Iron-deficient Cultures Research Articles

The remedy of lime-induced chlorosis in peanuts by pseudomonas sp. siderophores

Abstract Strains of siderophore producing Pseudomonas sp. were isolated from the rhizospheres of cotton (Gossypium herbaceum L.) and peanuts (Arachis hypogaea) (strains 1 and 11, respectively). The siderophores were produced in iron deficient culture medium. FeCl3 was added to the culture media at the end of the growth period in order to produce an iron pigment complex. These solutions were applied to the soil directly or after removal of bacteria and free salts by centrifugation and dialysis. Peanuts grown in 0.5 kg pots on a highly calcareous soil (63% CaCO3) and exhibiting severe iron chlorosis were used as test plants to the iron‐siderophore or iron‐siderophore‐bacteria activity as soil amendment for iron deficiency. The plants were treated with (1) FeEDDHAM10 mg 500 g‐1 soil; (2) iron pigment complex of the two isolates; and (3) bacterial suspension containing the iron pigment complex. Plants grown on untreated soil were used as a control. Significant increase of chlorophyll concentration of th...

Attenuation of Pasteurella multocida by culturing in iron-deficient culture medium; vaccine development

Attenuation of Pasteurella multocida by culturing in iron-deficient culture medium; vaccine development

Siderophore activity of pyoverdin for Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces an extracellular compound with yellowish green fluorescence, called pyoverdin, which functions as a siderophore. The production of pyoverdin, formerly called fluorescein, is concomitant with the production of another siderophore, pyochelin. Pyoverdin is produced by P. aeruginosa in several forms, some of which were separated on gel filtration columns and on reverse-phase, high-pressure liquid chromatography columns. An active form of iron-free pyoverdin was purified to homogeneity. The elution of pyoverdin from the columns was monitored for absorbance, fluorescence, and siderophore activities. These activities, iron binding, and the stimulation of bacterial iron transport indicated that pyoverdin can function as a siderophore for P. aeruginosa. The siderophore function of pyoverdin may be related to the pathogenicity of this bacterium because pyoverdin stimulated growth not only in iron-deficient culture medium, but also in defined medium containing transferrin and in human serum or plasma.

Activation and De Novo Synthesis of Hydrogenase in Chlamydomonas

Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg(2+), Ca(2+), and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.

Extracellular siderophores from Aspergillus ochraceous.

A large number of iron-chelating compounds (siderophores) were isolated from supernatants of iron-deficient cultures of a mold isolate, subsequently identified as Aspergillus ochraceous . Siderophores in their iron chelate form were purified to homogeneity by using Bio-Gel P2, silica gel, and C-18 bonded silica gel (reverse-phase) columns. Most of these compounds, as identified by 1H and 13C nuclear magnetic resonance spectroscopy and X-ray crystallography, belong to the ferrichrome family. The organism produces ferrirubin and ferrichrysin as the predominant and the second major compound (62 and 15% of the total siderophores), respectively. Ferrichrysin appears as the first siderophore in the medium on day 2 of growth. Several of the other siderophores are novel and ranged in quantities from 0.2 to 5% of the total. The trivial names asperchrome A, B1, B2, C, D1, D2, and D3 are proposed for these novel compounds, which are all members of the ferrichrome family, and all but the first one contain a common Orn1 - Orn2 - Orn3 - Ser1 -Ser2-Gly cyclic hexapeptide ring with three dissimilar ornithyl delta-N-acyl groups. Another compound which appeared late in the growth period was similar to fusarinine C ( fusigen ). All of these compounds showed growth factor activity to various extents in bioassays with Arthrobacter flavescens Jg-9. None of these compounds showed antibacterial activity against Escherichia coli or Bacillus megaterium.

