Iron Uptake Genes Research Articles

Generation of Genetic Tools for Gauging Multiple-Gene Expression at the Single-Cell Level.

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple-gene expression at the single-cell level has limited the understanding of phenotypic heterogeneity. To investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple-gene expression at the single-cell level has been generated. This tool, named pRGC, consists of a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP), and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single-cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population without the need for image processing for spectral cross talk correction. In addition, two pRGC variants have been generated for either (i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome that is suitable for long-term experiments in the absence of antibiotic selection or (ii) replication in bacterial genera other than Pseudomonas The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple-gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity.IMPORTANCE Within a bacterial population, single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial subpopulations with distinct phenotypes. The analysis of gene expression at the single-cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation, and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple-gene expression at the single-cell level by fluorescence microscopy without the need for advanced image-processing procedures. A proof of concept has been provided by investigating iron uptake and iron storage gene expression in response to iron availability in P. aeruginosa.

Phylogenetic genotyping, virulence genes and antimicrobial susceptibility of Escherichia coli isolates from cases of bovine mastitis.

The study described in this research communication used phylogenetic genotyping to identify virulence genes and antimicrobial susceptibility in Escherichia coli recovered from cases of bovine mastitis. From 385 mastitic milk samples, 30 (7.8%) isolates were confirmed as E. coli. Most isolates (80%) belonged to phylo-group A. These 30 E. coli isolates were also screened for 11 different virulence genes. The majority of isolates (63%) harbored no virulence gene. Only 11 (37%) isolates tested positive for two virulence genes, either the iron uptake gene iucD in 3 (10%) isolates or the serum resistance gene traT in 2 (7%) isolates or both traT and iucD in 6 (20%) isolates. The E. coli isolates showed highest susceptibility to gentamicin, meropenem, and pipracillin. Most isolates were resistant to ampicillin, cefotaxime and streptomycin. This study suggests that mastitis causing E. coli might originate from commensal bacteria and that the presence of these virulence genes, common in extra-intestinal pathogenic E. coli (ExPEC) strains could be attributed to high genetic variability of mastitis-causing E. coli.

IurV, Encoded by ORF VCA0231, Is Involved in the Regulation of Iron Uptake Genes in Vibrio cholerae.

The pathogen Vibrio cholerae has multiple iron acquisition systems which allow bacteria to exploit a variety of iron sources across the different environments on which it thrives. The expression of such iron uptake systems is highly regulated, mainly by the master iron homeostasis regulator Fur but also by other mechanisms. Recently, we documented that the expression of many of the iron-responsive genes is also modulated by riboflavin. Among them, the open reading frame VCA0231, repressed both by riboflavin and iron, encodes a putative transcriptional regulator of the AraC/XylS family. Nonetheless, the genes or functions affected by this factor are unknown. In the present study, a series of in silico analyses was performed in order to identify the putative functions associated with the product of VCA0231. The STRING database predicted many iron uptake genes as functional partners for the product of VCA0231. In addition, a genomic neighborhood analysis with the Enzyme Function Initiative tools detected many Pfam families involved in iron homeostasis genetically associated with VCA0231. Moreover, a phylogenetic tree showed that other AraC/XylS members known to regulate siderophore utilization in bacteria clustered together and the product of VCA0231 localized in this cluster. This suggested that the product of VCA0231, here named IurV, is involved in the regulation of iron uptake processes. RNAseq was performed to determine the transcriptional effects of a deletion in VCA0231. A total of 52 genes were overexpressed and 21 genes were downregulated in response to the iurV deletion. Among these, several iron uptake genes and other iron homeostasis-related genes were found. Six gene ontology (GO) functional terms were enriched in the upregulated genes, of which five were related to iron metabolism. The regulatory pattern observed in the transcriptomics of a subset of genes was independently confirmed by quantitative real time PCR analysis. The results indicate that IurV is a novel regulator of the AraC/XylS family involved in the repression of iron uptake genes. Whether this effect is direct or indirect remains to be determined.

Pre- sowing seed treatment with salicylic acid and sodium hydrosulfide confers Pb toxicity tolerance in maize (Zea mays L.)

