Iron-replete Conditions Research Articles

Capsular polysaccharides facilitate enhanced iron acquisition by the colonial cyanobacterium Microcystis sp. isolated from a freshwater lake.

Microcystis sp., especially in its colonial form, is a common dominant species during cyanobacterial blooms in many iron-deficient water bodies. It is still not entirely clear, however, how the colonial forms of Microcystis acclimate to iron-deficient habitats, and the responses of unicellular and colonial forms to iron-replete and iron-deficient conditions were examined here. Growth rates and levels of photosynthetic pigments declined to a greater extent in cultures of unicellular Microcystis than in cultures of the colonial form in response to decreasing iron concentrations, resulting in the impaired photosynthetic performance of unicellular Microcystis as compared to colonial forms as measured by variable fluorescence and photosynthetic oxygen evolution. These results indicate that the light-harvesting ability and photosynthetic capacity of colonial Microcystis was less affected by iron deficiency than the unicellular form. The carotenoid contents and nonphotochemical quenching of colonial Microcystis were less reduced than those of the unicellular form under decreasing iron concentrations, indicating that the colonial morphology enhanced photoprotection and acclimation to iron-deficient conditions. Furthermore, large amounts of iron were detected in the capsular polysaccharides (CPS) of the colonies, and more iron was found to be attached to the colonial Microcystis CPS under decreasing iron conditions as compared to unicellular cultures. These results demonstrated that colonial Microcystis can acclimate to iron deficiencies better than the unicellular form, and that CPS plays an important role in their acclimation advantage in iron-deficient waters.

Effects of high CO2 levels on the ecophysiology of the diatom Thalassiosira weissflogii differ depending on the iron nutritional status

AbstractIron availability in seawater, namely the concentration of dissolved inorganic iron ([Fe′]), is affected by changes in pH. Such changes in the availability of iron should be taken into account when investigating the effects of ocean acidification on phytoplankton ecophysiology because iron plays a key role in phytoplankton metabolism. However, changes in iron availability in response to changes in ocean acidity are difficult to quantify specifically using natural seawater because these factors change simultaneously. In the present study, the availability of iron and carbonate chemistry were manipulated individually and simultaneously in the laboratory to examine the effect of each factor on phytoplankton ecophysiology. The effects of various pCO2 conditions (∼390, ∼600, and ∼800 µatm) on the growth, cell size, and elemental stoichiometry (carbon [C], nitrogen [N], phosphorus [P], and silicon [Si]) of the diatom Thalassiosira weissflogii under high iron ([Fe′] = ∼240 pmol l−1) and low iron ([Fe′] = ∼24 pmol l−1) conditions were investigated. Cell volume decreased with increasing pCO2, whereas intracellular C, N, and P concentrations increased with increasing pCO2 only under high iron conditions. Si:C, Si:N, and Si:P ratios decreased with increasing pCO2. It reflects higher production of net C, N, and P with no corresponding change in net Si production under high pCO2 and high iron conditions. In contrast, significant linear relationships between measured parameters and pCO2 were rarely detected under low iron conditions. We conclude that the increasing CO2 levels could affect on the biogeochemical cycling of bioelements selectively under the iron-replete conditions in the coastal ecosystems.

The iron-responsive microsomal proteome of Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen. Siderophore biosynthesis and iron acquisition are essential for virulence. Yet, limited data exist with respect to the adaptive nature of the fungal microsomal proteome under iron-limiting growth conditions, as encountered during host infection. Here, we demonstrate that under siderophore biosynthetic conditions — significantly elevated fusarinine C (FSC) and triacetylfusarinine C (TAFC) production (p<0.0001), extensive microsomal proteome remodelling occurs. Specifically, a four-fold enrichment of transmembrane-containing proteins was observed with respect to whole cell lysates following ultracentrifugation-based microsomal extraction. Comparative label-free proteomic analysis of microsomal extracts, isolated following iron-replete and -deplete growth, identified 710 unique proteins. Scatterplot analysis (MaxQuant) demonstrated high correlation amongst biological replicates from each growth condition (Pearson correlation >0.96 within groups; biological replicates (n=4)). Quantitative and qualitative comparison revealed 231 proteins with a significant change in abundance between the iron-replete and iron-deplete conditions (p<0.05, fold change ≥2), with 96 proteins showing increased abundance and 135 with decreased abundance following iron limitation, including predicted siderophore transporters. Fluorescently labelled FSC was only sequestered following A. fumigatus growth under iron-limiting conditions. Interestingly, human sera exhibited significantly increased reactivity (p<0.0001) against microsomal protein extracts obtained following iron-deplete growth. Biological significanceThe opportunistic fungal pathogen Aspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus siderophore as a ‘Trojan horse’ to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome.

