Characterization of Iron Uptake from Ferrioxamine B by Chlorella vulgaris

Iron uptake from two Fe³⁺-hydroxamate siderophores, ferrioxamine B and Fe³⁺-rhodotorulate, by iron-stressed Chlorella vulgaris (ATCC strain 11468) was evaluated with some comparison to iron uptake from synthetic and organic acid ferric chelates. Iron-stress induced iron uptake from ferrioxamine B. Dissipation of the electrochemical gradient, via uncouplers, inhibited iron uptake. Respiratory inhibitors gave variable results, an indication that a direct link to respiration was not apparent. Vanadate inhibition of iron uptake indicated that an ATPase or phosphate intermediate could be involved in the uptake mechanism. Divalent cations manifested variable effects dependent on the cation and chelator used. These data confirm that C. vulgaris has an inducible iron-uptake system for Fe³⁺-hydroxamic acid siderophores which may involve a different mechanism than that observed for other chelates.     

Ternary Complex Formation Facilitates a Redox Mechanism for Iron Release from a Siderophore

While the naturally occurring reducing agents glutathione (GSH) and ascorbate (H₂A) alone are ineffective at reducing iron(III) sequestered by the siderophore ferrioxamine B, the addition of an iron(II) chelator, sulfonated bathophenanthroline (BPDS), facilitates reduction by either reducing agent. A mechanism is described in which a ternary complex is formed between ferrioxamine B and BPDS in a rapidly established pre-equilibrium step, which is followed by rate limiting reduction of the ternary complex by glutathione or ascorbate. Spectral, thermodynamic, and kinetic evidence are given for ternary complex formation. Ascorbate was found to be slightly more efficient at reducing the ternary complex than glutathione (k₄=2.1×10⁻³ M⁻¹ s⁻¹ and k₄=6.3×10⁻⁴ M⁻¹ s⁻¹, respectively) at pH 7. Reduction is followed by a rapid ligand exchange step where iron is released from ferrioxamine B to form tris-(BPDS)iron(II). The implications of these results for siderophore mediated iron transport and release are discussed.     

Fob1 and Fob2 Proteins Are Virulence Determinants of Rhizopus oryzae via Facilitating Iron Uptake from Ferrioxamine

Dialysis patients with chronic renal failure receiving deferoxamine for treating iron overload are uniquely predisposed for mucormycosis, which is most often caused by Rhizopus oryzae. Although the deferoxamine siderophore is not secreted by Mucorales, previous studies established that Rhizopus species utilize iron from ferrioxamine (iron-rich form of deferoxamine). Here we determined that the CBS domain proteins of Fob1 and Fob2 act as receptors on the cell surface of R. oryzae during iron uptake from ferrioxamine. Fob1 and Fob2 cell surface expression was induced in the presence of ferrioxamine and bound radiolabeled ferrioxamine. A R. oryzae strain with targeted reduced Fob1/Fob2 expression was impaired for iron uptake, germinating, and growing on medium with ferrioxamine as the sole source of iron. This strain also exhibited reduced virulence in a deferoxamine-treated, but not the diabetic ketoacidotic (DKA), mouse model of mucormycosis. The mechanism by which R. oryzae obtains iron from ferrioxamine involves the reductase/permease uptake system since the growth on ferrioxamine supplemented medium is associated with elevated reductase activity and the use of the ferrous chelator bathophenanthroline disulfonate abrogates iron uptake and growth on medium supplemented with ferrioxamine as a sole source of iron. Finally, R. oryzae mutants with reduced copies of the high affinity iron permease (FTR1) or with decreased FTR1 expression had an impaired iron uptake from ferrioxamine in vitro and reduced virulence in the deferoxamine-treated mouse model of mucormycosis. These two receptors appear to be conserved in Mucorales, and can be the subject of future novel therapy to maintain the use of deferoxamine for treating iron-overload.     

