Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity

Growth of Anabaena sp. strain 7120 (in the absence of chelators or added iron) was inhibited by the addition of 2.1 to 6.5 microM copper and was abolished by copper concentration of 10 microM or higher. When the copper was chelated to schizokinen (the siderophore produced by this organism in response to iron starvation), the toxic effects were eliminated. Analysis of culture filtrates showed that the cupric schizokinen remains in the medium, thereby lowering the amount of copper taken up by the cells. Although this organism actively transports ferric schizokinen, it apparently does not recognize the cupric complex. Thus, Anabaena sp. is protected from copper toxicity under conditions in which siderophore is being produced. For cells grown in low iron, the accumulation of extracellular schizokinen was observed to parallel cell growth and continue well into stationary phase. The actual iron status of the organism was monitored by using iron uptake velocity as an assay. Cultures grown on 0.1 microM added iron were found to be severely iron limited upon reaching stationary phase, thus explaining the continued production of schizokinen. These data show that the siderophore system in Anabaena spp. has developed primarily as a response to iron starvation and that additional functions such as alleviation of copper toxicity or allelopathic inhibition of other algal species are merely secondary benefits.     

Siderophore-mediated iron uptake in different strains of Anabaena sp.

Anabaena sp. strain 6411, which produces the dihydroxamate siderophore schizokinen to facilitate iron uptake, is also capable of using the related siderophore aerobactin. The two siderophores compete for the same iron transport system, but there is a markedly higher affinity for ferric schizokinen than for ferric aerobactin. The trihydroxamate siderophore ferrioxamine B is far less effective as an iron donor in this organism. Anabaena sp. strain 7120 appears to be closely related to strain 6411. It synthesizes schizokinen as its major siderophore and shows rates of iron uptake from ferric schizokinen, ferric aerobactin, and ferrioxamine B which are similar to those observed with strain 6411. Anabaena cylindrica Lemm. 7122 and 1611, on the other hand, differ from strain 6411. In contrast to schizokinen, the hydroxamate which they produce in response to iron starvation cannot be extracted with water from the organic layer and does not support the growth of the siderophore auxotroph Arthrobacter flavescens JG-9. Strain 7122 can use its endogenous siderophore or schizokinen to promote iron uptake, but at 50-fold-lower rates than are observed with Anabaena sp. strain 6411 or 7120.     

Active transport of ferric schizokinen in Anabaena sp

The cyanobacterium Anabaena sp. strain ATCC 27898 was found to utilize the siderophore schizokinen to accumulate iron from the environment. This organism had previously been shown to produce schizokinen under low-iron conditions, and we observed that the iron-transport capability is also increased in response to iron limitation. Uptake activity was specific for ferric schizokinen displayed kinetics typical of a protein-mediated process with an apparent Km of 0.04 microM and saturation at high concentrations of substrate. Light-driven transport was blocked by uncouplers and by ATPase inhibitors. Transport in dark-adapted cells was additionally blocked by inhibitors of respiration. We conclude that ATP serves as an energy source for the cellular uptake of ferric schizokinen.     

Multiple modes of iron uptake by the filamentous,siderophore‐producing cyanobacterium,Anabaena sp. PCC 7120

Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore‐ and non‐siderophore‐producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore‐producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self‐secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe′) via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore‐ and non‐siderophore‐mediated iron uptake. While assimilation of Fe′ and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe′ reduction and uptake is advantageous for low‐density cultures, while at higher densities siderophore uptake is preferred.     

Alr0397 is an outer membrane transporter for the siderophore schizokinen in Anabaena sp. strain PCC 7120

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.     

Iron mediated regulation of growth and siderophore production in a diazotrophic cyanobacterium Anabaena cylindrica

Iron mediated regulation of growth and siderophore production has been studied in a diazotrophic cyanobacterium Anabaena cylindrica. Iron-starved cells of A. cylindrica exhibited reduced growth (30%) when the cells were growing under N2-fixing conditions. In contrast, N03-, NO2-, NH4' and urea grown cells exhibited almost 50% reduction in their growth in the absence of iron as compared to their respective counterparts cultured in the presence of iron. However, at 60 microM of iron, A. cylindrica cells exhibited almost equal growth regardless of the nitrogen source available. Siderophore production in A. cylindrica was started after day 2nd of the cell growth and attained its optimal level on day 5th when the cells were at their mid-log phase. No siderophore production was, however, recorded on day 2nd at all the concentrations of iron tested. The production of siderophore in A. cylindrica further increased with increase in iron concentration and attained its optimum level on day 5th at 60 microM iron. A. cylindrica cells took at least 3 days for initiation of siderophore production and produced about 60% siderophore on day 5th even under iron-starved condition. A. cylindrica produced dihydroxamate type of siderophore.     

Isolation and purification of siderophores produced by cyanobacteria,Synechococcus sp. PCC 7942 andAnabaena variabilis ATCC 29413

New siderophores were isolated and purified from the spent growth medium of the cyanobacteriaSynechococcus sp. PCC 7942 (Anacystis nidulans R2) andAnabaena variabilis ATCC 29413 by solvent extraction and thin-layer chromatography. For each species the siderophore was released into the medium when the cells were grown at low iron concentrations and was not found in the medium of cells grown in iron-sufficient medium. Through a series of biological and chemical tests, combined with spectral analysis, the dihydroxamate nature of each siderophore was confirmed. The siderophores produced bySynechococcus sp. PCC 7942 andA. variabilis had distinct relative molecular masses of 310–313 Da and 520–525 Da, respectively. Neither of the two strains produced Arnow-positive extracellular organics, which indicate the excretion of extracellular catechol-type siderophores.     

Effect of Aluminum on the Production of Siderophore by Rhizobium sp. (Cicer arietinum)

Rhizobium sp. strain BICC 651 in the presence of 100 μM Al³⁺ produced a threefold higher level of siderophore than in the control culture under iron limitation during the stationary phase. Al³⁺ in increasing concentrations resulted in decreased growth, and the effect was alleviated by the addition of iron. Siderophore production decreased gradually in Al³⁺-treated culture as well as in the control with the addition of increasing concentrations of Fe³⁺, and at 50 μM Fe³⁺ the level of siderophore was practically undetectable. The siderophore binds Fe³⁺ and also Al³⁺. The outer membrane protein profiles of the bacteria grown in the presence or absence of Al³⁺ were indistinguishable. Received: 15 November 1999 / Accepted: 21 December 1999     

Effects of medium and trace metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain KC.

Under denitrifying conditions, Pseudomonas sp. strain KC transforms carbon tetrachloride (CT) to carbon dioxide via a complex but as yet undetermined mechanism. Transformation rates were first order with respect to CT concentration over the CT concentration range examined (0 to 100 micrograms/liter) and proportional to protein concentration, giving pseudo-second-order kinetics overall. Addition of ferric iron (1 to 20 microM) to an actively transforming culture inhibited CT transformation, and the degree of inhibition increased with increasing iron concentration. By removing iron from the trace metals solution or by removing iron-containing precipitate from the growth medium, higher second-order rate coefficients were obtained. Copper also plays a role in CT transformation. Copper was toxic at neutral pH. By adjusting the medium pH to 8.2, soluble iron and copper levels decreased as a precipitate formed, and CT transformation rates increased. However, cultures grown at high pH without any added trace copper (1 microM) exhibited slower growth rates and greatly reduced rates of CT transformation, indicating that copper is required for CT transformation. The use of pH adjustment to decrease iron solubility, to avoid copper toxicity, and to provide a selective advantage for strain KC was evaluated by using soil slurries and groundwater containing high levels of iron. In samples adjusted to pH 8.2 and inoculated with strain KC, CT disappeared rapidly in the absence or presence of acetate or nitrate supplements. CT did not disappear in pH-adjusted controls that were not inoculated with strain KC.     

Nitrogen starvation mediated by DL-7-azatryptophan in the cyanobacterium Anabaena sp. strain CA.

The addition of DL-7-azatryptophan (AZAT), a tryptophan analog, to continuous cultures of Anabaena sp. strain CA grown with 10 mM nitrate as the nitrogen source resulted in the differentiation of heterocysts. Analysis of the intracellular amino acid pools of Anabaena sp. strain CA after the addition of AZAT showed a marked decline in the intracellular glutamate pool and a slight increase in the levels of glutamine. The in vitro activity of glutamate synthase, the second enzyme involved in primary ammonia assimilation in Anabaena spp., was partially inhibited by the presence of AZAT at concentrations which are effective in triggering heterocyst formation (15% inhibition at 10 microM AZAT and up to 85% inhibition at 1.0 mM AZAT). Azaserine, a glutamine analog and potent glutamate synthase inhibitor, had no effect on the triggering of heterocyst development from undifferentiated batch and continuous cultures of Anabaena sp. strain CA. However, the presence of 1.0 microM azaserine significantly decreased the intracellular glutamate pool and increased the glutamine pool. The addition of AZAT also caused a decrease in the C-phycocyanin content of Anabaena sp. strain CA as a result of its proteolytic degradation. AZAT also had an inhibitory effect on the nitrogenase activity of Anabaena sp. strain CA. All these results suggest that AZAT causes a general nitrogen starvation of Anabaena sp. strain CA filaments, triggering heterocyst synthesis.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5μm and was completely inhibited at an iron concentration of 10μm. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

Effect of iron limitation on growth, siderophore production, and expression of outer membrane proteins of Vibrio cholerae.

