Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1×10⁶ insect cells produced up to 3 g opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.Abbreviations dpi days post infection - pfu plaque forming units - AcNPV Autographa californica nuclear polyhedrosis virus     

Acquisition of Fe from Various Natural Organic Matter Isolates by Aerobic Pseudomonad Bacteria

Iron (Fe) is an essential nutrient to most microorganisms. Aerobic microorganisms exhibit various strategies for acquiring Fe at near-neutral pH conditions, where Fe oxyhydroxides are insoluble. Although much research has focused on microbial acquisition of Fe from minerals, little is known about Fe acquisition from natural organic matter (NOM). Yet, in surface waters, soils and shallow sediments, Fe is often associated with natural organic matter (NOM), and this NOM-associated Fe could represent an important pool of Fe for microorganisms. Here, we investigated the growth of aerobic Pseudomonas mendocina on soil and surface water NOM samples containing Fe, under Fe-limited conditions. In the presence of NOM, bacteria grew to population sizes greater than in no-Fe-added controls, indicating that the bacteria were able to access Fe associated with NOM. Maximum population size correlated with the NOM-associated Fe concentration. In an additional experiment, Pseudomonas putida was able to acquire Fe from an NOM sample, demonstrating that this ability is not limited to P. mendocina. When Fe was added as 30 μ M FeEDTA plus NOM, together in the same reaction flasks, P. mendocina and P. putida growth was less than in the presence of 30 μM FeEDTA alone. The fact that Fe sources are not simply additive and that the presence of NOM inhibits growth in FeEDTA suggests that further study on the responses of bacteria to a combination of Fe sources is needed to understand the complexities of bacterial Fe acquisition in the subsurface.     

Responses of Two Coastal Algae (Skeletonema costatum and Chlorella vulgaris) to Changes in Light and Iron Levels1

Iron (Fe) is essential for phytoplankton growth and photosynthesis, and is proposed to be an important factor regulating algal blooms under replete major nutrients in coastal environments. Here, Skeletonema costatum, a typical red-tide diatom species, and Chlorella vulgaris, a widely distributed Chlorella, were chosen to examine carbon fixation and Fe uptake by coastal algae under dark and light conditions with different Fe levels. The cellular carbon fixation and intracellular Fe uptake were measured via ¹⁴C and ⁵⁵Fe tracer assay, respectively. Cell growth, cell size, and chlorophyll-α concentration were measured to investigate the algal physiological variation in different treatments. Our results showed that cellular Fe uptake proceeds under dark and the uptake rates were comparable to or even higher than those in the light for both algal species. Fe requirements per unit carbon fixation were also higher in the dark resulting in higher Fe: C ratios. During the experimental period, high Fe addition significantly enhanced cellular carbon fixation and Fe uptake. Compared to C. vulgaris, S. costatum was the common dominant bloom species because of its lower Fe demand but higher Fe uptake rate. This study provides some of the first measurements of Fe quotas in coastal phytoplankton cells, and implies that light and Fe concentrations may influence the phytoplankton community succession when blooms occur in coastal ecosystems.     

Functional characterization of a high-affinity iron transporter (PiFTR) from the endophytic fungus Piriformospora indica and its role in plant growth and development

Iron (Fe) is a micronutrient required for plant growth and development; however, most Fe forms in soil are not readily available to plants, resulting in low Fe contents in plants and, thereby, causing Fe deficiency in humans. Biofortification through plant-fungal co-cultivation might be a sustainable approach to increase crop Fe contents. Therefore, we aimed to examine the role of a Piriformospora indica Fe transporter on rice Fe uptake under low Fe conditions. A high-affinity Fe transporter (PiFTR) from P. indica was identified and functionally characterized. PiFTR fulfilled all criteria expected of a functional Fe transporter under Fe-limited conditions. Additionally, PiFTR expression was induced when P. indica was grown under low Fe conditions, and PiFTR complemented a yeast mutant lacking Fe transport. A knockdown (KD) P. indica strain was created via RNA interference to understand the physiological role of PiFTR. We observed that the KD-PiFTR-P. indica strain transported a significantly lower amount of Fe to colonized rice (Oryza sativa) than the wild type (WT) P. indica. WT P. indica-colonized rice plants were healthier and performed significantly better than KD-PiFTR-P. indica-colonized rice plants. Our study offers potential avenues for an agronomically sound amelioration of plant growth in low Fe environments.     