Mitochondrial iron supply is required for the developmental pulse of ecdysone biosynthesis that initiates metamorphosis in Drosophila melanogaster.

Synthesis of ecdysone, the key hormone that signals the termination of larval growth and the initiation of metamorphosis in insects, is carried out in the prothoracic gland by an array of iron-containing cytochrome P450s, encoded by the halloween genes. Interference, either with iron-sulfur cluster biogenesis in the prothoracic gland or with the ferredoxins that supply electrons for steroidogenesis, causes a block in ecdysone synthesis and developmental arrest in the third instar larval stage. Here we show that mutants in Drosophila mitoferrin (dmfrn), the gene encoding a mitochondrial carrier protein implicated in mitochondrial iron import, fail to grow and initiate metamorphosis under dietary iron depletion or when ferritin function is partially compromised. In mutant dmfrn larvae reared under iron replete conditions, the expression of halloween genes is increased and 20-hydroxyecdysone (20E), the active form of ecdysone, is synthesized. In contrast, addition of an iron chelator to the diet of mutant dmfrn larvae disrupts 20E synthesis. Dietary addition of 20E has little effect on the growth defects, but enables approximately one-third of the iron-deprived dmfrn larvae to successfully turn into pupae and, in a smaller percentage, into adults. This partial rescue is not observed with dietary supply of ecdysone's precursor 7-dehydrocholesterol, a precursor in the ecdysone biosynthetic pathway. The findings reported here support the notion that a physiological supply of mitochondrial iron for the synthesis of iron-sulfur clusters and heme is required in the prothoracic glands of insect larvae for steroidogenesis. Furthermore, mitochondrial iron is also essential for normal larval growth.

Effects of iron limitation and UV radiation on Phaeocystis antarctica growth and dimethylsulfoniopropionate, dimethylsulfoxide and acrylate concentrations

Environmental context Low iron concentrations and solar ultraviolet radiation can affect the growth of marine algae. We observed reduced growth and substantial increases in dissolved dimethylsulfoxide and cellular acrylate concentrations in low-iron cultures of a prevalent Southern Ocean algal species, Phaeocystis antarctica, with comparatively small increases observed for cellular dimethylsulfoniopropionate concentrations. Exposure of P. antarctica to high levels of ultraviolet and visible light had very little effect on concentrations of these compounds in culture, even under iron-limitation. Our results highlight the importance of iron to P. antarctica. Abstract Iron is a key nutrient regulating primary production in the Southern Ocean. We investigated the effect of iron limitation with and without exposure to ultraviolet radiation (UVR, 290–400nm) on concentrations of dimethylsulfoniopropionate (DMSP), dimethylsulfoxide (DMSO) and acrylate in axenic batch cultures of Phaeocystis antarctica, a dominant algal species in Antarctic waters. Cellular concentrations of DMSP and acrylate, and cell-number normalised dissolved DMSO concentrations were 1.4-, 11.5- and 6.9-fold higher in iron-limited cultures compared to iron-replete cultures, which we propose resulted from (1) increased reactions of DMSP and dimethylsulfide (DMS) with reactive oxygen species to produce DMSO and (2) increased DMSP cleavage under iron limitation to produce acrylate. Short-term exposure (4h) of iron-limited and iron-replete cultures to a range of photosynthetically active radiation (PAR) and UVR+PAR irradiances did not appreciably affect P. antarctica biomass or total DMSP, DMSO or acrylate concentrations, except at high UVR intensities, suggesting that iron limitation was the primary driver regulating growth and changes in concentrations of these compounds in P. antarctica. High millimolar cellular DMSP and acrylate concentrations under both iron-replete and iron-limited conditions indicated that these two compounds served as de facto antioxidants allowing P. antarctica to thrive under high UVR exposure and low iron concentrations. High dissolved acrylate concentrations indicate significant carbon removal possibly as part of an overflow mechanism during unbalanced growth.