利用市政污水培养Chlorella vulgaris生产生物柴油

为了考察利用南昌市政污水规模化培养富油微藻生产生物柴油,同时达到净化污水的目的,取南昌市青山湖污水处理厂未经任何处理的市政污水作为普通小球藻(Chlorella vulgaris)生长的培养液。监测了C.vulgaris在市政污水中连续培养10 d的特定生长率、生物质产量以及与之相关的市政污水中氨氮(NH4+-N)、总磷(TP)、化学需氧量(COD)、总悬浮固体(TSS)和挥发性悬浮固体(VSS)的清除情况。实验表明:营养物质的水平显著地影响了C.vulgaris的生长。C.vulgaris的生长率在培养8 d后达到最大,OD680为2.856,总的生物质产量日均最大积累速率为0.01 g/L,油脂含量为干质量的18%,油脂的平均日产量为0.001 g/L。培养10 d内NH4+-N、TP和COD的去除率分别为50.0%、32.1%和26.0%,TSS和VSS的日平均去除速率分别为0.01 g/L和0.006 1 g/L。     

The Relation between the Synthesis of Inorganic Polyphosphate and Phosphate Uptake by Chlorella vulgaris

During a period of phosphate starvation, the phosphate contentof cells of Chlorella vulgaris which had been grown in phosphate-richsolution, decreased. The levels of most phosphate fractionsdeclined, especially those of inorganic polyphosphates, whichat first accounted for about 5 per cent of the total phosphateand virtually disappeared after 36 h starvation. On return toa phosphate medium, phosphate was taken up at a much fasterrate than before starvation, with a striking increase in acid-solublepolyphosphate. The stimulated phosphate uptake and polyphosphateincrease have been shown to be specific effects of phosphatestarvation, occurred only when excess phosphate was suppliedand required light or air for the provision of energy. Therewas relatively little change in the concentrations of otherphosphate fractions, including orthophosphate. Inorganic polyphosphatewas found to be synthesized solely from phosphate absorbed fromthe medium. It is argued that polyphosphate synthesis is a consequenceof the stimulation of phosphate uptake, induced by the starvationperiod.     

Uptake of cadmium and zinc by the alga Chlorella vulgaris: II. Multi-ion situation

Many microorganisms are capable of sequestering and concentrating heavy metals from their aqueous environment. While much research has beep carried out on the uptake of single species of metal ions, little attention seems to have been given to the study of multimetal ion systems. A mathematical model has previously been developed to describe the uptake of individual metal species by a microorganism. The model proposes two sequential processes: an initial rapid uptake due to cellular surface adsorption and a subsequent slow uptake due to membrane transport of the metal into the cells. This article extends the treatment by considering the uptake of two metal species together, cadmium and zinc, under different experimental conditions. The results are discussed in terms of possible mechanistic interactions.     

Metabolism of Urea by Chlorella vulgaris

Urea metabolism was studied with nitrogen-starved cells of Chlorella vulgaris Beijerinck var. viridis (Chodat), a green alga which apparently lacks urease. Incorporation of radioactivity from urea-(14)C into the alcohol-soluble fraction was virtually eliminated in cell suspensions flushed with 10% CO(2) in air. This same result was obtained when expected acceptors of urea carbon were replenished by adding ornithine and glucose with the urea. Several carbamyl compounds, which might be early products of urea metabolism and a source of the (14)CO(2), were not appreciably labeled. If cells were treated with cyanide at a concentration which inhibited ammonia uptake completely and urea uptake only slightly, more than half of the urea nitrogen taken up was found in the medium as ammonia. Cells under nitrogen gas in the dark were unable to take up urea or ammonia, but the normal rate of uptake was resumed in light. Since 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not selectively inhibit this uptake, an active respiration supported by light-dependent oxygen evolution in these cells was ruled out. A tentative scheme for urea metabolism is proposed to consist of an initial energy-dependent splitting of urea into carbon dioxide and ammonia. This reaction in Chlorella is thought to differ from a typical urease-catalyzed reaction by the apparent requirement of a high energy compound, possibly adenosine triphosphate.     