Iron-Sulfur Centers and Activities of the Photosynthetic Electron Transport Chain in Iron-Deficient Cultures of the Blue-Green Alga Aphanocapsa

Cultures of the blue-green alga, Aphanocapsa, were grown under iron-limiting conditions and changes in concentration of redox components of the photosynthetic electron transport chain, particularly iron-sulfur centers, were monitored by spectroscopic methods. A moderate iron depletion (1/10 of the normal concentration) had little effect on photosynthetic electron transport reactions and growth. Nevertheless, the amount of membrane-bound non-heme iron decreased sharply, and ferredoxin was nearly totally replaced by a flavin-containing protein, flavodoxin. Severe iron-deficiency (1/100 of the normal concentration) was accompanied by growth inhibition and decreased rates of photosynthetic electron flow. The Photosystem I reaction center was most affected by iron depletion as evidenced by a decrease in the amounts of iron-sulfur centers A, B, and X. However, formation of other redox proteins, even those that do not contain iron, was also inhibited by severe iron deficiency.

Examination of hydroxamate-siderophore production by neritic eukaryotic marine phytoplankton

Species of neritic eukaryotic marine phytoplankton were investigated during 1982 for hydroxamate-type siderophore production under iron-sufficient and iron-deficient culture conditions. Three of the 5 Prorocentrum species examined produced siderophores. Prorocentrin, the extracellular hydroxamate-type siderophore isolated from P. minimum, was also produced by P. mariae-lebouriae and P. gracile. P. maximum and P. micans grew poorly in iron-deficient medium and did not produce intracellular or extracellular hydroxamate-type siderophores. Thalassiosira pseudonana and Dunaliella tertiolecta produced extracellular siderophores under iron-deficient conditions, but siderophore production was not detected in the other two species, Skeletonema costatum and Olisthodiscus luteus. Each species which produced extracellular Csaky-positive hydroxamate showed a similar pattern of production. Under iron-sufficient conditions there was no measurable siderophore found either intracellularly or extracellularly. Under iron-deficient culture conditions hydroxamate-type siderophore was produced 1 to 2 d after the cessation of growth in the stationary phase. Production was over a short period of time (1 to 2 d) and the siderophore did not remain in the medium. The rate of siderophore disappearance from the medium was similar to the rate of production. Each species which produced siderophores showed an increase in in vivo fluorescence coincidental with the disappearance of the extracellular siderophore from the culture medium. There was no corresponding increase in in vivo fluorescence in iron-sufficient cultures. It is suggested that in vivo fluorescence may be used as a screening procedure for determining hydroxamate-type siderophore production in eukaryotic phytoplankton. An hypothesis on the iron uptake mechanism is proposed.

Siderophore production by phytopathogenic microbial species

The production of siderophores (microbial iron transport agents) and siderophore-like substances was examined in the gram-negative bacterial plant pathogens, Agro-bacterium tumefaciens, Erwinia carotovora carotovora, Pseudomonas syringae pv. phaseolicola, and Erwinia amylovora, and in the saprophytic bacterium Agrobacterium radiobacter. This attribute was found to be widespread among the organisms surveyed. All but 2 of 27 different strains of A. tumefaciens and A. radiobacter investigated produced catechol-containing compounds during low-iron culture. One of these non-cat-echol-producing strains formed no detectable siderophore, and the other produced hydroxamate-like substances. The compound 2,3-dihydroxybenzoic acid as well as two water-soluble, ninhydrin-reacting catechols were found in iron-deficient cultures of E. c. carotovora 78. These phenolates enhanced the growth of the organism when stressed for iron. In response to iron-limiting growth, P. s. phaseolicola G50 produced substances which reacted with iron to give one purple and two orange-colored compounds. A mixture of these ferric complexes had growth factor activity for the organism when it was cultured in an iron-poor medium. Neither catechol nor hydroxamate-type siderophores were produced by E. amylovora Ea178 or Ea213. However, siderophore activity was detected in the supernatant fluids of low-iron cultures of both strains.

Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum.

The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined. Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium. The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures. The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes. The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities. These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa.

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.

