Microcystin-LR binds iron and iron promotes self-assembly

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Microcystin-LR binds iron and iron promotes self-assembly Laura Ceballos-Laita, Carlos Marcuello, Anabel Lostao, Laura Calvo-Begueria, Adrián Velázquez-Campoy, María Teresa Bes, María Francisca Fillat, and María Luisa Peleato Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05939 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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Environmental Science & Technology

Microcystin-LR

No metal

Fe3+

3

1

4 6

2 Monomers and dimers

oligomers

ACS Paragon Plus Environment


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Microcystin-LR binds iron and iron promotes self-assembly

2 3

Laura Ceballos-Laita1,3#, Carlos Marcuello2#, Anabel Lostao2,4, Laura Calvo-

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Begueria1,3, Adrián Velazquez-Campoy1,3,4, María Teresa Bes1,3, María F. Fillat1,3 and

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María-Luisa Peleato1,3

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1

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Microscopías Avanzadas, Instituto de Nanociencia de Aragón. Universidad de

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Zaragoza. (Spain). 3Institute for Biocomputation and Physics of Complex Systems

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(BIFI)-Joint Unit BIFI-IQFR (CSIC). Universidad de Zaragoza (Spain).4 Fundación

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*

Biochemistry Department, Universidad de Zaragoza, (Spain).

2

Laboratorio de

ARAID, Aragón, Spain.

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*Corresponding author:

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María Luisa Peleato

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Departamento de Bioquímica

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Facultad de Ciencias

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Pedro Cerbuna 12

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50009-Zaragoza, Spain

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Tf:+34976762479

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[email protected]

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#Both authors contributed equally to this work

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Abstract

24 25

The microcystin producing Microcystis aeruginosa PCC 7806 and its close strain, the

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non-producing Microcystis aeruginosa PCC 7005 grow similarly in the presence of 17

27

µM iron. Under severe iron deficient conditions (0.05 µM), the toxigenic strain grows

28

slightly less than in iron-replete conditions, while the non-producing microcystin strain

29

is not able to grow. Isothermal titration calorimetry performed at cyanobacterial cytosol

30

or meaningful environmental pHs values shows a microcystin-LR Kd for Fe2+ and Fe3+

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of 2.4 µM. Using Atomic Force Microscopy, 40% of microcystin-LR dimers were

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observed, and the presence of iron promoted its oligomerization up to 6 units.

33

Microcystin-LR binds also Mo6+, Cu2+ and Mn2+. Polymeric microcystin binding iron

34

may be related with a toxic cell colony advantage, providing enhanced iron

35

bioavailability and perhaps affecting the structure of the gelatinous sheath. Inside cells,

36

with microcystin implicated in the fitness of the photosynthetic machinery under stress

37

conditions, the toxin would be involved in avoiding metal-dependent Fenton reactions

38

when photooxidation causes disassembly of the iron-rich photosystems. Additionally, it

39

could be hypothesized that polymerization/depolymerization dynamics may be an

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additional signal that could trigger changes, for example in the binding of microcystin

41

to proteins.

42 43

Key words: Iron; Microcystin; Microcystis;

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1. Introduction

47 48

Microcystins are potent cyanotoxins that inhibit eukaryotic protein phosphatases 1 and

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2A, and produce adverse effects in water quality and potential human and animal

50

poisoning1. The toxic strains producing microcystins deviate a considerable amount of

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carbon and nitrogen to the synthesis of such secondary metabolites. For this reason it

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seems that microcystins must confer adaptative advantages to producer species.

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However, a coherent global model of its role in the cyanobacteria is not yet well

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established. Proteomic studies pointed to a different expression profile in toxic and non-

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toxic cells, affecting many proteins involved in photosynthesis, nitrogen and carbon

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metabolism, and redox control1,2. Microcystin was usually considered an endotoxin.

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Supplying radioactive inorganic carbon to M. aeruginosa cultures and following the fate

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of the intracellular radioactive microcystin pool, no export from the intracellular

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microcystin pool was observed3. On the other hand, the mcyH gene encoding an ABC

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transporter is essential for microcystin synthesis4, suggesting microcystin exportation

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out of the cell. In congruence with the toxin exportation, solid evidences linking

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microcystin presence and enhanced colony formation and size have been reported5, 6, 7.

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Cell-wall proteins could be implicated in the effect microcystin has on colony

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formation8,9, likely involving the microcystin-related protein C (MrpC)9. MrpC

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accumulates in microcystin-deficient cells, suggesting a role of microcystin in colony

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arrangement. Microcystin also causes upregulation of genes involved in polysaccharide

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synthesis7, involved in colony formation.

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Microcystin has been proposed to have allelopathic effects against other members of the

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ecosystem, but few data indicate harmful effects of microcystins at concentrations that

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are typical for the environment10. As a defence mechanism to microfauna, phylogenetic

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analysis suggested that the microcystin synthetase predated the metazoan lineage, thus

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dismissing the possibility that microcystins emerged as a means of defence against

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grazing11. Also, it has been suggested that microcystin may be involved in quorum

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sensing or act as a signalling molecule12. However, several studies have failed to find

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evidence for such a role13, 4.

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Regarding a possible physiological role inside the cell, even though microcystins have

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been involved in causing oxidative stress as an additional toxicity mechanism, an


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interesting piece of work proposes that the toxin increases the fitness of the toxic

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Microcystis strain under oxidative stress conditions14. Under stresses such as high light

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or carbon deficiency, microcystin synthesis becomes advantageous15,16. Improvement of

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competition for light has been also considered as a potential advantage provided by

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microcystin, but competition experiments with toxic and non-toxic Microcystis strains

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showed that non-toxic strains were better competitors for light than toxic strains17.

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Moreover, electron microscopy of immuno-gold labelled Microcystis cells indicates that

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two thirds of the intracellular microcystin is located around the thylakoids18, 19, 20. A role

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in the maintenance of the photosynthetic machinery, protecting against photoinhibition

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has been proposed21, in relation to oxidative stress protection14.

88 89

Microcystins have been described to bind to many proteins, sometimes in a bulk

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manner14,22,23. It has been proposed that there is a covalent interaction of the toxin with

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cysteine residues of proteins14, and in this way, an interesting proposal is that

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microcystin competes for binding sites in thioredoxin-regulated proteins during

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oxidative stress (low Fe)24. Lately, eukaryotic proteasome has been described as a target

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for microcystin, affecting its trypsin-like activity, and delaying protein degradation25.

95 96

The microcystin synthesis has been linked to iron metabolism for many years. Lyck and

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colleagues26 showed that during iron depletion, toxic strains of Microcystis maintained

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cell vitality much longer than the non-toxic strains. Moreover, Utkilen and Gjolme27

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found that toxic strains exhibited higher rates of iron uptake than non-toxic strains.

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They proposed that the microcystin is an intracellular chelator of Fe2+, as well as

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predicted that the synthesis of the toxin would be controlled by the amount of free iron

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present in the cells. Structural similarities between microcystin and bacterial

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siderophores28 lead also to propose a putative role as an extracellular iron-scavenging

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molecule.

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Another relevant aspect linking iron and microcystin is the regulation of the microcystin

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gene cluster. The mcy operon, responsible for microcystin synthesis seems to be

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regulated by Fur (ferric uptake regulator)29, an iron-dependent transcriptional regulator

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of a wide number of genes involved in iron metabolism and oxidative stress30. As a

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consequence, iron deficiency leads to an increased amount of microcystin27,31. Since a

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long time ago, several metal-microcystin complexes have been described32,33, and Klein

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and co-workers34 provided evidence for the formation of at least two Fe3+-microcystin-


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LR complexes, at acidic pHs.

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Previous results 32-34 formed the basis for examining microcystin-metal binding by ITC.

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As an extension, the potential influence on colony formation of microcystin-LR in the

115

presence of iron and other metals through a hypothesised oligomerization of

116

microcystin-metal complexes was examined..

117 118

2. Experimental

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2.1. Strains and culture conditions.

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This study was carried out with M. aeruginosa PCC 7806 and PCC 7005 provided by

121

the Pasteur Culture Collection. Cells were photoautotrophically grown in BG11

122

medium supplemented with 2 mM NaNO3 and 10 mM NaHCO3, as described

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previously by Rippka35, and as recommended by the Pasteur Institute. The culture

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medium was air bubbled at 25oC with continuous illumination (25 µmol of

125

photon/m2.s). Light was measured using a Quantum Sensor photometer (Skye

126

Instruments, SKP 200). Iron-deficient (0.5 µM iron) and iron-replete cultures were

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started from an equal aliquot from an iron-replete culture in late exponential phase.

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After washing two times with iron-deficient media or iron-replete medium respectively,

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cells were inoculated and grown in batch conditions (1 L Roux flasks with initial

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Abs700nm= 0.15). This experiment was performed 3 times, and measurements were done

131

in duplicated samples.

132 133

2.2. Cell counting

134

The cell density was determined daily both spectrophotometrically and by counting

135

cells with a Neubauer cell. To avoid cell aggregation, the samples were treated with

136

KOH following described methodology36.

137 138

2.3. Analytical methods

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Samples of 1 mL and 5 mL were collected for chlorophyll a and protein determination,

140

respectively. Chlorophyll a was quantified according to Mackinney37 and protein

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contents were estimated by the bicinchoninic acid method (BCATM Protein Assay


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Reagent Kit from Pierce). Microcystin-LR was quantified using MicroCystest®

143

(Zeulab). HPLC procedure is indicated in Supporting Information section.

