regulator Fep1 represses the expression of iron uptake genes. The repressor function of Fep1 requires the presence of the Tup11 or Tup12 transcriptional co-repressor. Under low iron conditions, two regulatory mechanisms occur. First, the iron transport genes are highly induced. Second, there is a transcription factor cascade implicating the heteromeric CCAAT-binding complex that turns off a set of genes encoding iron-utilizing proteins, presumably to avoid a futile expenditure of energy in producing iron-using proteins that lack the necessary cofactor to function. Thus, collectively, these regulatory responses to variations in iron concentrations ensure that iron is present within cells for essential biochemical reactions, yet prevent the accumulation of iron or iron-using proteins to deleterious levels.

The Actinobacillus actinomycetemcomitans Ribose Binding Protein RbsB Interacts with Cognate and Heterologous Autoinducer 2 Signals

Autoinducer 2 (AI-2) produced by the oral pathogen Actinobacillus actinomycetemcomitans influences growth of the organism under iron limitation and regulates the expression of iron uptake genes. However, the cellular components that mediate the response of A. actinomycetemcomitans to AI-2 have not been fully characterized. Analysis of the complete genome sequence of A. actinomycetemcomitans (www.oralgen.lanl.gov) indicated that the RbsB protein was related to LuxP, the AI-2 receptor of Vibrio harveyi. To determine if RbsB interacts with AI-2, the bioluminescence of the reporter strain V. harveyi BB170 (sensor 1-, sensor 2+) was determined after stimulation with partially purified AI-2 from A. actinomycetemcomitans or conditioned medium from V. harveyi cultures in the presence and absence of purified six-His-tagged RbsB. RbsB efficiently inhibited V. harveyi bioluminescence induced by both A. actinomycetemcomitans AI-2 and V. harveyi AI-2 in a dose-dependent manner, suggesting that RbsB competes with LuxP for AI-2. Fifty percent inhibition occurred with approximately 0.3 nM RbsB for A. actinomycetemcomitans AI-2 and 15 nM RbsB for V. harveyi AI-2. RbsB-mediated inhibition of V. harveyi bioluminescence was reversed by the addition of 50 mM ribose, suggesting that A. actinomycetemcomitans AI-2 and ribose bind at the same site of RbsB. The RbsB/AI-2 complex was thermostable since A. actinomycetemcomitans AI-2 could not be recovered by heating. This was not due to heat inactivation of A. actinomycetemcomitans AI-2 since signal activity was unaffected by heating in the absence of RbsB. Furthermore, an isogenic A. actinomycetemcomitans mutant that was unable to express rbsB was deficient in depleting A. actinomycetemcomitans AI-2 from solution relative to the wild-type organism. Inactivation of rbsB also influenced the ability of the organism to grow under iron-limiting conditions. The mutant strain attained a cell density of approximately 30% that of the wild-type organism under iron limitation. In addition, real-time PCR showed that the expression of afuABC, encoding a major ferric ion transporter, was reduced by approximately eightfold in the rbsB mutant. This phenotype was similar to that of a LuxS-deficient mutant of A. actinomycetemcomitans that is unable to produce AI-2. Together, our results suggest that RbsB may play a role in the response of A. actinomycetemcomitans to AI-2.

Iron limitation induces SpoT‐dependent accumulation of ppGpp in Escherichia coli

In Escherichia coli the beta-lactam mecillinam specifically inhibits penicillin-binding protein 2 (PBP2), a peptidoglycan transpeptidase essential for maintaining rod shape. We have previously shown that PBP2 inactivation results in a cell division block and that an increased concentration of the nucleotide ppGpp, effector of the RelA-dependent stringent response, confers mecillinam resistance and allows cells to divide as spheres in the absence of PBP2 activity. In this study we have characterized an insertion mutation which confers mecillinam resistance in wild-type and DeltarelA strains but not in DeltarelADeltaspoT strains, devoid of ppGpp. The mutant has an insertion in the fes gene, coding for enterochelin esterase. This cytoplasmic enzyme hydrolyses enterochelin-Fe(3+) complexes, making the scavenged iron available to the cells. We show that inactivation of the fes gene causes iron limitation on rich medium plates and a parallel SpoT-dependent increase of the ppGpp pool, as judged by the induction of the iron-regulated fiu::lacZ fusion and the repression of the stringently controlled P1(rrnB)::lacZ fusion respectively. We further show, by direct ppGpp assays, that iron starvation in liquid medium produces a SpoT-dependent increase of the ppGpp pool, strongly suggesting a role for iron in the balance of the two activities of SpoT, synthesis and hydrolysis of (p)ppGpp. Finally, we present evidence that ppGpp exerts direct or indirect positive control on iron uptake, suggesting a simple homeostatic regulatory circuit: iron limitation leads to an increased ppGpp pool, which increases the expression of iron uptake genes, thereby alleviating the limitation.

The Schizosaccharomyces pombe corepressor Tup11 interacts with the iron-responsive transcription factor Fep1.

The Schizosaccharomyces pombe fep1(+) gene encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron concentrations. This transcriptional response is altered only in strains harboring a combined deletion of both tup11(+) and tup12(+) genes. This suggests that Tup11 is capable of negatively regulating iron transport gene expression in the absence of Tup12 and vice versa. The tup11(+)- and tup12(+)-encoded proteins resemble the Saccharomyces cerevisiae Tup1 corepressor. Using yeast two-hybrid analysis we show that Tup11 and Fep1 physically interact with each other. The C-terminal region from amino acids 242 to 564 of Fep1 is required for interaction with Tup11. Within this region, a minimal domain encompassing amino acids 405-541 was sufficient for Tup11-Fep1 association. Deletion mapping analysis revealed that the WD40-repeat sequence motifs of Tup11 are necessary for its interaction with Fep1. Analysis of Tup11 mutants with single amino acid substitutions in the WD40 repeats suggested that the Fep1 transcription factor interacts with a putative flat upper surface on the predicted beta-propeller structure of this motif. Further analysis by in vivo coimmunoprecipitation showed that Tup11 and Fep1 are physically associated. In vitro pull-down experiments further verified a direct interaction between the Fep1 C terminus and the Tup11 C-terminal WD40 repeat domain. Taken together, these results describe the first example of a physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes.