Response of the cyanobacterium, Oscillatoria tenuis, to low iron environments: the effect on growth...

Arch Microbiol (1992) 157:349-354 Archives of

Hicrobiology © Springer-Verlag 1992

Response of the cyanobacterium, Oscillatoria tenuis, to low iron environments: the effect on growth rate and evidence for siderophore production Christopher M. Brown and Charles G. Trick

Department of Plant Sciences, University of Western Ontario, London, Ontario, N6A 5B7, Canada

Received August 20, 1991/Accepted December 4, 1991

Abstract. Cyanobacteria vary in their ability to grow in media containing low amounts of biologically available iron. Some strains, such as Oscillatoria tenuis, are well adapted to thrive in low-iron environments. We in- vestigated the mechanism of iron scavenging in O. tenuis and found that this cyanobacterium has a siderophore- mediated iron transport system that differs significantly from the traditional hydroxamate-siderophore transport system reported from other cyanobacteria. Unlike other cyanobacteria, this strain produces two types of siderophores, a hydroxamate-type siderophore and a catechol- type siderophore. Production of these two siderophores is expressed at two different iron levels in the medium, suggesting two different iron regulated uptake systems. We compared the production of each siderophore with the growth rate of the culture and found that the production of the catechol siderophore enhances the growth rate of the cyanobacterium, whereas the cells maintain lower than maximal growth rates when only the hydroxamate-type siderophore is being produced.

Key words" Catechol - Cyanobacteria - Hydroxamate - Oscillatoria - Iron - Siderophore

Cyanobacteria, like most microorganisms, have a demon- strated iron requirement for cell processes such as respi- ration, photosynthesis, and pigment biosynthesis (Oquist 1974; Peschek 1979; Sandmann 1985; Rogers 1987). When present in an environment with low amounts of available iron, many microorganisms respond by producing low-molecular-weight, ferric-specific ligands, termed siderophores, to scavenge and transport iron into the cells. Iron-siderophore transport systems have been well established in many microorganisms (Boyer et al. 1987; Winkelmann et al. 1987) and their production has

Offprint requests to: C. G. Trick

Abbreviation. EDDA, ethylene diamine di-(o-hydroxyphenylacetic acid)

been considered as a mechanism by which aquatic microorganisms, such as cyanobacteria, obtain iron from low iron aquatic environments.

Cyanobacteria have been exemplified as the proto- typical siderophore producers from the aquatic environment. Estep et al. (1975) and Murphy et al. (1976) were the first to report siderophore production in the cyanobacteria and suggested that the presence of siderophore- mediated iron-transport might assist the producing strain in dominating other phytoplankton in aquatic ecosystems characterized by low-iron availability. Armstrong and Van Baalen (1979); Kerry et al. (1988) and others (Boyer et al. 1987) contributed evidence that siderophore production and release was not a constitutive process in the cyanobacteria but was regulated by the level of available ferric ion. While the presence of extracellular siderophores has been established in a wide selection of cyanobacteria (Boyer et al. 1987; Kerry et al. 1988), the molecular structure of only one siderophore from a cyanobacterium has been determined (Simpson and Neilands 1976).

In a previous report (Kerry et al. 1988), it was found that the ability of cyanobacterial strains to grow in an iron-EDDA bioassay was highly variable. This variability was attributed to the presence or absence of a high affinity iron scavenging system in the tested strain, most likely, a siderophore-mediated iron transport system. Results of the bioassay indicated that filamentous cyanobacteria, often associated with highly alkaline, low-iron environments (Spirulina sp. and Oscillatoria sp.) (Reynolds 1984), grew at the lowest tested levels of available iron. These strains could grow at lower ferric ion concentrations than the siderophore- producing strains of Syn- echococcus sp. that we subsequently examined. In this paper, the question is posed: do these filamentous cyanobacteria have a mechanism to acquire iron which is distinct from the proposed siderophore-mediated iron- transport system of the unicellular cyanobacteria? In this study the production of siderophores by Oscillatoria tenuis was examined. In contrast to reports concerning unicellular cyanobacteria, O. tenuis produces more than one siderophore and the release of each siderophore is shown to be regulated at different levels of available iron.