144 145

2.4. Isothermal titration calorimetry (ITC)

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Metal binding to microcystin-LR was assessed in a high-sensitivity isothermal titration

147

calorimeter Auto-iTC200 (MicroCal, GE Healthcare). Assays were performed with a

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final concentration of 150 µM metal and 10 µM microcystin-LR. For Fe3+ binding

149

assays, a stock solution of Fe-NTA (nitrilotriacetic acid) was prepared according to

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previously described procedure38. The solution was made anoxic by purging with

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nitrogen and stored in a sealed vial until needed39. Experiments were carried out at 25°C

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in 150 mM sodium phosphate (pH 7), 100 mM Hepes (pH 7.6) or 50 mM Tris-HCl (pH

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8, 8.5) buffers, as indicated in each experiment. Assays with Fe2+ were performed with

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fresh made solutions in the presence of 1.5 mM ascorbate for maintaining reducing

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conditions. Microcystin-LR solution (10 µM) in the calorimetric cell was titrated with

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metal solution (150 µM). Control experiments (either dilution of microcystin-LR or

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dilution of metal ions, Fe2+, Fe3+, Mo6+, Mn2+, Cu2+, Ca2+. Zn2+, Co2+, Ni2+) were

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performed under the same experimental conditions. The heat evolved after each ligand

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injection was obtained from the integrated calorimetric signal. The heat due to the

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binding reaction was obtained as the difference between the reaction heat and the

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corresponding heat of dilution, the latter estimated as a constant heat throughout the

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experiment, and included as an adjustable parameter in the analysis. The dissociation

163

constant (Kd) and the interaction enthalpy (∆H) were obtained through non-linear

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regression of experimental data to a model considering a single ligand-binding site. A

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brief description of the procedure has been included in Supporting Information. Data

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were analyzed using software developed in our laboratory implemented in Origin 7

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(OriginLab).

168 169

2.5. Cross-Linking of Microcystin-LR

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1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is a zero-length crosslinker

171

agent widely used to reveal protein-protein specific interactions. It is generally used as a

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carboxyl activating agent for the coupling of primary amines to yield amide bonds.

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Microcystin-LR cross-linking reactions (30 µL 1 mg/mL) were performed in the


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presence of 2.7 mM FeCl2, or FeCl3. The mixture was incubated with 10 mM 1-Ethyl-3-

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(3-dimethylaminopropyl)carbodiimide, EDC, (freshly dissolved in H2O) and 10 mM

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Sulfo-NHS (N-hydroxysulfosuccinimide) in 50 mM acetic-acetate buffer pH 5.5, for 30

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min at 25 ºC. Adding 20 mM 2-mercaptoethanol stopped the reaction. Other metals

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tested (Mn2+, Zn2+, Cu2+) were used at the same concentration as iron.

179 180

2.6. Atomic force microscopy imaging

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Sample-nanoflat surface immobilization is a vital step to carry out atomic force

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microscopy (AFM) measurements without dragging biomolecules through the tip

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movement on the sample. 1 cm2 cleaved muscovite mica pieces (Electron Microscopy

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Sciences) were incubated with 1 µg/mL of microcystin-LR samples, for 10 min at room

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temperature. The surfaces were washed three times using the solvent to remove not well

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attached molecules, introduced in a liquid cell and covered with the same washing

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solution ready for measurements. Microcystin-LR molecules in the different

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experimental conditions were measured in different solvents, 20 % methanol (v/v),

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milli-Q water and PBS, pH 7.4. The immobilization of biomolecules on mica was

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previously evaluated observing clearly they preserve their functionality40.

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AFM measurements were performed with a Cervantes Fullmode SPM (Nanotec

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Electrónica S.L, Spain). AFM images were taken using the force based Jumping Mode

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under very small applied forces barely touching the molecules in a non-intrusive

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manner41. V-shaped silicon nitride cantilevers with spring constants from 0.01 to 0.03

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N/m (Bruker Probes, MSNL lever Probes) calibrated using the thermal noise method

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were used. Image processing was performed with the WSxM software42. Percentages

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corresponding for the different oligomeric features were calculated taking several

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sample areas and zooming with the WSxM functions to identify the monomeric units in

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the features without losing data as reported43. The quantification was carried out by

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counting a minimum of 250 oligomers/species from at least 10 different locations of

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each sample. At the same time, each type of sample was measured –at least- by

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triplicate to ensure reproducibility. Each associate was analyzed in detail using the

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zoom and the profile functions of the WSxM program.

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The error was calculated from the results dispersion analysis of different AFM images

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corresponding to several areas of the sample.