IscR of Rhodobacter sphaeroides functions as repressor of genes for iron-sulfur metabolism and represents a new type of iron-sulfur-binding protein.

IscR proteins are known as transcriptional regulators for Fe–S biogenesis. In the facultatively phototrophic bacterium, Rhodobacter sphaeroides IscR is the product of the first gene in the isc-suf operon. A major role of IscR in R. sphaeroides iron-dependent regulation was suggested in a bioinformatic study (Rodionov et al., PLoS Comput Biol 2:e163, 2006), which predicted a binding site in the upstream regions of several iron uptake genes, named Iron-Rhodo-box. Most known IscR proteins have Fe–S clusters featuring (Cys)3(His)1 ligation. However, IscR proteins from Rhodobacteraceae harbor only a single-Cys residue and it was considered unlikely that they can ligate an Fe–S cluster. In this study, the role of R. sphaeroides IscR as transcriptional regulator and sensor of the Fe–S cluster status of the cell was analyzed. A mutant lacking IscR is more impaired in growth under iron limitation than the wild-type and exhibits significantly increased ROS levels in iron-replete and iron-deplete conditions. Expression studies reveal that R. sphaeroides IscR in its cluster-bound form functions as transcriptional repressor of genes involved in iron metabolism by direct binding to the promoter region of genes preceded by the motif. A total of 110 genes are directly or indirectly affected by IscR. Furthermore, IscR possesses a unique Fe–S cluster ligation scheme with only a single cysteine involved.

Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur- and PerR-transcriptomes.

BackgroundThe genome of Campylobacter jejuni contains two iron activated Fur-family transcriptional regulators, CjFur and CjPerR, which are primarily responsible for regulating iron homeostasis and oxidative stress respectively. Both transcriptional regulators have been previously implicated in regulating diverse functions beyond their primary roles in C. jejuni. To further characterize their regulatory networks, RNA-seq was used to define the transcriptional profiles of C. jejuni NCTC11168 wild type, Δfur, ΔperR and ΔfurΔperR isogenic deletion mutants under both iron-replete and iron-limited conditions.ResultsIt was found that 202 genes were differentially expressed in at least one mutant under iron-replete conditions and 331 genes were differentially expressed in at least one mutant under iron-limited conditions. The CjFur and CjPerR transcriptomes characterized in this study were compared to those previously identified using microarray profiling and found to be more extensive than previously understood. Interestingly, our results indicate that CjFur/CjPerR appear to co-regulate the expression of flagellar biogenesis genes in an opposing and iron-independent fashion. Moreover the ΔfurΔperR isogenic deletion mutant revealed that CjFur and CjPerR can compensate for each other in certain cases, suggesting that both regulators may compete for binding to specific promoters.ConclusionsThe CjFur and CjPerR transcriptomes are larger than previously reported. In particular, deletion of perR results in the differential expression of a large group of genes in the absence of iron, suggesting that CjPerR may also regulate genes in an iron-independent manner, similar to what has already been demonstrated with CjFur. Moreover, subsets of genes were found which are only differentially expressed when both CjFur and CjPerR are deleted and includes genes that appear to be simultaneously activated by CjFur and repressed by CjPerR. In particular the iron-independent co-regulation of flagellar biogenesis by CjFur/CjPerR represents a potentially novel regulatory function for these proteins. These findings represent additional modes of co-regulation by these two transcriptional regulators in C. jejuni.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1661-7) contains supplementary material, which is available to authorized users.

The mitogen-activated protein kinase CgHog1 is required for iron homeostasis, adherence and virulence in Candida glabrata.