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The p roduc t i on of s iderophores is also compared to the growth rate of the cells in culture.

Materials and methods

Strains and media

Two strains were obtained from the Culture Collection of Algae at the University of Texas at Austin. Oscillatoria tenuis (UTEX 428) was rendered axenic by physical manipulation through soft agar (Brown 1991). The other strains, Synechococcus leopoliensis (UTEX 625) (= PCC 6301) and Synechococcus sp. PCC 7492 (Ana- cystis nidulans R2), a gift from Dr N. Straus (University of Toronto) were obtained axenic. All cultures were maintained on BG- 11 agar plates. Cultures were grown at 25 °C under a light flux of 70 gmol photons - m- 2. s - 1.

BG-11 medium (Rippka et al. 1979) was modified by replacing ferric ammonium citrate with FeC13.6 H20, and Tris-HC1 was added to a final concentration of 0.5 g - 1-1 to maintain the pH at 7.4. All water and nutrient stocks used in the preparation of media were treated with Chelex-100 resin to remove residual iron (Price et al. 1989). The concentration of FeC13 • HzO added to the media was altered to produce a range of iron concentrations, from 4.1 x 10 -4 M to 5.4 x 10 -1° M, with each iron concentration differ- ing by approximately one order of magnitude fi'om the next. In all experiments, cells were preconditioned for at least 10 transfers at the desired iron concentration to ensure that cellular storage of iron did not interfere with the experiments.

Measurement of growth

Growth studies were performed using batch cultures of O. tenuis in BG-11 medium. Growth was monitored daily using optical density, measured at 720 nm. The natural logarithm of these measurements was plotted as a function of time, and growth rates were determined from the slopes of the plots.

Cell concentrations were determined using a hemocytometer. Trichomes were passed through a syringe to disrupt filaments into shorter fragments. At least four aliquots of each culture were enumerated and within each aliquot, four 1 mm z areas were examined, until a total area of at least 16 mm z was enumerated or until a standard error of less than 5% of the mean cell number was obtained. Cell numbers were obtained indirectly by dividing filament lengths by a previously established average cell length of 2.5 gm (data not shown). Filament lengths were measured using a calibrated eyepiece.

Detection of siderophores

The presence of a putative high affinity iron transport system was detected using a modified EDDA bioassay system (Kerry et al. 1988). EDDA plates were prepared by adding ethylene diamine di-(o-hydroxyphenylacetic acid) (EDDA) to BG-11 medium containing 0.41 ~tM FeC13 and 1% agar. In the absence of EDDA, 0.41 gM FeC13 would support cyanobacteria growth. All of the iron is chelated with the addition of 10 gg/ml EDDA. Bioassays were carried out by spreading strains out on plates containing increasing amounts of EDDA, from 0 to 300 gg/ml. Plates were incubated at 20 °C under a light flux of 70 gmol photons • m- 2. s- i. Growth was monitored for 10 days. Cells that could adapt to the EDDA concentration grew and retained chlorophyll, while cells unable to adapt became chlorotic and did not grow.

Quantification of siderophores

Siderophore concentrations were quantified in cell-free culture supernatants using selected colorimetric tests. All cell-free supernatants were reduced to 1/5th volume by lyophilization prior to testing. Hydroxamate siderophores were detected using a modifica- tion of the test of Csaky (1948), as described by Gillam et al. (1981), with hydroxylamine as a standard. Catechol-type siderophores were measured using a test developed by Arnow (1937) or by Rioux et al. (1983), with 2,3 dihydroxybenzoic acid (2,3 DHBA) as a standard. The assay of Rioux et al. (1983), unlike the Arnow test, is not selective for catechols that are unsubstituted at either the 3- or 4-positions, since it is based on the reduction, in acidic conditions, of Fe 3 + (in excess over the catechol) to Fe z + by the strongly reducing hydroxyl groups on the catechol.