In the present study, impact of salicylic acid (SA), sodium hydrosulfide (NaHS) and concomitant application of SA and NaHs seed priming was investigated in alleviation of the lead stress adverse effects on growth parameters, total chlorophyll content, dicarbonyl stress, and lead-induced iron deficiency in maize. Maize seeds were soaked in 0.5 mM SA and 0.5 mM NaHS individually and in 0.25 mM SA and 0.25 mM NaHS concomitantly for 12 h. The 6 day old plants were subjected to 2.5 mM Pb(NO3)2 for 9 days. Lead stress caused a significant decrease in growth parameters and total chlorophyll and shoot iron contents, whereas increased lead and methylglyoxal accumulation significantly. The cysteine and methionine contents elevated in shoots of lead stressed plants; probably due to redirection of sulfur assimilation requirement for lead detoxification. The SA, NaHS and SA+NaHS applications modulated glyoxalase I activity and resulted in remarkable reduction in methylglyoxal accumulation during lead stress. Lead induced iron deficiency was reverted under SA, NaHS and SA+NaHS seed priming in shoots, probably through reduction in lead uptake and increase in nitric oxide content. Lead imposition activated iron starvation pathway via elevation in methionine content and expression of iron uptake and hemostasis-related genes including Yellow Stripe1 (ZmYS 1), S-adenosylmethionine synthase (ZmSAMS) and 2′-deoxymugineic acid synthase (ZmDMAS1) in roots. However, the ZmSAMS and ZmDMAS1 transcript levels did not change under lead exposure in shoots. The SA, NaHS and SA+NaHS seed primed plants displayed downregulation of ZmSAMS and ZmDMAS1 in shoots and roots under lead stress. In conclusion, seed priming with SA and NaHS could improve lead tolerance in maize via reduction in the Pb uptake, consequently lowering lead toxicity in the food chain.

Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds

Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34540), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.

Uncovering mechanisms of greengage wine fermentation against acidic stress via genomic, transcriptomic, and metabolic analyses of Saccharomyces cerevisiae.

Acid stress is one of the most common adverse conditions during fermentation of fruit wines, and the acid tolerance of yeasts is, therefore, critical for fruit wine production. However, the biological mechanism underlying the acquired tolerance of yeasts against acid stress is poorly understood. We have previously obtained an evolved Saccharomyces cerevisiae strain ET008-c54 with increased tolerance against acid stress, and potentially, it serves as a promising yeast strain for greengage wine fermentation. In the current study, we further revealed the alterations responsible for the adaptation of ET008-c54 to low pH by whole-genome re-sequencing, transcriptomic, and metabolic analyses. Results confirmed the outstanding fermenting performance of ET008-c54 at low pH as compared with the parental ET008. More specifically, the growth rate of ET008-c54 at low pH was increased by 6.24 times and the fermentation time was shortened by 70%. Differences were also observed in the physiology of the strains through ergosterol, H+-ATPase activity, and aroma determinations. By integrating both RNA-seq and whole-genome re-sequencing data, we demonstrated some metabolic pathways in ET008-c54, namely ergosterol synthesis and ferrous iron uptake, in which several acid-responsive genes were involved being upregulated. Also, upregulation of the pathways responsible for aroma compound formation, including fatty acid ethyl ester synthesis and aromatic amino acid biosynthesis, was identified. Thus, the enhanced fermentation ability of ET008-c54 at low pH should be, at least partly, contributed by the altered gene expressions associated with the aforementioned pathways. By elucidating the biological mechanism of yeasts against acid stress, this current study allows better-defined targets for future studies of genetic improvement of wine yeasts and enhancement of the fermentation processes. KEY POINTS: • Metabolic analysis confirmed the excellent fermentation performance of ET008-c54. • Acid tolerance genes for ergosterol synthesis and ferrous iron uptake were upregulated. • Aroma genes for fatty acid ethyl ester and aromatic amino acid synthesis were upregulated.