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3. Results

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3.1. A toxic Microcystis endures iron deficiency more effectively than a non-toxic strain

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Toxic and non-toxic M. aeruginosa (PCC7806 and PCC7005 respectively) were

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transferred from an iron-replete medium in middle exponential phase (samples were

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washed twice) to an iron-deficient or iron-replete BG11 medium. The consequences of

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iron depletion in toxic (PCC 7806) and non-toxic (PCC 7005) M. aeruginosa strains

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were much more evident in the non-toxic strain. Figure 1 shows that growth of cells is

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quite different (number of cells is shown in Fig 1A, total protein in cells is shown in

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Figure 1B, and chlorophyll in Figure 1C). The toxic strain was virtually unaffected by

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the low iron availability in the culture medium while the growth of the non-toxic cells is

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almost zero when iron is limited (Fig.1A and 1B). Microcystin-containing cells were

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only slightly affected in chlorophyll content, while the non-toxic strain exhibited a very

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low level of the pigment (Figure 1C) under the same conditions, due to the fact that iron

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deficiency impairs chlorophyll biosynthesis, causing bleaching. Our results indicated

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that the toxic strain, M. aeruginosa PCC7806, has some kind of advantage either

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concerning intracellular iron storage or a more efficient iron uptake. Under iron-replete

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conditions, the non-toxic strain, PCC7005 performed slightly better (Fig.1A and 1B).

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3.2. Microcystin-LR binds iron and other metals

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The ability of the microcystin-producing strain to overcome iron deficiency led us to

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investigate the binding activity microcystin-LR-iron. We were especially interested in

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measurements at physiological and environmental meaningful pHs, for instance pH of

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cytosol or pH of alkaline environments, with low iron availability. A different

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experimental approach than the previous described in the literature32-34,44 was chosen:

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isothermal titration calorimetry (ITC). In order to investigate if microcystin-LR plays a

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role by interacting with metals, assays were performed with several metal ions, Fe2+ and

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Fe3+ in particular, and binding dissociation constants were measured. According to these

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assays, microcystin-LR binds Fe2+, Fe3+, Mo6+ (Figure 2), Cu2+ and Mn2+ (Table 1); on

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the other hand, in our experimental conditions microcystin-LR does not specifically

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interact with Ca2+, Zn2+, Co2+ and Ni2+ (no interaction was observed in our calorimetric


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experiments; however, although we could observe a weak interaction of microcystin-LR

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with Zn2+, it was too weak to report it). Titrations were performed at different pHs (7.0,

241

7.6, 8.0 and 8.5), and the higher affinity for iron was found at pH 7. Microcystin-LR

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exhibits a similar Kd, 2.4 µM, when binding Fe3+ and Fe2+; however, a more negative

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enthalpy in the case of Fe2+ would suggest a more specific interaction with the reduced

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metal form. Other metals interacting with microcystin-LR exhibited dissociation

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constants in the low-to-moderate micromolar range (Table 1). The interaction of other

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metals at different pHs (e.g., Cu, Mn, Mo) has been measured. In the case of Cu and Mn

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it is observed that the interaction affinity decreases with the pH, while for Mo it is

248

observed that the interaction affinity increases with pH. A complete set of all the data of

249

the interactions observed by calorimetry is shown in Supporting Information (Figure

250

1S).

251 252

3.3. Microcystin-LR oligomerizes, being Fe3+ the main promoter of chain formation

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After confirming that microcystin-LR interacts with iron and other metals, it was

254

considered interesting to study the complex, and HPLC of the samples were performed.

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In order to study changes in microcystin-LR in the presence of iron, studies of

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chromatographic mobility of the different samples were carried out. Figure 2S in

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Supporting Information section shows HPLC reverse phase chromatograms of cross-

258

linked pure microcystin-LR in the presence and absence of Fe3+, after subtraction of the

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EDC-NHS baseline. A single peak was obtained when microcystin-LR and EDC-NHS

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cross-linked microcystin-LR were run, whereas several peaks and a shoulder in the

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main peak were observed when microcystin-LR was EDC-NHS treated in the presence

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of Fe3+. These results suggest that different species of microcystin-LR (with different

263

hydrophobicity) were generated in the presence of iron.

264 265

3.4. Oligomerization patterns analyzed by AFM imaging

266

The different molecular species of microcystin-LR observed by HPLC was studied

267

using an experimental approach that allows visualizing single molecules: Atomic Force

268

Microscopy Imaging (AFM). This technique would confirm unequivocally the presence

269

of microcystin-LR oligomers, and ascertain the influence of metals on microcystin-LR

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oligomerization. The effect of incubating microcystin-LR with different metal cations in

271

20 % methanol was investigated and the results of the cross-linked samples are


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summarized in Table 2. A clear set of images were obtained for all the measurements,

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and despite the small size of the microcystin-LR molecules, the species were

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distinguishable at the single molecule level. Control experiments were also performed

275

in milli-Q water and PBS buffer, pH 7.4 (Table 2). The results indicated that

276

microcystin-LR appears mainly as a monomer, and only a maximum of 10 % of the

277

molecules are dimerized independently of the three liquid media used (Figure 3, image

278

A).