Isolation of siderophores

Cells were removed from the culture media by first collecting most of the cell mass on glass fiber filters. The supernatants were passed through a 1-gin pore-size membrane filter to remove the remaining cells. Cell-free supernatants were adjusted to pH 3 to release iron from the siderophores and the solutions were passed over a column (2.5 cm x 40 cm) of Amberlite XAD-2 resin. The column was rinsed with distilled water (pH 3) to remove salts and unbound material, and the adsorbed material was eluted from the column with several volumes of 100% methanol. The eluant was concentrated by rotary evaporation to near dryness and stored at - 2 0 °C.

Aqueous siderophore samples were separated on a 1 x 40 cm Bio-Gel P-2 molecular exclusion column. This column had a void volume of 7 ml and a bed volume of approximately 30 ml. The flow rate was adjusted to 0.5 ml/min using a peristaltic pump. Collected subfractions were applied to Whatman 250 gm silica gel thin layer plates and resolved using either 7:3 :: methanol:water or 6:3:1 :: butanol:acetic acid:water as solvents. Siderophores were visualized by spraying plates with a solution of 1% FeC13 in ethanol (Neilands 1984).

Results

Growth on iron-EDDA competition bioassay plates

The growth of three separate strains of cyanobac te r ia on BG-11 plates con ta in ing 4.1 x 10 -7 M FeC13 was moni - tored (Table 1). The a m o u n t of i ron avai lable to the bioassay species was modified by reducing the concent ra - t ion of free ferric ion with the add i t ion of the artificial chelator, EDDA. Synechococcus sp. U T E X 625, selected as a cont ro l s train since we have been unab le to f ind any evidence of a high affinity i ron-scavenging system in this s train (data no t shown), showed a marked reduc t ion of growth a nd survival on the bioassay plates, indicated by the chlorotic status of the inocula ted cells on the add i t ion of m i n i ma l a m o u n t s of E D D A (10 ~g/ml). Synecho coccus sp. PCC 7942 (Anacystis nidulans R2), a k n o w n siderophore p roducer (Kerr et al. 1988), became chlorot ic at 100 ~tg/ml E D D A and failed to thrive at 150 gg/ml. In compar ison , Oscillatoria tenuis was capable of ma in - ta in ing chlorophyl l p roduc t ion and growth on BG-11 plates con ta in ing concen t ra t ions of E D D A of 350 gg/ml, the highest concen t ra t ion examined. F r o m the E D D A bioassay results, O. tenuis has a high-affinity i ron- t rans - por t system that enables it to compete for i ron from low avai lable- i ron envi ronments .

Table 1. Growth of three cyanobacterial strains on BG-11 medium containing 0.41 btM FeC13 and increasing additions of the artificial chelator, EDDA

EDDA Osc i l la tor ia Synechococcus sp. Synechococcus sp. [gg ml- 1] tenuis PCC 7942 UTEX 625

0 + + + + + + 10 + + + + + 50 + + + + 0

100 + + + 0 350 + + 0 0

+ + strong growth, cells remain pigmented + growth evident, cells become chlorotic

0 no growth

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Growth rate of O. tenuis in medium containing different levels of iron