小球藻的玻璃化超低温保存法

先用含0.5 m01·L-1甘油和0.4 mol·L-1蔗糖的预处理液处理20min,然后用含30%蔗糖 15%乙二醇 10%二甲基亚砜 BBMG培养液的玻璃化液处理,在0℃下预冻60min后,将小球藻投入液氮.此法存活率较高,可达到60.14%,小球藻种质保存效果较好.通过试验初步建立了小球藻玻璃化法超低温保存的技术程序.     

Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris

ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the V_max. Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half V_max value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half V_max value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn²⁺ and Mg²⁺ were highly sigmoidal. The activator concentration at half V_max values for Mn²⁺ and Mg²⁺ were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.     

网织红细胞铁摄取机制的研究进展

从细胞及分子水平重点讨论了有关网织红细胞铁代谢的两个问题. a.网织红细胞如何摄取载铁蛋白结合铁？分七个主要步骤总结了这一摄取过程的研究新进展. b.在内吞小体内,铁从载铁蛋白释放后如何穿越内吞小体膜进入细胞质？ 论述了有关的机制,包括铁通道假说,铁载体,H^＋-ATP酶介导的铁转位,自由基和过氧化物的作用. 结语部分概述了目前这一领域中需要进一步研究的一些问题.     

Characteristics of Sulfite Transport by Chlorella vulgaris

The rate of short-term accumulation of [³⁵S]sulfite in Chlorellavulgaris cells was found to be strongly dependent on the pHof the medium. The rate increased with decreased pH, and theincrease in rate closely paralleled the increase in the concentrationof the un-ionized form of sulfite. When the pH of the mediumwas increased, fast accumulation ceased immediately. The rateof accumulation showed a strong temperature dependence, withan apparent temperature coefficient of 1.93 per 10°C rise,between 10 and 25°C. Because pK_a values of sulfite shiftwith temperature, the rates were corrected by dividing by theconcentration of the un-ionized form of sulfite present at therespective temperatures. The temperature coefficient was thenfound to decrease to 1.45. When cells which had been allowedto accumulate [³⁵S]sulfite for 20 min were transferred to amedium containing no sulfite, more than 50% of the accumulated[³⁵S] was released into the medium in 20 min. Our results arecompatible with a simple diffusion model of SO₂ transport intoChlorella cells. (Received September 26, 1996; Accepted January 20, 1997)     

Uptake of Uracil by Synchronous Chlorella fusca

Uptake of radioactive uracil by light-dark synchronized Cblorella fusca Shihira and Krauss was studied. For the characterization of the uptake system autospores were used and the following results obtained. Autospores kept in the dark accumulated uracil against a concentration gradient in a process having an observed activation energy of 10 keal/mol in the 10–40°C interval. Addition of glucose to the reaction suspension did not affect the uptake, but, 100 γM dinitrophenol inhibited the process by 90%. Abrupt changes in rate were found upon changing the conditions from light to dark and vice versa, and the rates measured in light were about 2.5 times larger than those found in the dark. Initial rates measured in the dark followed saturation kineties with half maximal rate found at 0.25 γM uracil, and with an apparent maximal rate of 1.7.10^-10 mol/10 min . 10⁷ cells. The effect of 14 pyrimidines on uptake was tested, and it was found that uracils which were substituted in the 5′ or 5′+ 6′ positions were strongly inhibitory. Of these, thymine and dihydrouracil were tested and shown to inhibit uracil uptake competitively. Initial uptake rates, measured in the dark with 1.0 γM uracil, were recorded at intervals during the 24 h synchronous cycle. The uptake rate per ml culture was constant during the first 9 h, thereafter increasing to reach a peak value at 14 h. This peak was followed by a strong increase from 18 h onwards, this increase being concomitant with the sporulation process, and closely followed its time course.     