Abscisic acid alleviates iron deficiency by promoting root iron reutilization and transport from root to shoot in Arabidopsis

Abscisic acid (ABA) has been demonstrated to be involved in iron (Fe) homeostasis, but the underlying mechanism is largely unknown. Here, we found that Fe deficiency induced ABA accumulation rapidly (within 6 h) in the roots of Arabidopsis. Exogenous ABA at 0.5 μM decreased the amount of root apoplastic Fe bound to pectin and hemicellulose, and increased the shoot Fe content significantly, thus alleviating Fe deficiency‐induced chlorosis. Exogenous ABA promoted the secretion of phenolics to release apoplastic Fe and up‐regulated the expression of AtNRAMP3 to enhance reutilization of Fe stored in the vacuoles, leading to a higher level of soluble Fe and lower ferric–chelate reductase (FCR) activity in roots. Treatment with ABA also led to increased Fe concentrations in the xylem sap, partially because of the up‐regulation of AtFRD3, AtYSL2 and AtNAS1, genes related to long‐distance transport of Fe. Exogenous ABA could not alleviate the chlorosis of abi5 mutant resulting from the significantly low expression of AtYSL2 and low transport of Fe from root to shoot. Taken together, our data support the conclusion that ABA is involved in the reutilization and transport of Fe from root to shoot under Fe deficiency conditions in Arabidopsis.     

LOCALIZATION OF IRON WITHIN CENTRIC DIATOMS OF THE GENUS THALASSIOSIRA1

The cellular iron (Fe) quota of centric diatoms has been shown to vary in response to the ambient dissolved Fe concentration; however, it is not known how centric diatoms store excess intracellular Fe. Here, we use synchrotron X‐ray fluorescence (SXRF) element mapping to identify Fe storage features in cells of Thalassiosira pseudonana Hasle et Heimdal and Thalassiosira weissflogii G. A. Fryxell et Hasle grown at low and high Fe concentrations. Localized intracellular Fe storage features, defined as anomalously high Fe concentrations in regions of relatively low phosphorus (P), sulfur (S), silicon (Si), and zinc (Zn), were twice as common in T. weissflogii cells grown at high Fe compared to low‐Fe cells. Cellular Fe quotas of this strain increased 2.9‐fold, the spatial extent of the features increased 4.6‐fold, and the Fe content of the features increased 14‐fold under high‐Fe conditions, consistent with a vacuole storage mechanism. The element stoichiometry of the Fe features is consistent with polyphosphate‐bound Fe as a potential vacuolar Fe storage pool. Iron quotas increased 2.5‐fold in T. pseudonana grown at high Fe, but storage features contained only 2‐fold more Fe and did not increase in size compared to low‐Fe cells. The differences in Fe storage observed between T. pseudonana and T. weissflogii may have been due to differences in the growth states of the cultures.     

The arbuscular mycorrhizal fungus Rhizophagus irregularis uses a reductive iron assimilation pathway for high‐affinity iron uptake

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high‐affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1‐2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane‐localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045 mM Fe, while RiFTR2 was upregulated under Fe‐deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe‐limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis.     

Molecular evidence for phytosiderophore‐induced improvement of iron nutrition of peanut intercropped with maize in calcareous soil

Peanut/maize intercropping is a sustainable and effective agroecosystem that evidently enhances the Fe nutrition of peanuts in calcareous soils. So far, the mechanism involved in this process has not been elucidated. In this study, we unravel the effects of phytosiderophores in improving Fe nutrition of intercropped peanuts in peanut/maize intercropping. The maize ys3 mutant, which cannot release phytosiderophores, did not improve Fe nutrition of peanut, whereas the maize ys1 mutant, which can release phytosiderophores, prevented Fe deficiency, indicating an important role of phytosiderophores in improving the Fe nutrition of intercropped peanut. Hydroponic experiments were performed to simplify the intercropping system, which revealed that phytosiderophores released by Fe‐deficient wheat promoted Fe acquisition in nearby peanuts and thus improved their Fe nutrition. Moreover, the phytosiderophore deoxymugineic acid (DMA) was detected in the roots of intercropped peanuts. The yellow stripe1‐like (YSL) family of genes, which are homologous to maize yellow stripe 1 (ZmYS1), were identified in peanut roots. Further characterization indicated that among five AhYSL genes, AhYSL1, which was localized in the epidermis of peanut roots, transported Fe(III)–DMA. These results imply that in alkaline soil, Fe(III)–DMA dissolved by maize might be absorbed directly by neighbouring peanuts in the peanut/maize intercropping system.     

Regulation of nitrogenase activity by covalent modification in Chromatium vinosum

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH ₄ ⁺ . Activity of the Fe protin component of nitrogenase required both Mn²⁺ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.Abbreviation HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid     

Nfu facilitates the maturation of iron‐sulfur proteins and participates in virulence in Staphylococcus aureus

The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron‐sulfur (Fe‐S) clusters, which are required for functional Fe‐S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe‐S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe‐S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as an Fe‐S cluster carrier, which aids in the maturation of Fe‐S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non‐incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe‐S cluster metabolism as an attractive antimicrobial target.     