The growth rate of O. tenuis was calculated from daily cell counts for cells grown in BG-11 medium containing iron ranging from 4.2x 10 - s M to 3.1 x 1 0 - 9 M. At the highest levels of added iron (4.2 x 10 -5 and 4.7 x 1 0 - 6 M FeC13) growth rates were identical, at approximately 0.6 day-1 (Fig. 1). The growth rate of O. tenuis declin- ed from 0.57 + 0.3 day-1 to 0.39 + 0.03 day-1 as the iron concentration was decreased from 4.2x 10 -5 to 3.1 x 10-8 M. However, cells grown in medium supplied with lower levels of iron (5.1 x 10-9 M) exhibited growth rates that recovered to 0.8 +_ 0.11 day -1, significantly (P < 0.05) higher than cells grown at the moderate iron concentrations (4.1 x 10 .7 and 3.1 x 10 -8 M). Growth rates of the cells grown in the medium with the lowest amount of added iron (3.1 x 10 .9 M) appeared to be higher than the iron sufficient cultures (4.2 x 10-5 M), al though this difference was not significant (P < 0.05).

Effect of added iron on the production of siderophores

There was no detectable accumulation of either type of siderophore in the extracellular medium of cells grown in chemostats at the highest amount of added iron (Table 2). At added iron concentration of 4.1 x 10- 7 and

1.2

1.0

I 0 . 8

2>-,

" U 0.6

0.4

0.2 i

0.0

_L T

i i i

2 ~ 2 2 2

c4

[FeC%] (M) Fig. 1. The growth rate of Oscillatoria tenuis as a function of the amount of FeC13 added to the medium. All cultures were adapted to the experimentaliron level for at least 10 transfers; (n = 3, ± SE)

Table 2. Concentration of extracellular siderophores from spent medium of Oscillatoria tenuis cultures grown under different iron concentrations

Iron Extracellular Extracellular concentration hydroxamate siderophore catechol siderophore [FeC13] M (pg hydroxylamine cell- 1) (fM 2,3-DHBA equi-

valent cell- 1)

4.1 x 10 .5 ND" ND b 4.7 x 10 -6 ND ND 4.1 x 10 - 7 1.93 ± 0.52 12.3 ± 3.4 3.1 x 10 _8 1.94 _+ 0.11 13.0 +_ 3.9 3.1 x 10 -9 3.2 ± 0.67 35.2 ± 5.0

" Limit of detection is 0.1 pg hydroxylamine equivalents cell- ~ b Limit of detection is 1.0 fM 2,3-DHBA equivalents cell- 1

3.1x 10 .8 M the concentration of hydroxamate- type siderophores in the culture supernatant increased to concentrations in the range of 2 x 10- 6 gg hydroxylamine equivalents per cell. A further reduction in the amount of available iron (3.1 x 10 .9 M FeC13) resulted in an apparent increase in the concentration of measurable siderophore (3.2 +__ 0.67 x 10 .6 gg equivalents hydroxylamine per cell). This increase was not significantly different than the siderophore product ion at the two higher iron concentrations (P >.05). Thus, hydroxamate-type siderophore production, once initiated at 4.1 x 10 .6 M FeC13, remains constant with reduction in the

amount of available iron. The concentration of extracellular catechol-type side-

rophore was undetectable at the highest iron concentrations and detectable, but low, at the intermediate iron concentrations. Cells grown at the lowest level of iron examined (3.1 x 1 0 . 9 M FeC13) showed a three-fold increase in the amount of measurable, extracellular, catechol-type siderophore compared with cells grown at 3.1 x 10 -8 M FeC13. Examinat ion by TLC verified that the extracellular material providing a catechol-positive reaction at the two iron levels were identical, confirming that the difference in levels of extracellular catechol-type siderophore is due to the elevated release of catechol at the lowest iron levels, rather than the production of a different catechol-type siderophore.