Candida glabrata has emerged as a major fungal pathogen over the last two decades, although our understanding of its survival strategies inside the mammalian host remains rudimentary. An important requirement for survival in vivo is the ability to acquire critical nutrients such as iron from host niches of varied iron content. In the present study, we demonstrate for the first time that C. glabrata cells respond to high external iron levels via activation of two stress-responsive mitogen-activated protein kinases, CgHog1 and CgSlt2, and lack of either kinase results in sensitivity to the high-iron medium. Furthermore, we show that CgHOG1 deletion led to perturbed iron homeostasis (elevated intracellular iron content and high mitochondrial aconitase activity), reduced survival in macrophages and attenuated virulence in the murine model of disseminated candidiasis. Consistently, several genes implicated in iron acquisition and storage displayed deregulated expression in the Cghog1∆ mutant. Genome-wide transcriptional profiling analysis revealed upregulation of genes implicated in DNA repair, RNA processing and autophagy, and downregulation of genes related to cellular respiration and organonitrogen compound metabolism under iron-limiting conditions. In contrast, genes involved in the respiratory electron transport chain were induced under iron-replete conditions. Gene expression microarrays also identified a set of iron-responsive regulon in C. glabrata. Lastly, we present evidence for the iron-regulated expression of the major adhesin-encoding EPA1 gene, decreased histone deacetylase activity in a high-iron environment and increased adherence of iron-surplus-medium-grown C. glabrata cells to epithelial cells. Together, our findings yield novel insights into iron abundance-based regulation of transcriptional and mitogen-activated protein kinase signaling pathways in C. glabrata.

Pseudomonas fluorescens: Iron-responsive proteins and their involvement in host infection

For pathogenic bacteria, the ability to acquire iron is vital to survival in the host. In consequence, many genes involved in iron acquisition are associated with bacterial virulence. Pseudomonas fluorescens is a bacterial pathogen to a variety of farmed fish. However, the global regulatory function of iron in pathogenic P. fluorescens is essentially unknown. In this study, in order to identify proteins affected by iron condition at the expression level, we performed proteomic analysis to compare the global protein profiles of P. fluorescens strain TSS, a fish pathogen, cultured under iron-replete and iron-deplete conditions. Twenty-two differentially expressed proteins were identified, most of which were confirmed to be regulated by iron at the mRNA level. To investigate their potential involvement in virulence, the genes encoding four of the 22 proteins, i.e. HemO (heme oxygenase), PspB (serine protease), Sod (superoxide dismutase), and TfeR (TonB-dependent outermembrane ferric enterobactin receptor), were knocked out, and the pathogenicity of the mutants was examined in a model of turbot (Scophthalmus maximus). The results showed that compared to the wild type, the hemO, pspB, and tfeR knockouts were significantly impaired in the ability to survive in host serum, to invade host tissues, and to cause host mortality. Immunization of turbot with recombinant TfeR (rTfeR) and PspB induced production of specific serum antibodies and significant protections against lethal TSS challenge. Further analysis showed that rTfeR antibodies recognized and bound to TSS, and that treatment of TSS with rTfeR antibodies significantly impaired the infectivity of TSS to fish cells. Taken together, these results indicate for the first time that in pathogenic P. fluorescens, iron affects the expression of a large number of proteins including those that are involved in host infection.

Tenellin acts as an iron chelator to prevent iron-generated reactive oxygen species toxicity in the entomopathogenic fungus Beauveria bassiana.

Iron is an essential element for life. However, the iron overload can be toxic. Here, we investigated the significant increase of tenellin and iron-tenellin complex production in ferricrocin-deficient mutants of Beauveria bassiana. Our chemical analysis indicated that the ferricrocin-deficient mutants T1, T3 and T5 nearly abolished ferricrocin production. In turn, these mutants had significant accumulation of iron-tenellin complex in their mycelia at 247-289 mg g(-1) cell dry weight under iron-replete condition. Both tenellin and iron-tenellin complex were not detected in the wild-type under such condition. Mass analysis of the mutants' crude extracts demonstrated that tenellin formed a 3:1 complex with iron in the absence of ferricrocin. The unexpected link between ferricrocin and tenellin biosynthesis in ferricrocin-deficient mutants could be a survival strategy during iron-mediated oxidative stress.

Iron- and hemin-dependent gene expression of Porphyromonas gingivalis.

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron-dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron- and hemin-dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole-genome microarrays of P.gingivalis genes were used to compare the levels of gene expression under iron-replete and iron-depleted conditions as well as under hemin-replete and hemin-depleted conditions. Under iron-depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron-depleted conditions to those from hemin-depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron-dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron-regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron-stressed bacterium than the iron-replete P.gingivalis cells. The major iron-regulated proteins encoded by PG1019-20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole-genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P.gingivalis.

Genome mining and functional genomics for siderophore production in Aspergillus niger.

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism.