拟南芥bHLH Ib转录因子调控FIT的转录

FIT是调控拟南芥铁稳态的一个关键调控因子，它在转录水平上受到缺铁诱导，但其背后的调控机制还不甚清楚。该研究以拟南芥bHLH38和FIT的单、双过表达植物及bHLH Ib四突变体植物为材料，采用缺铁(-Fe)处理实验和定量RT-PCR的方法从RNA角度分析了FIT转录水平的变化。结果表明：(1)在铁充足时，bHLH38过表达植物中FIT的转录水平显著高于其在野生型中的水平。(2)在bHLH Ib四突变体植物中FIT的转录水平不受缺铁诱导。(3)FIT单过表达不能激活内源FIT的转录，而在加铁(+Fe)条件下bHLH38和FIT的双过表达则可以激活内源FIT的转录。(4)在缺铁条件下，所有植物中FIT的转录水平均与野生型中的FIT水平无明显差异。基于以上结果认为，bHLH Ib转录因子是缺铁诱导FIT转录的必要条件，而非充分条件。该研究结果为深入了解植物通过多种途径共同维持铁稳态提供了新的见解。     

Effects of different iron supply to pregnant sows (Sus scrofa domestica L.) on reproductive performance as well as iron status of new-born piglets

The present study aimed to investigate the effects of different iron (Fe) supply to sows during gestation on their reproductive performance and placental Fe load. Additionally, the Fe status of the corresponding offspring was assessed. Twenty multiparous sows were fed from insemination to farrowing with isoenergetic and isonitrogenic balanced diets differing in Fe content. The diet low in Fe (Group ?Fe) was mainly composed of soybean meal and maize meal and had a Fe content of 114 mg/kg DM. For the diet high in Fe (Group +Fe), the diet was supplemented with Fe(II)SO₄ · 7H₂O to a total Fe content of 256 mg/kg. Blood characteristics (haemoglobin, haematocrit, mean corpuscular haem concentration, total Fe-binding capacity, transferrin saturation) of all sows were measured at the beginning and at the end of gestation. Daily Fe retention was calculated at the day of farrowing. After birth, reproductive performance (litter size, piglet weight, litter weight), placental Fe content and Fe blood characteristics of the piglets were determined. Apparent daily Fe retention tended to be greater in Group +Fe (p < 0.1). Blood parameters of the sows did not show any variations between feeding groups, neither at the beginning nor at the end of pregnancy, whereas placental Fe content was lower in Group ?Fe (p < 0.05). In addition, Fe supply during gestation improved litter size (p < 0.01) and litter weight (p < 0.05). Although all sows were supplied according to the current Fe recommendations, a significant decline in reproductive performance of Group ?Fe was recognised. Therefore, it was concluded that the re-evaluation of the gross Fe requirements of pregnant sows is inevitable to accommodate the current feeding recommendations.     

Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron–sulfur clusters

Biological assembly of iron–sulfur (Fe–S) clusters is mediated by complex systems consisting of multiple proteins. Escherichia coli possesses two distinct systems called the ISC and SUF machineries encoded by iscSUA‐hscBA‐fdx‐iscX and sufABCDSE respectively. Deletion of both pathways results in absence of the biosynthetic apparatus for Fe–S clusters, and consequent lethality, which has hampered detailed genetic studies. Here we report that modification of the isoprenoid biosynthetic pathway can offset the indispensability of the Fe–S cluster biosynthetic systems and show that the resulting Δisc Δsuf double mutants can grow without detectable Fe–S cluster‐containing proteins. We also constructed a series of mutants in which each isc gene was disrupted in the deletion background of sufABCDSE. Phenotypic analysis of the mutants revealed that Fdx, an essential electron‐transfer Fe–S protein in the ISC machinery, is dispensable under anaerobic conditions, which is similar to the situation with IscA. Furthermore, we found that several suppressor mutations in IscU, an Fe–S scaffold protein responsible for the de novo Fe–S cluster assembly, could bypass the essential role of the chaperone system HscA and HscB. These findings pave the way toward a detailed molecular analysis to understand the mechanisms involved in Fe–S cluster biosynthesis.     

Genes for iron–sulphur cluster assembly are targets of abiotic stress in rice,Oryza sativa

Iron–sulphur (Fe–S) cluster assembly occurs in chloroplasts, mitochondria and cytosol, involving dozens of genes in higher plants. In this study, we have identified 41 putative Fe–S cluster assembly genes in rice (Oryza sativa) genome, and the expression of all genes was verified. To investigate the role of Fe–S cluster assembly as a metabolic pathway, we applied abiotic stresses to rice seedlings and analysed Fe–S cluster assembly gene expression by qRT‐PCR. Our data showed that genes for Fe–S cluster assembly in chloroplasts of leaves are particularly sensitive to heavy metal treatments, and that Fe–S cluster assembly genes in roots were up‐regulated in response to iron toxicity, oxidative stress and some heavy metal assault. The effect of each stress treatment on the Fe–S cluster assembly machinery demonstrated an unexpected tissue or organelle specificity, suggesting that the physiological relevance of the Fe–S cluster assembly is more complex than thought. Furthermore, our results may reveal potential candidate genes for molecular breeding of rice.