Isolation and characterization of siderophores

The variable levels of the ectracellular siderophore- reactive complexes led us to investigate the nature and

352

extent of the extracellular siderophores in relation to the amount of iron added to the medium. The cell-free spent medium of cultures grown in media containing 3.1 x 10 -v M FeC13 was acidified to pH 3 and applied to a XAD-2 column. This resin collects and retains both the hydroxamate- and the catechol-reactive fractions from the spent-cell medium (data not shown). The XAD-2 collected fraction was eluted with methanol and concentrated prior to separation by gel filtration on a P-2 column. The column fractions were analyzed by TLC and by the chemical tests for the two types of siderophores (Fig. 2). By applying chemical tests for hydroxamate-type and catechol-type siderophores to each column fraction, two very prominent iron-binding compounds could be resolved (Table 3). Column fraction b reacted positively to the Csaky test and provided a single iron-binding compound on TLC, with a R s of 0.68 in methanol:water (7:3). Column fractions a and c contained compounds reactive to Arnow and catechol tests and identical TLC profiles, each containing two prominent iron-binding compounds (R s of 0.76 and 0.65 in butanol :propanol :wa- t e r : : 1:1:1). In contrast, the Arnow-reactive complex

1.0

543nrn (Csaky test) • • b d u = 51Onto (Riou× test) ~ : C / ~

.<

._>

Z 10 12 14. 16 18 20 22 24 26 28 30 32 34 .36 38 4.0 4-2 #4" 46 4,8 50

froction

Fig. 2. Fractions collected from the P-2 column molecular partition- ing of the concentrated extracellular organics from the spent, cell-free medium of low-iron grown O. tenuis. Chemical tests for each fraction involved the Csaky test for hydroxamate-type siderophores and the chemical test of Rioux et al. (1983) for catechol-type siderophores

d contained a single iron-reactive compound (R s of 0.76 in butanol :propanol:water :: 1:1:1). While the compound having the higher Ry was found in all P-2 fractions reactive to the catechol test, the compound with the lower R I occurred only in the first two catechol-reactive fractions (a, c). Neither compound was found in fractions that did not show a catechol-positive reaction.

In a similar manner, cell-free exudates were examined at all experimental iron concentrations. Iron sufficient conditions (4.2 x 10 -s and 4.2 x 10 -6 M FeCI3)provided no Csaky- or catechol-reactive subfractions from the P-2 column fractionation and no indication of ferric-binding compounds by TLC observation. Spent media from cells grown at the intermediate iron concentrations (4.1 x 10 - 7 and 3.1 x 10-8 M FeC13) provided profiles similar to the profile from 3.1 x 1 0 - 9 M FeC13, but with the relative reactivity of fraction b in excess of fractions a, c and d.

Discussion

If we base induction of siderophore biosynthesis on the presence of siderophores in the extracellular medium, then we can note that the maximal production of hydroxamate-type siderophore is provided by cells grown at 4.1 x 10- v M FeC13. A further reduction in the amount of added iron does not result in a further stimulation in the level of hydroxamate-type siderophore production. In contrast the production of catechol-type siderophores, indicated by the amount of measurable extracellular siderophore, is low at most iron concentrations, but elevated production levels occur only at the lowest added iron concentrations examined.

There is a hazard in considering the level of siderophore production to be related to the level of siderophore in the extracellular medium. It is highly likely that the cells are producing siderophores at levels of iron greater than that which provides an accumulation of the metabo- lic product. At these iron levels, however, the production of siderophores is more closely linked to the biological iron demands of the cells so that no net accumulation of the siderophore occurs. This may be particularly true for the hydroxamate-type siderophore from Oscillatoria tenuis. Product ion of this siderophore occurs over the

Table 3. Relative mobilities offerric- binding compounds concentrate on XAD-2 resin and partitioned by gel permeation chromatography (P-2)

Fraction Siderophore reactivity Rf Solvent system

A Arnow and 2 compounds Butanol:propanol:water catechol positive Csaky negative (0.76, 0.65) (1 : 1 : 1)

B Csaky positive 1 compound Methanol:water Arnow and catechol negative (0.68) (7 : 3)

C Arnow and 2 compounds Butanol:propanol:water catechol positive Csaky Negative (0.76, 0.65) (1 : 1 : 1)

D Arnow and 1 compound Butanol:propanol:water catechol positive Csaky negative (0.76) (1 : 1 : 1)

353

region of iron levels that is controlling the growth of the cells. Thus the hydroxamate-type siderophore production is more "constitutive" than is the catechol-type siderophore. The growth rate of cells is enhanced to levels equal to the iron-satiated control when there is an accumulation in the amount of siderophore in the extracellular medium.