Vibrio cholerae strains secrete a phenolate-type siderophore when grown in low-iron medium. The siderophore was detected as early as 3.5 h after downshift to iron-poor medium, and it continued to accumulate in the medium as the cells entered stationary phase. Two clinical isolates and an environmental isolate were examined for the amount of siderophore produced. The environmental isolate produced more siderophore and continued to secrete it at concentrations of iron that repressed synthesis in the clinical isolates. Concomitant with production of siderophore, at least six new proteins were seen in the outer membranes of iron starved cells. One of the proteins was large (200,000 Mr [220K]) and appeared to be loosely associated with the outer membrane. The other five proteins had approximate Mr values of 77K, 76K, 75K, 73K, and 62K. The 62K protein, like the 40K major outer membrane protein, was heat modifiable. One or more of these proteins may be a component of the receptor for the iron-siderophore complex.     

Isolation and purification of canadaphore, a siderophore produced by Helminthosporium carbonum

A new siderophore was isolated and purified from the spent growth medium of the fungus Helminthosporium carbonum by solvent extraction and reverse phase high pressure liquid chromatography. This new molecule has been assigned the name Canadaphore. Canadaphore was detected in culture filtrates after 15 days of growth and production was maximal after growth of H. carbonum to maximal stationary phase in modified Fries basal medium. Production of Canadaphore was completely suppressed when the organism was grown in medium supplemented with iron. Mass spectral analysis yielded a molecular mass of 680 Daltons for the iron-Canadaphore complex and 627 Daltons for the iron-free molecule. Spectroscopic analysis indicates that Canadaphore is a siderophore of the hydroxamate type.     

Hydroxamate Recognition During Iron Transport from Hydroxamate-Iron Chelates

Kinetics of radioactive iron transport from three structurally different secondary hydroxamate-iron chelates (schizokinen-iron, produced by Bacillus megaterium ATCC 19213; Desferal-iron, produced by an actinomycete; and aerobactin-iron, produced by Aerobacter aerogenes 62-1) revealed that B. megaterium SK11 (a mutant which cannot synthesize schizokinen) has a specific transport system for utilization of ferric hydroxamates with a recognition capacity based on the chemical structure of the hydroxamate. Both Desferal and schizokinen enhanced iron uptake in this organism; however, Desferal-iron delivered only one-sixth the level of iron incorporated from the schizokinen-iron chelate. Desferal-iron did not generate the rapid rates of iron transport noted with schizokinen-iron at elevated iron concentrations. Assays containing large excesses of either iron-free Desferal or iron-free schizokinen suggested that the iron-free hydroxamate may compete with the ferric hydroxamate for acceptance by the transport system although the system has greater affinity for the iron chelate. Aerobactin-iron did not stimulate iron uptake in B. megaterium SK11 and aerobactin inhibited growth of this organism, indicating that B. megaterium SK11 cannot efficiently process the aerobactin-iron chelate.     

Iron requirement and siderophore production in Bradyrhizobium strains isolated from Acacia mangium

Fourteen Bradyrhizobium strains isolated from nitrogen-fixing Acacia mangium trees which originated from different geographical areas were analysed for their iron requirement and siderophore production. All strains were affected by starvation but responded differently to it. The increase in bacterial cell yield in response to iron supplementation, as well as the strain's sensitivity towards the synthetic iron-chelator 2,2-dipyridyl, suggested a discrimination of these strains into two groups. Four strains in one group, including a reference strain, produced siderophore(s) when grown under starvation. Other strains belonging to the second group were characterized by a lower iron requirement, a higher sensitivity to 2,2-dipyridyl, and did not apparently demonstrate any siderophore production. Two of the siderophore-producer strains, as well as a Rhizobium reference strain, excreted citrate, which was under iron regulation. Citrate was shown to facilitate iron incorporation in strains of either group.     

Anaerobic iron uptake by Escherichia coli.