279

The carbodiimide used (EDC) is a zero-length cross-linker; it causes direct conjugation

280

of closely positioned carboxylates (–COOH) to primary amines (–NH2) without

281

becoming part of the final cross-link (amide bond) between such interacting target

282

molecules. EDC showed the dimerization of a portion of microcystin-LR molecules,

283

with an average of about 40 % (Figure 3, image D). The height found for microcystin-

284

LR molecules in the samples with EDC is a bit higher due to the bound cross-linker, 2.0

285

nm (Figure 3, images D, E, F) with respect to 1.0 nm on average for the untagged

286

molecules (Figure 3, images A, B, C). This result can be corroborated using the Z-

287

height profile function on each feature (Figure 4, images B, C, E, F). Oligomerization

288

develops in the same plane through the building of chains, and no other type of

289

molecular architecture was detected. The zoom function made possible to unequivocally

290

attribute the association pattern of each oligomeric chain. It is important to remark that

291

the measurement in the Z-height (height profiles) presents a high accuracy, with

292

uncertainty lower than 1 nm, whereas the XY size of the feature, the width, is less

293

accurate due to the well documented broadening effect related to the AFM tip dilation45.

294

Despite this broadening effect, XY width can be compared in relative terms and it can

295

be observed that dimers have approx. twice the width of the monomer features (Figure

296

3, images B, E).

297

The presence of Fe2+ ions during the cross-linking process induces the addition of a

298

third molecule to the dimers to form trimers up to 21 % referred to total peptide (Figure

299

4, images A, B, C). The maximum degree in the oligomerization process was achieved

300

when Fe3+ was added. This agent induces the formation of chains with six microcystin-

301

LR units (Figure 4, images G, H, I). Visualization of AFM topography images shows

302

that a considerable 28% of the analyzed features were hexameric chains. This result

303

corresponds to the fact that a 62 % of the molecules are building these chains, whereas

304

no trimeric or tetrameric arrangements were found in that sample.


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Other divalent metals such as Mn2+ or Zn2+ used in the cross-linking procedure resulted

306

in no polymerization of microcystin-LR and even inhibition of dimerization. Strands of

307

microcystin-LR with a maximum of four molecules appeared when Cu2+ was present

308

during the cross-linking (Figure 4, images D, E, F). The percentage of these longer

309

chains reaches 10 % considering features and rises up 26 % if the data is translated to

310

total peptide.

311 312 313

4. Discussion

314

The selective pressure of iron limitation has led to sophisticated systems to overcome

315

the deprivation of such essential element46 endowing cyanobacteria to compete very

316

favourably for iron or adapt to its deficiency. As part of these adaptive mechanisms,

317

microcystin was proposed long time ago to provide a selective advantage to toxin-

318

producer cells under iron deficient conditions27. Over time, many other functions for

319

microcystins have been proposed, some of them clearly demonstrated, such as its role in

320

colony formation5-7 or in protection against oxidative stress14,47 and protection to

321

photoxidation16,17,49. Besides, many data in the literature pointed also to a function

322

related with iron metabolism26,27,33-34,44. In this work, we show that using iron-limited

323

BG11 medium, the toxic strain has overcome iron deficiency without serious influence

324

on its growth, while the non-toxic strain is affected with severely reduced growth

325

(Figure 1A and 1B). Fujii et al.48 described that the siderophore-independent iron uptake

326

rates were similar in the toxic strain PCC 7806 and in a genetically modified strain

327

unable to produce microcystin, but the growth rates measured for the microcystin

328

deficient strain were lower than those of the wild-type strain, even under conditions

329

where similar iron uptake rates were expected48. In addition, previous data pointed that

330

the microcystin-producing strain of M. aeruginosa (CYA 228/1) acquires iron about 2

331

fold faster than the non-toxic strain CYA 4327. Recent reports pointed to the fact that

332

microcystin-producing strains have been shown to outcompete their nontoxic

333

counterparts under iron-limiting conditions47, in good agreement with the data presented

334

in this paper. The ecophysiological advantage of microcystin production under iron-

335

limiting conditions could be justified if microcystin can act as iron-chelator or iron

336

storage molecule. Our data indicate that microcystin-LR binds iron and other cations at

337

physiological or environmentally meaningful pHs. According to calorimetric titrations