Characterization of the crude samples and comparison of concentrated fractions by TLC confirms that O. tenuis produces multiple siderophore. At the intermediate iron concentrations, the predominant extracellular siderophore is a hydroxamate-type siderophore. As the added iron concentrations are decreased, hydroxamate-production is maintained, while a second set of siderophores is released. Two different catechol-reactive ironbinding compounds are expressed, along with the hydroxamate- type siderophore.

Based on the EDDA bioassay O. tenuis clearly can grow at lower iron concentrations than other cyanobacteria (Kerry et al. 1988). In this paper we have verified that this strain produces and releases extracellular siderophores in response to low iron environments, indicative of a high affinity iron transport system. However, since Synechococcus sp. PCC 7942 has been shown to produce a siderophore (Kerry et al. 1988), the presence of the iron-siderophore transport system alone, does not dis- tinguish O. tenuis from the other cyanobacteria that are less successful in the bioassay. The iron scavenging system of O. tenuis differs from the other cyanobacteria in two predominant ways. Firstly, O. tenuis produces multiple siderophores. Secondly, while most cyanobacteria examined produce hydroxamate-type of siderophore, one of the siderophores produced by O. tenuis is a catechol- type siderophore.

It is clear that O. tenuis produces more than one siderophore, each regulated at a different level of iron in the medium. While this is the first report of multiple siderophore production in a cyanobacterium, other microorganisms have been shown to produce more than one siderophore. For example, two hydroxamate-type siderophores, ferrichrome and ferrichrome A, are produced by the smut fungus Ustilago sphaerogena under iron-deficient growth conditions (Ecker et al. 1982). Ecker et al. (1982) suggest that a second hydroxamate siderophore (ferrichrome A), a more powerful chelator, is excreted only when the first siderophore (ferrichrome) fails to provide enough iron to the cell. This stepwise mechanism of siderophore production has also been observed in cultures of F. roseurn (Eckert et al. 1982), Azotobacter vinelandii (Page and Huyer 1984), and Escherichia coli (Bagg and Neilands 1987). The rationali- zation of the production of more than one siderophore by any microorganism is that the cells encounter more than a single type of habitat. A parallel explanation may be applied to O. tenuis. Cells grown at moderate iron concentrations produce only the water-soluble hydroxamate-type siderophore. Cells grown at the lowest iron concentrations, while producing not only the hydroxamate-type but also the catechol-type siderophore, undergo various physiological changes (Brown 1991), including the icreased gas vacuolization of the trichomes (Douglas et al. 1986). This results in the accumulation of trichomes

at the surface and the formation of trichome aggreates. Since diffusion of iron into the aggregates would be restricted, an alteration in the type of siderophore may be required.

The fact that O. tenuis also produces a catechol-type siderophore at the lowest iron concentrations may also validate the need for the two siderophore system. Cate- chol siderophores have a high affinity for iron and form very strong ferric complexes that are stable in alkaline environments. In contrast, these compounds are not very water soluble and would participate in iron exchange at the cell surface layer. It is anticipated that the role of the catechol siderophore is to scavenge iron from the solubilization of ferric hydroxides associated with the trichome surface or restricted to the intratrichome spaces. This physical model may help explain why filamentous algae such as Oscillatoria sp. and Spirulina sp. are the most successful in the EDDA bioassay.

Experiments are under way to discern and establish the chemical structure of these three iron-regulated, iron-binding compounds and to establish the physiological mechanism that enables the cells, when producing all three iron-binding compounds, to reestablish the cell growth at maximal values.

Acknowledgement. The authors wish to thank the Natural Sciences and Engineering Research Council of Canada for research support.

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