Purification, properties and synthesis of delta-aminolaevulinate dehydratase from Neurospora crassa.

Delta-aminolaevulinate dehydratase, the second and rate-limiting enzyme of the haem-biosynthetic pathway, was purified 300-fold from induced cultures of Neurospora crassa. The native enzyme has a mol.wt. of about 350000, whereas the salt-treated enzyme after incubation at 37 degrees C for 10 min has a mol.wt. of about 232000. The mol.wt. of the subunit is about 38000. Antibodies to the purified enzyme were raised in rabbits. By using radiolabelling and immunoprecipitation techniques it was shown that addition of iron and laevulinate to iron-deficient cultures brings about a significant increase in the synthesis of the enzyme, and protoporphyrin, the penultimate end product of the pathway, represses enzyme synthesis.

Characterization of protoporphyrin as the physiological regulator of δ-aminolevulinate dehydratase in Neurospora crassa

In Neurospora crassa, the activity of δ-aminolevulinate dehydratase, the second and rate-limiting enzyme of the heme-biosynthetic pathway, is low in normal cells compared to the activity detected in plants, animals and bacteria. The activity is almost undetectable when Neurospora crassa is grown under iron-deficient conditions. The enzyme activity increases strikingly on addition of iron to iron-deficient cultures. This increase can be blocked by the addition of protoporphyrin, the penultimate product of the heme-biosynthetic pathway, to the cultures. The question whether iron directly acts at the genetic level or acts merely by removing protoporphyrin, converting the latter into heme prosthetic groups of hemoproteins, has been investigated by studying the effect of inhibition of heme synthesis on the induction of δ-aminolevulinate dehydratase. It has been found that treatments with levulinic acid or cyanide which inhibit the formation of the porphyrin moiety, induce δ-aminolevulinate dehydratase, whereas treatments which inhibit at a step after protoporphyrin formation (iron-deficiency and cobalt treatment) repress the enzyme. The endogenous levels of protoporphyrin are strictly controlled: a decrease below the optimum level causing induction and an increase above the optimum level leading to repression of δ-aminolevulinate dehydratase. Levulinic acid and cyanide can induce the enzyme in iron-deficient cultures in the absence of added iron, indicating that the metal iron acts only by converting protoporphyrin to heme fixed in hemoproteins in Neurospora crassa. Therefore it is suggested that protoporphyrin is the physiological regulator of δ-aminolevulinate dehydratase in Neurospora crassa.

Siderophores produced by saprophytic bacteria as stimulants of germination of conidia of Colletotrichum musae

The addition of suspensions of washed cells of a Pseudomonas sp. (isolate UV3) grown in 1 10 nutrient broth to drops containing conidia of Colletotrichum musae stimulated their germination and appressorium formation. No stimulation was observed when the bacterium was grown in full strength nutrient broth. Similarly, bacteria grown in a synthetic medium (A) based on glucose, ammonium sulphate, phosphates and trace elements were not stimulatory unless iron was first removed from the medium. Filtrates from iron deficient cultures were themselves highly stimulatory. Pseudomonas UV3 produced a yellow compound in medium A and 1 10 nutrient broth. Of the fractions separated from culture filtrates on medium A only those containing this yellow compound were stimulatory to germination of conidia of C. musae. The compound was extracted with chloroform: phenol and purified by chromatography on columns of Bio-beads SM2 and Bio-gel P4. The compound gave a positive reaction to the test for hydroxamates, an apparent mol. wt of 500 to 600 daltons and ornithine and alanine were the principal hydrolysis products. Addition of ferric ions to aqueous solutions resulted in a shift of absorption from 384 to 404 run and a change of colour from light yellow to red/brown. It was concluded that the compound was a hydroxamate siderophom. The purified siderophore was highly stimulatory to germination of C. musae over the concentration range 75 to 300 μg ml −1. The addition of iron reduced the stimulation suggesting that iron uptake by conidia is not involved. Leachate made from bananas was found to contain 0·065 μg ml −1 Fe, and when the leachate was used to prepare media this quantity was found insufficient to suppress the siderophore formation by UV3, suggesting that siderophores could be produced by saprophytic bacteria on plant surfaces.