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338

microcystin-LR specifically interacts with Fe2+, Fe3+, Mo6+, Cu2+ and Mn2+. The

339

stoichiometry of the interaction is 1:1 and the dissociation constants are in the low-to-

340

moderate micromolar range. Interestingly, Fe2+ and Fe3+ display similar dissociation

341

constants (Kd 2.4 µM); however, a more negative enthalpy in the case of Fe2+ is

342

observed. Apparently microcystin-LR does not specifically interact with Ca2+, Zn2+,

343

Co2+ and Ni2+, although unspecific interactions cannot be ruled out since we have

344

observed a weak interaction with Zn2+. Klein et al.34 found electrochemical evidence for

345

the complexation of Fe3+ by microcystin-LR at acidic pHs, but not evidence for Fe2+.

346

However, Saito et al.33 using cryospray ionization Fourier transform ion cyclotron

347

resonance mass spectroscopy (CSI-FTIC-MS) identified a microcystin-Fe2+ complex,

348

but not complex formation of microcystin with Fe3+. On the other hand, Zn2+, Cu2+ and

349

Mg2+ were observed previously to form complexes with microcystin-LR32,33.

350

We have classified the interaction of microcystin with the metal ions assessed in our

351

work as having low-to-moderate binding affinity, in comparison with the standard

352

affinity ranking in biomolecular interactions: low affinity (Kd above 100 µM), moderate

353

affinity (Kd from 10 nM to 100 µM), and high affinity (Kd below 10 nM). Of course,

354

regarding iron chelation, siderophores usually exhibit extremely high affinities (Kd in

355

the range from 10-50 M to 10-30 M)50,51. However, in some cases the binding affinity of

356

the siderophore for iron is not as high as that in the more common siderophores, with

357

reported Kd is in the range from 5 to 300 µM52. Our results are in agreement with some

358

published results, but in disagreement with other ones. Humble et al.32 reported

359

dissociation constants of 10 µM approximately for Cu2+ and Zn2+ binding to

360

microcystin-LR at pH 7.5, 100 mM KCl, with no significant differences with

361

microcystin-LW and microcystin-LF. They concluded that microcystin-LR is a

362

medium-strength ligand for these metal ions. This is a statement equivalent to our

363

classification of microcystin-LR as having low-to-moderate binding affinity towards

364

metal ions. On the other hand, Yan et al.44 measured much smaller dissociation

365

constants for the binding of the same metal ions to microcystin-LR at pH 7 (10 nM for

366

Cu2+ and 0.01 nM for Zn2+). Klein et al.34 reported the formation of a complex between

367

microcystin-LR and Fe3+ following a two-step pathway, with a labile, rapidly forming

368

initial complex followed by a more stable, slowly forming complex with dissociation

369

constant of 0.1 pM in 60% v/v methanol/water, but no complex was observed between

370

microcystin-LR and Fe2+. Importantly, this dissociation constant corresponds to the


12


Page 14 of 32

371

formation of the more stable complex, while a much larger dissociation constant would

372

correspond to the first, labile complex representing the direct encounter between

373

microcystin-LR and iron, which must be that one observed by ITC.

374

Using AFM we achieved the direct visualization of the molecular arrangements of the

375

samples using “zero-length” cross-linkers, such as EDC. The cross-linker helps to

376

mimic the conditions for the oligomerization in vivo53. The first observation was that the

377

nature of the solvent did not affect the association pathway of microcystin-LR.

378

Regarding the metals, Mn2+ and Zn2+ not only did not promote association but also even

379

seemed to behave as inhibitors of dimerization. Fe2+ ions were able to bind a monomer

380

to a dimer composing a certain portion of trimers, so it could play a role as inductor in

381

the microcystin-LR strand formation. On the other hand, Cu2+ ions seem to promote the

382

formation of tetrameric chains binding dimers among themselves. Other metals as Mn2+

383

binds microcystin-LR but did not promote oligomerization, while Cu2+ induced a

384

considerable amount of trimers and tetramers. Finally, we can see that Fe3+ is a potent

385

inducer of microcystin-LR oligomerization. Ferric ions make a majority of the

386

molecules to form hexameric chains (more than 60%). Figure 3S in Supporting

387

Information summarizes the observed oligomerization in the presence of Fe2+ and Fe3+.

388

These chains show an irregular conformation, and do not appear as rigid nanotubes (Fig.

389

4 images G, H). This effect is favoured by the degree of freedom of the chains provided

390

by the electrostatic adsorption on the mica surface. Microcystin hexamers are not rigid,

391

and certain angles between monomers are observed (Figure 4), maybe related with the

392

flexibility of the arginine side-chain in microcystin-LR54, if arginine is involved in the

393

bond.