Bacterial Compound N,N-Dimethylhexadecylamine Modulates Expression of Iron Deficiency and Defense Response Genes in Medicago truncatula Independently of the Jasmonic Acid Pathway

Plants face a variety of biotic and abiotic stresses including attack by microbial phytopathogens and nutrient deficiencies. Some bacterial volatile organic compounds (VOCs) activate defense and iron-deficiency responses in plants. To establish a relationship between defense and iron deficiency through VOCs, we identified key genes in the defense and iron-deprivation responses of the legume model Medicago truncatula and evaluated the effect of the rhizobacterial VOC N,N-dimethylhexadecylamine (DMHDA) on the gene expression in these pathways by RT-qPCR. DMHDA increased M. truncatula growth 1.5-fold under both iron-sufficient and iron-deficient conditions compared with untreated plants, whereas salicylic acid and jasmonic acid decreased growth. Iron-deficiency induced iron uptake and defense gene expression. Moreover, the effect was greater in combination with DMHDA. Salicylic acid, Pseudomonas syringae, jasmonic acid, and Botrytis cinerea had inhibitory effects on growth and iron response gene expression but activated defense genes. Taken together, our results showed that the VOC DMHDA activates defense and iron-deprivation pathways while inducing a growth promoting effect unlike conventional phytohormones, highlighting that DMHDA does not mimic jasmonic acid but induces an alternative pathway. This is a novel aspect in the complex interactions between biotic and abiotic stresses.

Asymptomatic carriers of Neisseria meningitidis and Moraxella catarrhalis in healthy children.

There is a close correlation between asymptomatic oropharyngeal colonization by bacterial pathogens and paediatric respiratory diseases. Evaluation of the frequency of asymptomatic carriers of Neisseria meningitidis and Moraxella catarrhalis in healthy children was the main aim of the current study. In this cross-sectional study, 123 oropharyngeal swabs were collected from children between 2 and 6 years old in kindergartens of Ilam, Iran. Moraxella catarrhalis and N. meningitidis were identified using phenotypic and genotypic assays. In addition, the occurrence of the virulence factors (ctrA and uspA1) and iron uptake (tbpA) genes was evaluated by PCR. Results showed that 21 M. catarrhalis isolates and 17 N. meningitidis isolates were identified by conventional microbiological and biochemical methods, but the RT-PCR assay detected that 18 and 8 isolates were positive for M. catarrhalis and N. meningitidis, respectively. The tbpA gene was positive in all N. meningitidis and M. catarrhalis isolates. Seven isolates were positive for the ctrA gene in N. meningitidis and seven isolates were positive for the uspA1 gene in M. catarrhalis. These pathogenic bacteria often occurred as asymptomatic carriage of N. meningitidis in children from large families with low economic status, which reflects the importance of the environment and socio-economic level of families in the distribution of these potentially pathogenic bacteria in the oropharynx of children. Monitoring for the carriage of potential pathogenic bacteria in the nasopharynx of healthy children is important as this can predispose to infectious diseases; common exposure to human respiratory bacterial pathogens is a further risk factor.

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.

Mimosine facilitates metallic cation uptake by plants through formation of mimosine–cation complexes

Iron deficiency conditions as well as iron supplied as a Fe(III)-mimosine complex induced a number of strategy I and strategy II genes for iron uptake in leucaena. Leucaena leucocephala (leucaena) is a tree-legume that can grow in alkaline soils, where metal-cofactors like Fe(III) are sparingly available. Mimosine, a known chelator of Fe(III), may facilitate Fe(III) uptake in leucaena by serving as a phytosiderophore. To test if mimosine can serve as a phytosiderophore, three sets of experiments were carried out. First, the binding properties and solubility of metal-mimosine complexes were assessed through spectrophotometry. Second, to study mimosine uptake in plants, pole bean, common bean, and tomato plants were supplied with mimosine alone and metal-mimosine complexes. Third, the expression of strategy I (S1) and strategy II (S2) genes for iron uptake from the soil was studied in leucaena plants exposed to different Fe(III) complexes. The results of this study show that (i) mimosine has high binding affinity for metallic cations at alkaline pH, Fe(III)-mimosine complexes are water soluble at alkaline pH, and that mimosine can bind soil iron under alkaline pH; (ii) pole bean, common bean, and tomato plants can uptake mimosine and transport it throughout the plant; and (iii) a number of S1 and S2 genes were upregulated in leucaena under iron-deficiency condition or when Fe(III) was supplied as a Fe(III)-mimosine complex. These findings suggest that leucaena may utilize both S1 and S2 strategies for iron uptake; and mimosine may play an important role in both strategies.