Iron-responsive chromatin remodelling and MAPK signalling enhance adhesion in Candida albicans.

Recent cumulative data show that various transcription factors are recruited to the chromatin in an iron-responsive manner to affect diverse cellular functions in the pathogenic fungus Candida albicans. Here we identified groups of iron-responsive genes in C. albicans by chromatin remodelling analysis at gene promoters, using micrococcal nuclease (MNase) digestion followed by deep sequencing. Chromatin in the promoter regions of iron uptake and utilization genes showed repressed and active configuration, respectively, under iron-replete conditions. GO Term enrichment analysis of genes with differentially remodelled chromatin, in respective promoter locales, suggested that many genes involved in adhesion are also iron-responsive. C. albicans was observed to be more self-adherent (twofold increase) and formed higher biofilm mass (77% increase) in the presence of iron. Furthermore, we identified various known and novel adhesion-related genes with iron-dependent active chromatin profiles that are indicative of potential upregulation under iron-replete conditions. Transcription factor Cph1 that is activated upon Cek1 phosphorylation also showed an active chromatin profile under iron-replete conditions and cells showed iron-responsive Cek1 MAPK phosphorylation in the presence of iron. Thus, iron affects diverse biological functions by modulating chromatin profiles of large gene sets and by signalling through Cek1 MAPK in C. albicans.

Root associated iron oxidizing bacteria increase phosphate nutrition and influence root to shoot partitioning of iron in tolerant plant Typha angustifolia

Typha angustifolia is a heavy metal tolerant plant that grows in a uranium mine tailings highly contaminated with iron. In this study three iron oxidizing microbes (FeOBs) isolated from Typha rhizoplane were investigated for their role in plant growth promotion (PGP). Their effect on iron nutrition in Typha under iron replete and excess condition was also evaluated. The PGP activities of the FeOBs were studied by measuring their influence on plant growth. To investigate the mechanism of growth promotion their ability to solubilize phosphate, and to produce Indole acetic acid and siderophores were studied. The influence of the FeOBs on root to shoot partitioning of iron was tested by measuring total iron content in roots and shoots treated with microbes. The FeOBs were named as Paenibacillus cookii JGR8, (MTCC12002), Pseudomonas jaduguda JGR2 (LMG25820) and Bacillus megaterium JGR9 (MTCC12001). The siderophore producers, influenced iron accumulation in the plant root. Additionally P. pseudoalcaligenes JGR2 increased shoot iron content overcoming the root- shoot barrier that allows Typha to exclude metals from its shoot. Among the PGP mechanisms tested, ability to solubilize phosphate appeared to be most significant for increasing the plant biomass. FeOBs that produce siderophore increased iron content in plant and therefore can be of immense biotechnological importance. However Biomass increase was directly correlated with increased phosphate acquisition and not with enhanced iron accumulation in Typha.

The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance ofPseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that is refractory to a variety of current antimicrobial therapeutic regimens. Complicating treatment for such infections is the ability of P. aeruginosa to form biofilms, as well as several innate and acquired resistance mechanisms. Previous studies suggest iron plays a role in resistance to antimicrobial therapy, including the efficacy of an FDA-approved iron chelator, deferasirox (DSX), or Gallium, an iron analog, in potentiating antibiotic-dependent killing of P. aeruginosa biofilms. Here, we show that iron-replete conditions enhance resistance of P. aeruginosa nonbiofilm growth against tobramycin and tigecycline. Interestingly, the mechanism of iron-enhanced resistance to each of these antibiotics is distinct. Whereas pyoverdine-mediated iron uptake is important for optimal resistance to tigecycline, it does not enhance tobramycin resistance. In contrast, heme supplementation results in increased tobramycin resistance, while having no significant effect on tigecycline resistance. Thus, nonsiderophore bound iron plays an important role in resistance to tobramycin, while pyoverdine increases the ability of P. aeruginosa to resist tigecycline treatment. Lastly, we show that iron increases the minimal concentration of tobramycin, but not tigecycline, required to eradicate P. aeruginosa biofilms. Moreover, iron depletion blocks the previous observed induction of biofilm formation by subinhibitory concentrations of tobramycin, suggesting iron and tobramycin signal through overlapping regulatory pathways to affect biofilm formation. These data further support the role of iron in P. aeruginosa antibiotic resistance, providing yet another compelling case for targeting iron acquisition for future antimicrobial drug development.

Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

ABSTRACTPseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections.

Impact of Iron Availability on &amp;lt;i&amp;gt;Bacillus amyloliquefaciens&amp;lt;/i&amp;gt; Growth

Bacillus amyloliquefaciens is a biocontrol agent whose genome has been sequenced. Within the genome of B. amyloliquefaciens are genes associated with iron chelation, but these genes are not found within all sequenced strains. The impact of iron availability on the B. amyloliquefaciens physiology was examined in this study. B. amyloliquefaciens ATCC 23843 was cultured under ironreplete and iron-deplete conditions for 48 hours, at 37°C. Final growth yields were dependent on iron concentration. Cultures grown in the absence of detectable iron were restricted in growth, but reached their highest yields at 48 hours. Iron restriction was confirmed by the presence of iron chelators in the filtrates. In contrast, B. amyloliquefaciens ATCC 23843 cultures grown with ferric ammonium citrate as the iron source research reached the highest yields at 24 hours. Iron chelator production was not detected in the ferric ammonium samples. A significant decrease in turbidity was observed for these cultures, which coincided with elevated spore production in B. amyloliquefaciens ATCC 23843. A decrease in turbidity was also observed on blood agar, where hemolysis was readily evident. We propose that iron impacts numerous physiological responses and further studies will elucidate the complex regulatory mechanisms governed by iron availability.

Iron, Oxidative Stress, and Virulence: Roles of Iron-Sensitive Transcription Factor Sre1 and the Redox Sensor ChAp1 in the Maize Pathogen Cochliobolus heterostrophus

The gene SRE1, encoding the GATA transcription factor siderophore biosynthesis repressor (Sre1), was identified in the genome of the maize pathogen Cochliobolus heterostrophus and deleted. Mutants were altered in sensitivity to iron, oxidative stress, and virulence to the host. To gain insight into mechanisms of this combined regulation, genetic interactions among SRE1 (the nonribosomal peptide synthetase encoding gene NPS6, which is responsible for extracellular siderophore biosynthesis) and ChAP1 (encoding a transcription factor regulating redox homeostasis) were studied. To identify members of the Sre1 regulon, expression of candidate iron and oxidative stress-related genes was assessed in wild-type (WT) and sre1 mutants using quantitative reverse-transcription polymerase chain reaction. In sre1 mutants, NPS6 and NPS2 genes, responsible for siderophore biosynthesis, were derepressed under iron replete conditions, whereas the high-affinity reductive iron uptake pathway associated gene, FTR1, was not, in contrast to outcomes with other well-studied fungal models. C. heterostrophus L-ornithine-N(5)- monooxygenase (SIDA2), ATP-binding cassette (ABC6), catalase (CAT1), and superoxide dismutase (SOD1) genes were also derepressed under iron-replete conditions in sre1 mutants. Chap1nps6 double mutants were more sensitive to oxidative stress than either Chap1 or nps6 single mutants, while Chap1sre1 double mutants showed a modest increase in resistance compared with single Chap1 mutants but were much more sensitive than sre1 mutants. These findings suggest that the NPS6 siderophore indirectly contributes to redox homeostasis via iron sequestration, while Sre1 misregulation may render cells more sensitive to oxidative stress. The double-mutant phenotypes are consistent with a model in which iron sequestration by NPS6 defends the pathogen against oxidative stress. C. heterostrophus sre1, nps6, Chap1, Chap1nps6, and Chap1sre1 mutants are all reduced in virulence toward the host, compared with the WT.

Role Of Transferrin In Recovery From Phlebotomy In Iron Replete Or Iron Deficient Mice