394

A possible consequence of the ability of microcystin-LR to form complexes with iron

395

can be related to the toxic cells colony advantage. In microcystin containing colonies,

396

microcystin can act as siderophore, with increased colony size and extracellular

397

polysaccharides7, providing enhanced iron availability versus the environment. Iron-

398

promoted oligomerization has been observed in vitro, but the data leads to speculation

399

regarding a possible role of microcystin-LR chains in the structure of the colony

400

gelatinous sheath. Gan et al.7, found that depletion of extracellular microcystin

401

concentrations caused small colonies. However, the authors indicated that induction of

402

extracellular polysaccharides by microcystin exposure is the major mechanism

403

explaining the increased colony size in presence of the toxin. On the other hand, Amano


13

Page 15 of 32


404

et al.55 described that added extracellular polysaccharides to M. aeruginosa (UTEX LB

405

2061) did not promote colony formation or aggregation of cells. When the

406

polysaccharides where added with iron 6.8 mg/L, colonies were formed. Unfortunately,

407

the strain used in such experiments does not produce microcystin but the authors infer

408

that the extracellular polysaccharides themselves would not act as binders for M.

409

aeruginosa cells and only when high amount of iron is present colony formation

410

occurs56.

411

Microcystin was inmunolocated around the thylakoids18-20. Many hypotheses may arise,

412

for instance microcystin would prevent metal-dependent Fenton reactions when

413

photooxidation causes disassembly of photosystems and iron-containing photosynthetic

414

components release iron. In the other hand, low iron or high light, as well as other

415

environmental stresses, causes oxidative stress in cyanobacteria46, and the ROS-induced

416

microcystin binding to proteins14 may be related with iron-induced changes in the

417

oligomerization status of the peptide.

418

The role of a polymeric microcystin binding iron in an intracellular scenario may be

419

related

420

conditions16,17,48,56,57. Beyond the consideration of the long-standing idea of microcystin

421

acting as an intracellular siderophore or chelator for storing iron27, it seems clear that

422

microcystin may have a relevant role in the adaptation of photosynthesis to

423

environmental conditions. Several studies pointed to an active role of microcystins in

424

preventing light photoxidation48,56,57, and providing ecophysiological fitness under

425

environmental stresses. Moreover, the localization of exogenous microcystin-LR taken

426

by a non-producing cyanobacterium, Synechocystis PCC6803, located the toxin in the

427

thylakoid membranes, causing a decrease in photosystem II activity58. Phycobiliproteins

428

may be the major proteins that have binding sites for these oligopeptides59, and

429

curiously, a molar ratio near to 6 was observed for microcystins to the phycobilin (αβ)

430

monomer59.

431

Either low iron or high light, as well as other environmental stresses, causes in

432

cyanobacteria oxidative stress46, and the ROS-induced microcystin binding to proteins14

433

may be related with iron-induced changes in the oligomerization status of the peptide.

434

When photooxidation causes disassembly of the iron-rich photosystems, the

435

oligomerization may occur, avoiding metal-dependent Fenton reactions. Also, the large

with

the

fitness

of

the

photosynthetic

machinery


under

stress

14


Page 16 of 32

436

subunit of RubisCO showed a lower susceptibility to proteases in the presence of

437

microcystin14, maybe keeping up the machinery under stress conditions. Metal-driven

438

oligomerization of microcystin may represent an additional signalling: as iron is

439

released from disassembly of photosynthetic machinery, the equilibrium is shifted

440

towards the oligomerization state, which is achieved through metal binding.

441

Finally, our work would inspire future experiments for elucidating the possible role of

442

the microcystin as an ecophysiological molecule, either as iron concentrator agent or as

443

involved in reversible inhibition of the photosystem II. Microcystin present in the

444

surface of the cells and gelatinous sheaths surrounding colonies may function as a

445

capturing iron agent from the environment, able to concentrate the scarce metal around

446

the colony and playing a role in the structure of the sheath. Microcystin, iron and

447

extracellular polysaccharides should be related with MrpC9, necessary to piece the

448

microcystin-role puzzle. Inside the cells, microcystin would protect photosystem II and

449

photosynthetic machinery against photoxidation, triggered by iron release and

450

subsequent oligomerization. However, to better understand the implications of this

451

study, in the near future is necessary to study the ability of other microcystin variants as

452

well as nodularin to oligomerize in the presence of iron or other metals.

453 454 455

Acknowledgements

456

This work was funded by the Spanish Ministerio de Educación y Ciencia (BFU2006-

457

03454 and BFU2009-07424). L.C.B. was partially supported by a fellowship from the

458

BIFI (Zaragoza, Spain). A.L. and A.V-C thank ARAID for financial support. C.M. is

459

indebted to Gobierno de Aragón for receiving a PhD fellowship. We thank to Laura

460

Vela, Irene Gregorio and María del Mar Pascual for performing initial experiments on

461

microcystin cross-linking; we also thank J. L. Díez and I. Echániz for technical support.