Identification of Some Virulence Genes of E. coli Isolated from Humans and Broilers in Alexandria Governorate

Avian pathogenic Escherichia coli (APEC) and human uropathogenic Escherichia coli (UPEC) may encounter similar difficulties during the establishment of infection in extraintestinal sites. They may share similar virulence genes and the ability to cause disease. In the present study, 12 APEC isolates and 16 UPEC isolates were compared by their virulence genes content, The two groups showed a significant overlap of virulence genotypes including genes encoding adhesins (fimH, fimAvMT78, and papC), which were the most prevalent genes among both APEC and human UPEC isolates. Genes related to large transmissible R plasmid known as pTJ100 plasmid (isS, iutA, sitA, and traT) were more dominant among APEC than UPEC isolates, iron-related (feoB gene) that mediates ferric iron uptake and ompT gene which encodes outer membrane proteins and presented with high incidence in APEC isolates, while tsh gene was absent in the two groups. these results indicated that the examined virulence genes occurred among both APEC and human UPEC isolates but with differences in their prevalence, these similarities give a hypothesis that APEC probably serves as a source or as a reservoir of virulence genes for human ExPEC and may be able to cause extraintestinal disease in humans.

Rapid and direct molecular detection of Streptococcus pneumoniae and Haemophilus influenzae isolated in oropharynx and nasal cavity of children.

There is a direct correlation between asymptomatic oropharyngeal colonization by pathogenic bacteria and the prevalence of paediatric respiratory infections. Streptococcus pneumoniae and Haemophilus influenzae are common cause of serious bacterial infections such as meningitis in children. This study aimed to monitor healthy children to detect asymptomatic carriers of S. pneumoniae and H. influenzae. In the present cross-sectional study, real-time PCR assay was developed and evaluated in comparison with culture for direct detection of S. pneumoniae and H. influenzae in 123 oropharynx and nasal cavity specimens from healthy children in Ilam, Iran. In addition, virulence factor (ply and hpd) and iron uptake (tbpA and piuA) genes were evaluated by PCR. Our results demonstrated that among all isolates only 14 S. pneumoniae and eight H. influenzae were identified by phenotypic methods, whereas 37 and 21 S. pneumoniae and H. influenzae were identified, respectively, by real-time PCR assay. All S. pneumoniae and H. influenzae isolates possessed tbpA and piuA genes. Also, 81% (30/37) of S. pneumoniae isolates were positive for ply gene, which encoded pneumolysin, as well as 90% (19/21) of H. influenzae isolates were positive for hpd gene. Simultaneous colonization of S. pneumoniae and H. influenzae could indicate the importance of monitoring of healthy children to identification of carriers.

Distinct TP53 Mutation Subtypes Differentially Influence Cellular Iron Metabolism

The most commonly mutated gene in all human cancers is the tumor suppressor gene TP53; however, in addition to the loss of tumor suppressor functions, mutations in TP53 can also promote cancer progression by altering cellular iron acquisition and metabolism. The primary objective of this work was to determine how TP53 mutation status influences the molecular control of iron homeostasis. The effect of TP53 mutation type on cellular iron homeostasis was examined using cell lines with inducible versions of either wild-type TP53 or a representative mutated TP53 gene from exemplary “hotspot” mutations in the DNA binding domain (R248, R273, and R175) as well as H193Y. The introduction of distinct TP53 mutation types alone was sufficient to disrupt cellular iron metabolism. These effects were mediated, at least in part, due to differences in the responsiveness of iron regulatory proteins (IRPs) to cellular iron availability. IRPs are considered the master regulators of intracellular iron homeostasis because they coordinate the expression of iron storage (ferritin) and iron uptake (transferrin receptor) genes. In response to changes in iron availability, cells harboring either a wild-type TP53 or R273H TP53 mutation displayed canonical IRP-mediated responses, but neither IRP1 RNA binding activity nor IRP2 protein levels were affected by changes in iron status in cells harboring the R175H mutation type. However, all mutation types exhibited robust changes in ferritin and transferrin receptor protein expression in response to iron loading and iron chelation, respectively. These findings suggest a novel, IRP-independent mode of iron regulation in cells expressing distinct TP53 mutations. As TP53 is mutated in nearly half of all human cancers, and iron is necessary for cancer cell growth and proliferation, the studies have implications for a wide range of clinically important cancers.