Iron Transport in Microalgae: the Isolation and Biological Activity of a Hydroxamate Siderophore from the Blue-Green Alga Agmenellum quadruplicatum

Summary: Supernatants from iron-deficient cultures of the marine, coccoid blue-green alga Agmenellum quadruplicatum strain PR-6 contained a chloroform-soluble iron-binding hydroxamate which was stimulatory for the growth of Arthrobacter jlavescens JG-9. Production of this material was maximal at 1 μM-Fe, was temperature dependent, and was increased by adding NH4 +. At the absorption maximum of 430 nm the At of the purified ferric complex was 32. The iron-free compound yielded nitroso dimers on periodate oxidation and was 60% as active on a weight basis for growth of Arth. flavescens as ferrioxamine B. At concentrations as low as 1 ng ml−l the purified hydroxamate stimulated growth of iron-depleted Agm. quadruplicatum in a chemically defined medium. Growth factor activity for Arth. flavescens JG-9 was also found in the supernatants and/or cell extracts of other blue-green algae and several diatoms grown under low-iron conditions.

Site of action of iron in the induction of δ-aminolevulinate dehydratase in Neurospora crassa

Summary δ-Aminolevulinate (ALA) dehydratase, the second and rate limiting enzyme of the heme biosynthetic pathway in the mold Neurospora crassa is induced maximally in 30 min by the addition of iron to iron-deficient cultures. The induction of the enzyme is blocked by cycloheximide, protoporphyrin, 8-azaguanine and cordycepin. Iron also brings about an increase in poly(A)-containing RNA synthesis under conditions of induction. The iron dependent increase in poly(A)-containing RNA synthesis is blocked by protoporphyrin. It is suggested that at the time intervals examined, bulk of the messenger RNA synthesized in response to iron addition represents the messenger for ALA dehydratase.

Production of enterochelin by Escherichia coli 0111

The major neutral iron-transporting compound produced by Escherichia coli 0111/K58/H2 has been isolated from iron-deficient cultures of the organism and compared with the corresponding compound, enterochelin, produced by E. coli K12. The product contained serine and 2,3-dihydroxybenzoic acid and formed a complex with Fe 3+. Since the PMR spectra of the products from the two strains were identical, it was concluded that E. coli 0111 also secreted enterochelin under iron-deficient conditions. although it was not possible to establish the optical configuration of the serine residues in the molecule, the CD spectra of the metal free and Fe 3+, complexes were found to be of the same sign and magnitude. The spectra show that metal binding results in considerable conformational changes in the enterochelin molecule. The biological properties of the two compounds appear to be identical as judged by their ability to abolish the bacteriostatic effect of serum on E. coli 0111.

The effect of p-aminosalicyclic acid on iron transport and assimilation in mycobacteria

p-Aminosalicylic acid inhibits growth of Mycobacterium bovis BCG and Mycobacterium smegmatis more effectively if cells are growing with a sufficiency of iron (> 1 μg Fe/ml) in the medium than if cells are deficient in iron (<0.1 μg Fe/ml). In iron-deficient cultures formation of mycobactin, an ionophore for iron transport, is strongly inhibited by p-aminosalicylic acid. Uptake of iron into cell suspensions is also inhibited and the activity of several iron-containing enzymes declines in cells exposed to p-aminosalicylic acid during their growth. p-Aminosalicylic acid is about 50 times more effective towards a mutant of M. smegmatis which required mycobactin under iron-deficient growth conditions than towards the wild-type parent. p-Aminosalicylate is taken up into cells by an active process independent of the salicylate uptake system, possibly by the route used for assimilation of p-aminobenzoate. (This could account for why p-aminobenzoic acid, but not salicylic acid, antagonizes the action of p-aminosalicylic acid.) With iron-deficient cells, salicylate assimilation is about 50 times greater than either p-aminosalicylate or p-aminobenzoate but with iron-sufficient cells and with the mycobactin mutant salicylate uptake is negligible whereas p-aminobenzoate and p-aminosalicylate uptakes are unaffected. p-Aminosalicylic acid at 3.3 mM (500 μg/ml) partially inhibits the uptake of both p-aminobenzoate and, if it is occuring, that of salicylate as well. As p-aminosalicylic acid is always more effective when the intracellular concentration of salicylic acid is low, it probably acts as an anti-metabolite of salicylic acid, not, however, by inhibiting the conversion of salicylic acid to mycobactic, but probably somewhere along the metabolic pathway of iron uptake.