Flocculation of Chlorella vulgaris by Lactobacillus casei

Summary The flocculation of Chlorella vulgaris by Lactobacillus casei was studied to determine whether the latter could act as a suitable flocculant for the removal of Chlorella from algal ponds. The flocculating activity of the Lactobacilli was caused by the bacterial cells themselves, and not by diffusible products of bacterial metabolism. Diffusible products of algal metabolism inhibited flocculation. For algae resuspended in water, the best flocculation occurred at pH values less than 3.5 where the charges on the bacterial and algal cells were opposite. For flocculation at least one bacterium was required for every algal cell; in terms of cell concentrations,10 mg/l of bacteria were required to flocculate an algal suspension of 1,000 mg/l. The mechanism of flocculation implied by the results is that positively charged cells of L. casei adsorb to the surface of negatively charged cells of C. vulgaris neutralizing the charge and thus destabilizing the algal suspension. Because of the low pH required and because diffusible products of algal metabolism inhibit the flocculation, it is unlikely that L. casei could be usefully employed as a flocculant of Chlorella from algal ponds.     

The Assimilation of Acetate by Chlorella vulgaris

The acetate metabolism of autotrophically grown, acetate-adaptedand acetate-grown cells is compared. All oxidize acetate rapidlyand assimilate about half of the acetate added in short-termexperiments. Kinetic analysis of the incorporation of ¹⁴C-acetatereveals citrate as a primary product of acetate assimilationin all cells in darkness. Malate formed from acetate-I-¹⁴C byacetate-grown cells is asymmetrically labelled in a manner consistentwith a primary incorporation of acetate into malate by a malatesynthase reaction. The chief difference between autotrophic and acetate-grown cellsis the faster rate at which the latter incorporate acetate carboninto compounds outside the tricarboxylic acid cycle. In particular,incorporation into protein and carbohydrate is much faster inacetate-grown cells and it is suggested that enzymes catalysingreactions leading away from the tricarboxylic acid cycle mayincrease in activity in acetate-grown cells. Light greatly stimulates acetate incorporation into lipide andalso increases the synthesis of protein and carbohydrate.     

Uptake of cadmium and zinc by the alga Chlorella vulgaris: part 1. Individual ion species

The ability of algae and bacteria to accumulate heavy metals from the surrounding environment is a widely recognized phenomenon that has a number of important implications. This work reports on the development of a quantitative model that addresses the basic mechanisms inherent in many uptake processes. The model postulates two mechanisms: an initial rapid metal ion uptake due to attachment onto the cell wall followed by a relatively slow uptake due to membrane transport of the metal into the cell. The mathematical model has been tested using the alga Chlorella vulgaris in the presence of cadmium and zinc in solution under various experimental conditions.     

Evaluation of Active Oxygen Effect on Photosynthesis of Chlorella vulgaris

The relationship between O₂ and an active oxygen scavenging system in Chlorella vulgaris var.vulgaris (IAM C-534) was investigated. When Chlorella vulgaris was exposed to 2% O₂, only traces of active oxygen scavenging enzymes were found. When the Chlorella vulgaris was treated with 20% or 50% O₂, it was shown that the level of enzyme activity increased as the O₂ concentration increased. An increase in enzyme activity was not found in any specific enzyme but in all of the enzymes, but the level of glutathione and ascorbate remained the same in all the cases. In addition, the photosynthetic efficiency also decreased as the concentration of O₂ was increased. These results suggest that an O₂ enriched environment can lead to an increase in the production of active oxygen species such as O_bullet2 and H₂O₂ and to a decrease in the photosynthetic efficiency in Chlorella vulgaris. The hydroxyl radical (^bulletOH) was detected directly in the Chlorella vulgaris suspension with a spin trapping reagent. It was also clear that the increase in the ^bulletOH intensity as the visible light intensity increased was unrelated to the O₂ concentration. It was suggested that the conditions for producing ^bulletOH and the other active oxygen species were different, and that two types of oxygen stress should exist in the Chlorella vulgaris.