Investigation of Iron uptake and virulence gene factors (fur, tonB, exbD, exbB, hgbA, hgbB1, hgbB2 and tbpA) among isolates of Pasteurella multocida from Iran

Iron is an essential compound in metabolic pathway of wide range of organisms. Because of limited free iron supply in mammalian and avian hosts, bacteria have applied various ways to acquire iron. In this study, the frequency of 8 iron acquisition factors was examined among 63 avian and ovine Pasteurella multocida field isolates and their vaccine strains using PCR method. Five candidate genes (fur, tonB, exbD, exbB and hgbA) were identified among all isolates. For the first time, 2 loci (hgbB1 and hgbB2) of the hgbB gene were identified, which were previously reported as 1 gene. Also, it was found that 5 ovine and 1 avian isolates possessed all the virulence factors, which could also be considered for evaluating the frequency of other virulence factors. More studies need to be conducted on the frequency of all other virulence factors among these isolates, which can provide basic information for improvement or substitution of current vaccinal strains. Overall, as the new designed sets of primers showed more potential in detecting the corresponded genes, researchers can consider them in further studies.

Abstract 4365: Induction of distinct p53 mutation types differentially influences the control of cellular iron metabolism

Abstract The most commonly mutated gene in all human cancers is the tumor suppressor gene p53, but in addition to loss of tumor suppressor functions, mutations in p53 can also promote cancer progression by altering cellular iron acquisition and metabolism. The primary objective of this work was to determine how p53 mutation status influences the molecular control of iron homeostasis. Though hundreds of p53 mutations have been identified, the majority occur within the DNA binding domain and can be subdivided into two broad classes: contact (e.g., R273H) or conformational (e.g., R175H). By generating cell lines with inducible versions of some of the most common conformational and contact-type p53 mutations we have established that introduction of specific p53 mutation types alone is sufficient to disrupt cellular iron metabolism. We also demonstrate these effects are mediated at least in part due to differences in the responsiveness of Iron Regulatory Proteins (IRP) to cellular iron availability. IRP are considered the master regulators of intracellular iron homeostasis because they coordinate the expression of iron storage (ferritin) and iron uptake (transferrin receptor) genes. In response to changes in iron availability cells harboring either a wild-type p53 or contact (R273H) p53 mutation display canonical IRP-mediated responses, but neither IRP1 RNA binding activity nor IRP2 protein levels are affected by changes in iron status in cells harboring the conformational type mutant (R175H). Yet, both contact and conformational mutants exhibit robust changes in ferritin and transferrin receptor protein expression in response to iron loading and iron chelation, respectively. These findings suggest a novel, IRP-independent mode of iron regulation in cells expressing conformational type p53 mutants. Preliminary proteomics data suggests that lack of IRP responsiveness in cells expressing conformational p53 mutations may result from impaired iron-sulfur cluster biogenesis. These findings are currently being explored as a means of exploiting distinct p53 mutation types to more favorably induce iron-mediated cell death via the activation of ferroptosis. As p53 is mutated in nearly half of all human cancers, and iron is necessary for cancer cell growth and proliferation, the studies have implications for a wide range of clinically important cancers. Citation Format: Eshan Dandekar, Aishwarya Srinivasan, Stephen Clarke, Mckale Montgomery. Induction of distinct p53 mutation types differentially influences the control of cellular iron metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4365.

Renibacterium salmoninarum iron-acquisition mechanisms and ASK cell line infection: Virulence and immune response.

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron-acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT-qPCR to assess the effects of normal and iron-limited conditions on iron-uptake genes controlled by IdeR and related to iron acquisition in Chilean R.salmoninarum strain H-2 and the type strain DSM20767T . Further evaluated was the in vitro immune-related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R.salmoninarum grown under iron-limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H-2 than DSM20767T . These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF-α, IL-1β, TLR1 and INF-γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron-limited conditions, especially H-2. These observations indicate that iron-acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R.salmoninarum. In conclusion, treatments that block iron-uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R.salmoninarum, which causes significant aquaculture losses.

Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation.

Iron is required for growth and metabolism by virtually all organisms. The human fungal pathogen Candida albicans has evolved multiple strategies to acquire iron. The Cap2/Hap43 transcriptional regulator, essential for robust virulence of C.albicans, controls iron homeostasis gene expression by promoter binding and repression of iron utilization genes. The expression of iron uptake genes is also dependent on Cap2, although Cap2 was not recruited to its promoters. Cap2, bearing the conserved bipartite HAP4L-bZIP domain, also contains multiple blocks of amino acids that form the highly conserved carboxyl-terminal region. In this study, we sought to identify the requirements of the different domains for Cap2 function. We constructed a series of mutants bearing either point mutations or deletions in the conserved domains and examined Cap2 activity. Deletion of the highly conserved extreme C-terminal region did not impair expression of Cap2 mutant protein but impaired cell growth and expression of iron homeostasis genes under iron-depleted conditions. Mutations in the amino-terminal HAP4L and basic leucine zipper (bZIP) domains also impaired growth and gene expression. Furthermore, chromatin immunoprecipitation (ChIP) assays showed that the HAP4L domain and the bZIP domain are both essential for Cap2 recruitment to ACO1 and CYC1 promoters. Unexpectedly, the C-terminal conserved region was also essential for Cap2 promoter recruitment. Thus, our results suggest that Cap2 employs multiple evolutionarily conserved domains, including the C-terminal domain for its transcriptional activity.IMPORTANCE Iron is an essential micronutrient for living cells. Candida albicans, the predominant human fungal pathogen, thrives under diverse environments with vastly different iron levels in the mammalian host. Therefore, to tightly control iron homeostasis, C.albicans has evolved a set of transcriptional regulators that cooperate to either upregulate or downregulate transcription of iron uptake genes or iron utilization genes. Cap2/Hap43, a critical transcriptional regulator, contains multiple conserved protein domains. In this study, we carried out mutational analyses to identify the functional roles of the conserved protein domains in Cap2. Our results show that the bZIP, HAP4L, and the C-terminal domain are each required for Cap2 transcriptional activity. Thus, Cap2 employs multiple, disparate protein domains for regulation of iron homeostasis in C.albicans.

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.

The Gcn2-eIF2α pathway connects iron and amino acid homeostasis in Saccharomyces cerevisiae.

In eukaryotic cells, amino acid biosynthesis is feedback-inhibited by amino acids through inhibition of the conserved protein kinase Gcn2. This decreases phosphorylation of initiation factor eIF2α, resulting in general activation of translation but inhibition of translation of mRNA for transcription factor (TF) Gcn4 in yeast or ATF4 in mammals. These TFs are positive regulators of amino acid biosynthetic genes. As several enzymes of amino acid biosynthesis contain iron-sulfur clusters (ISCs) and iron excess is toxic, iron and amino acid homeostasis should be co-ordinated. Working with the yeast Saccharomyces cerevisiae, we found that amino acid supplementation down-regulates expression of genes for iron uptake and decreases intracellular iron content. This cross-regulation requires Aft1, the major TF activated by iron scarcity, as well as Gcn2 and phosphorylatable eIF2α but not Gcn4. A mutant with constitutive activity of Gcn2 (GCN2c ) shows less repression of iron transport genes by amino acids and increased nuclear localization of Aft1 in an iron-poor medium, and increases iron content in this medium. As Aft1 is activated by depletion of mitochondrial ISCs, it is plausible that the Gcn2-eIF2α pathway inhibits the formation of these complexes. Accordingly, the GCN2c mutant has strongly reduced activity of succinate dehydrogenase, an iron-sulfur mitochondrial enzyme, and is unable to grow in media with very low iron or with galactose instead of glucose, conditions where formation of ISCs is specially needed. This mechanism adjusts the uptake of iron to the needs of amino acid biosynthesis and expands the list of Gcn4-independent activities of the Gcn2-eIF2α regulatory system.