Die Regulation der Synthese der Riboflavinsynthetase durch Eisen in flavinogenen Hefen

Summary The influence of iron on the synthesis of riboflavin (RF) and riboflavin synthetase (RFS) activity in the cells of some flavinogenic and non-flavinogenic yeasts was investigated. It is shown that during the cultivation of Pichia guilliermondii, Torulopsis candida and Schwanniomyces occidentalis in iron-deficient media the induction of RF oversynthesis is correlated with an increase in the RFS activity. This phenomenon did not occur in iron-deficient cultures of weak RF producers such as P. ohmeri, Candida utilis and C. pulcherrima. The incubation of iron-rich cells of T. candida with α,α′-bipyridine or o-phenanthroline chelaters which possess a high affinity to iron also causes a marked increase of RFS activity and the flavino-genesis rate. Cycloheximide in low concentration abolishes this effect; the same does amino-acid starvation of the cells of the methionine-deficient mutant of T. candida, MET-2. The addition of Fe (II) ions to the suspension of young iron-deficient cells prevents the increase of RFS activity. The Fe (II) ions and the autolysates of iron-rich cells do not inhibit the RFS activity in crude extracts of iron-deficient cells of this species. It is concluded that iron participates in the regulation of RFS synthesis in the cells of flavinogenic yeasts.

Structure of the decarboxylated derivative of the chromophore from the fluorescent peptide produced by iron-deficient Azotobacter vinelandii

Iron-deficient cultures of Azotobacter vinelandii contain a yellow-green fluorescent peptide which, upon hydrolysis, yield several amino-acids and a chromophoric residue which retains the spectral properties of the parent peptide. The decarboxylated derivative of the chromophore (I), which retains the spectral behaviour, has been determined by X-ray crystallographic study. Crystals are monoclinic, space group P21/c, with four molecules of C13H12N3O3Cl.2H2O in the unit cell of dimensions a= 10·342(1), b= 6·986(1), c= 21·525(3)A, β= 115·78(2)°. The structure was solved by direct methods from diffractometer data and refined by block-diagonal least-squares techniques to R 8·0% for 1331 independent reflections. The molecules are piled one above the other between hydrogen-bonded spirals of chloride ions and water molecules.

Delta-aminolaevulinate dehydratase, the regulatory enzyme of the haem-biosynthetic pathway in Neurospora crassa.

The activity of delta-aminolaevulinate dehydratase is very low in the mould Neurospora crassa compared with the activities detected in bacterial and animal systems. The enzyme is inducible in iron-deficient cultures by addition of iron and is repressed by protoporphyrin. The properties of the purified enzyme indicate its allosteric nature and susceptibility to feedback inhibition by coproporphyrinogen III. Neurospora extracts also contain a protein inhibitor of the enzyme and a small-molecule activator, which appears to be associated with the enzyme. The regulatory function of this enzyme in vivo is correlated with the accumulation of delta-aminolaevulinic acid in normal cultures of N. crassa. The decay curve of the iron-induced enzyme in vivo shows a biphasic pattern, with one of the components showing a half-life of 4-5 min.