The most common reason for deferral of blood donation is low hemoglobin concentration and anemia. Furthermore, repeated donations, especially in young females, may lead to iron deficiency, drop of hemoglobin, and consequent iron deficiency anemia. Recent data suggests that iron deficiency, in the absence of anemia, may be associated with symptoms in otherwise healthy individuals. Our previous findings suggested that additional transferrin ameliorates anemia in mouse models of β-thalassemia; this effect appears driven by an increase in the number of circulating red blood cell (RBC) by extending RBC survival (Li Nat Med 2010). We hypothesized that exogenous transferrin increases RBC count and enables a more rapid recovery after phlebotomy. To test this hypothesis, C57 mice were fed an iron replete (35ppm iron) and iron deficient (2.5ppm iron) diet for 3 months to simulate the human condition. Mice were phlebotomized an average of 220ul of blood weekly for four weeks. Additionally, all mice received intraperitoneal (IP) injection of either of 10mg of human apo-transferrin (apo-Tf) (200 ul volume) or 200ul PBS daily Monday – Friday for 4 weeks (in total 20 days of injection). Mice (n=4/group) were sacrificed and samples analyzed at the end of the 4 weeks. RBC survival was evaluated using an in vivo biotin avidin assay previously described (Li Nat Med 2010). We analyzed all RBC indices using ADVIA 120 (Bayer). MCV decreased in phlebotomized mice on 2.5ppm compared to 35ppm diet (p=0.012) and after apo-Tf injection (p<0.05 in 2.5ppm diet, and p<0.001 in 35ppm diet). Reticulocyte counts increased by 50% in apo-Tf treated compared to PBS treated phlebotomized mice on 2.5ppm diet (p=0.006). Apo-Tf injections also resulted in an increase in RBC counts in phlebotomized mice on 35ppm diet (p=0.056). Furthermore, using antibodies to CD44 and TER119 in flow cytometry (Chen PNAS 2010), we assessed the proportion of erythroid precursors at all stages of terminal erythroid differentiation. Apo-Tf injections result in more erythroid precursors in spleen and bone marrow (p<0.05) in phlebotomized mice on 2.5ppm or 35ppm diets without changes in the degree of apoptosis or quantity of reactive oxygen species. Mice on 2.5ppm diet had smaller spleens relative to 35ppm diet (0.002 vs. 0.004 spleen / body weight, p=0.05) that increased as a consequence of apo-Tf injection (0.003 vs. 0.002 spleen / body weight, p=0.02). No differences in spleen size or spleen / body weight is observed in apo-Tf treated mice on 35ppm diet. Immunohistochemistry of spleen samples using anti-TER119 antibodies revealed more extramedullary erythropoiesis in all apo-Tf treated mice. Lastly, no difference in RBC survival was observed in mice as a consequence of diet or injection. Taken together, our data suggest that exogenous apo-Tf in recurrently phlebotomized mice on iron deficient diet stimulates erythropoiesis without a direct effect on RBC survival. This data provides initial evidence that exogenous apo-Tf accelerates recovery from phlebotomy especially in iron replete conditions, without evidence of harm in iron deficiency. The molecular mechanisms underlying the effect of iron availability and iron delivery on different stages of erythroid differentiation requires further analysis. Disclosures:No relevant conflicts of interest to declare.

The Transcriptional Landscape of Campylobacter jejuni under Iron Replete and Iron Limited Growth Conditions

The genome-wide Campylobacter jejuni transcriptional response under iron replete and iron limited conditions was characterized using RNA-seq. We have identified 111 novel C. jejuni 5’UTRs and mapped 377 co-transcribed genes into 230 transcriptional operons. In contrast to previous microarray results, the C. jejuni iron stimulon is less extensive than previously believed and consists of 77 iron activated genes and 50 iron repressed genes. As anticipated, the iron repressed genes are primarily those involved in iron acquisition or oxidative stress defense. Interestingly, these experiments have revealed that iron is an important modulator of flagellar biogenesis with almost all the components of the flagella found to be iron activated. Given that motility is a well-known C. jejuni colonization factor, this suggests that there is an important regulatory coupling of flagellar biogenesis and iron level in C. jejuni. In addition we have identified several consensus mutations in the C. jejuni NCTC11168 strain that are widespread in the Campylobacter research community and which may explain conflicting phenotypic reports for this strain. Comparative analysis of iron responsive genes with the known Fur regulon indicates that many iron responsive genes are not Fur responsive; suggesting that additional iron regulatory factors remain to be characterized in C. jejuni. Further analysis of the RNA-seq data identified multiple novel transcripts including 19 potential ncRNAs. The expression of selected ncRNAs was confirmed and quantified with qRT-PCR. The qRT-PCR results indicate that several of these novel transcripts are either Fur and/or iron responsive. The fact that several of these ncRNAs are iron responsive or Fur regulated suggests that they may perform regulatory roles in iron homeostasis.