Assimilation and uptake of iron in anaerobic cultures of Escherichia coli were supported by iron supplied as ferrienterobactin, ferrichrome, and ferrous ascorbate; however, as in the aerobic cultures, ferrichrome A was a poor iron source. Albomycin inhibited both aerobically and anaerobically grown cells. The siderophore outer membrane receptor proteins FepA and FhuA were produced under anaerobic iron-deficient conditions. Anaerobic transport of ferrienterobactin and ferrichrome was inhibited by KCN and dinitrophenol. The Km for ferrienterobactin uptake in anaerobically grown cells was 0.8 microM, and the Vmax was 38 pmol/min per mg, compared with 0.1 microM and 80 pmol/min per mg, respectively, in aerobically grown cells.     

Siderophore-Mediated Aluminum Uptake by Bacillus megaterium ATCC 19213

The bacterium Bacillus megaterium ATCC 19213 is known to produce two hydroxamate siderophores, schizokinen and N-deoxyschizokinen, under iron-limited conditions. In addition to their high affinity for ferric ions, these siderophores chelate aluminum. Aluminum was absorbed by B. megaterium ATCC 19213 through the siderophore transport receptor, providing an extra pathway for aluminum accumulation into iron-deficient bacteria. At low concentrations of the metal, siderophore-mediated uptake was the dominant process for aluminum accumulation. At high concentrations of aluminum, passive transport dominated and siderophore production slowed the passive transport of aluminum into the cell. Siderophore production was affected by the aluminum content in the media. High concentrations of aluminum increased production of siderophores in iron-limited cultures, and this production continued into stationary phase. Aluminum did not stimulate siderophore production in iron-replete cultures. The production of siderophores markedly affected aluminum uptake. This has direct implications on the toxicity of heavy metals under iron-deficient conditions.     

INFLUENCE OF IRON LIMITATION AND NITROGEN SOURCE ON GROWTH AND SIDEROPHORE PRODUCTION BY CYANOBACTERIA1

To characterize the mobilization and uptake of iron by cyanobacteria, 14 species were screened for ability to scavenge iron in a competitive system. The cyanobacteria exhibited a range of growth responses to iron limitation which could be separated into three groups, and a representative species from each group was chosen for further study. Effects of iron-limitation on growth and siderophore production of Anacystis nidulans R2, Anabaena variabilis ATCC 29413, and Plectonema boryanum UTEX 581 were determined. Both A. nidulans R2 and A. variabilis showed a reduced rate of growth with decreased available iron concentration (PFe 17–19). Growth rates increased with further reduction in the level of available iron (pFe 20 to pFe 21). The increase in growth rate occurred at the same available iron concentration as the initiation of extracellular siderophore production. In contrast, the growth of P. boryanum decreased with decreasing available iron levels. No siderophore production was detected from P. boryanum cultures. The growth kinetics of siderophore-producing species differ from traditional nutrient-limited growth kinetics and clearly reflect the presence of a high affinity, siderophore-mediated iron transport system in A. nidulans R2 and A. variabilis. Iron-limited growth kinetics more similar to traditional nutrient-limited growth kinetics were found in P. boryanum. The available nitrogen source influenced amount of siderophore produced and concentration of available iron which induced siderophore production. Siderophores were produced at high iron concentrations (pFe 18) when A. variablilis cultures were grown in the absence of combined nitrogen source. When nitrate was supplied to the culture, iron concentrations had to be reduced to pFe 20 before siderophores were produced. Cells grown on nitrogen also produced greater than two times the amount of siderophore compared with nitrate grown cells. This may be indicative of an increased demand for iron by nitrogen fixing A. variabilis Cultures.     

Isolation and characterization of the siderophore N-deoxyschizokinen from Bacillus megaterium ATCC 19213

N-Deoxyschizokinen, a novel siderophore, was isolated from stationary phase cultures of Bacillus megaterium ATCC 19213 and identified as 4-[(3(acetylhydroxyamino)propyl)amino]-2-[2-[(3-(acetylamino)propyl)amino]-2-oxoethyl]-2-hydroxy-4-oxo-butanoic acid. The siderophore was purified by HPLC and its structure determined using ¹H and ¹³C NMR, ¹H-¹H COSY and electrospray mass spectroscopy. The monohydroxamate siderophore has the same carbon skeleton as schizokinen but the hydroxyl group on one hydroxamate is replaced by a hydrogen. A detailed ¹H NMR study of schizokinen, N-deoxyschizokinen and their imides, schizokinen A and N-deoxyschizokinen A is presented.