462

The research was partially funded by Gobierno de Aragón and European Social Fund

463

(Grupo Consolidado B-18).

464 465

Supporting Information:

466

-Determination of dissociation constants by isothermal titration calorimetry


15

Page 17 of 32


467

-Microcystin HPLC analysis

468

-Figure 1S: Calorimetric titrations for the interaction of microcystin LR with different

469

metal ions at several pH values.

470

-Figure 2S: HPLC of cross-linked microcystin-LR.

471

-Figure 3S: Summary of the main oligomeric chain species composed by microcystin-

472

LR under different conditions.

473 474 475 476 477

1.

478

expression in different strains of the bloom-forming cyanobacterium Microcystis

479

aeruginosa. Mol. Cell. Proteomics 2011, 10: M110 003749.

References Alexova, R.; Haynes, P.A.; Ferrari, B.C.; Neilan, B.A. Comparative protein

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Tonietto, A.; Petriz, B.A.; Araujo, W.C., Mehta, A.; Magalhaes, B.S.; Franco,

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O.L. Comparative proteomics between natural Microcystis isolates with a focus on

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Rohrlack, T.; Hyenstrand, P. Fate of intracellular microcystins in the

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Pearson, L.A.; Hisbergues, M.; Borner, T.; Dittmann, E.,; Neilan, B.A.

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Inactivation of an ABC transporter gene, mcyH, results in loss of microcystin

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Dohren, H.; Borner, T. Altered expression of two light-dependent genes in a

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by Microcystis PCC7806 under High Light. J. Phycol. 2011, 47: 1241-1246.

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Kardinaal, W.E.; Tonk, L.; Janse, I.; Hol, S.; Slot, P.; Huisman, J.; Visser, P.M.

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Microcystis. Appl. Environ. Microbiol. 2007, 73: 2939-2946.

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Shi, L.; Carmichael, W.W.; Miller, I. Immonugold Localization of Hepatotoxins

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in Cyanobacterial Cells. Arch. Microbiol. 1995, 163: 7-15.

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Young, F.M.; Thomson, C.; Metcalf, J.S.; Lucocq, J.M.; Codd, G.A.

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Immunogold localisation of microcystins in cryosectioned cells of Microcystis. J.

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Struct. Biol. 2005, 151: 208-214.

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Gerbersdorf, S.U. An advanced technique for immuno-labelling of microcystins

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in cryosectioned cells of Microcystis aeruginosa PCC 7806 (cyanobacteria):

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implementations of an experiment with varying light scenarios and culture densities.

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Downing, T.G.; Phelan, R.R.; Downing, S. A potential physiological role for

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cyanotoxins in cyanobacteria of arid environments. J. Arid. Environ. 2015, 112: 147-

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Vela, L.; Sevilla, E.; Gonzalez, C.; Bes, M.T.; Fillat, M.F.; Peleato, M.L.

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Exploring the interaction of microcystin-LR with proteins and DNA. Toxicol. In Vitro

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Meissner, S.; Fastner, J.; Dittmann, E. Microcystin production revisited:

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Alexova, R.; Dang, T.C.; Fujii, M.; Raftery, M.J.; Waite, T.D.; Ferrari, B.C.;

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Neilan, B.A. Specific global responses to N and Fe nutrition in toxic and non-toxic

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Microcystis aeruginosa. Environ. Microbiol. 2015, 18: 401-413.

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LR. Toxins 2015, 7: 2221-2231.

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Microcystis aeruginosa CYA 228/1 (Chroococcales, Cyanophyceae). Phycologia 1996,

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aeruginosa PCC7806. Environ. Microbiol. 2008, 10: 2476-2483.

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cyanotoxins in cyanobacteria of arid environments. J. Arid Environ. 2015, 112, 147-

691

151.

692 693

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701

Microcystis PCC 7806. Toxicon 2008, 51, (3), 388-397.

702 703


23

Page 25 of 32


704 705

Figure legends

706 707

Figure 1. Microcystis aeruginosa strains PCC 7806 (microcystin-producer) and PCC

708

7005 (microcystin non-producer) growth. Growth was determined in iron-replete and

709

iron-deficient culture medium. Panel A: number of cells/mL. Panel B: total soluble

710

protein. Panel C: chlorophyll a content.

711 712

Figure 2: Microcystin-metal interaction monitored by ITC. Microcystin-LR solution (10

713

µM) was titrated with metal solution (100-150 µM) at 25°C: (A) Fe2+ in 100 mM

714

phosphate buffer, 1.5 mM ascorbate, pH 7; (B) Fe3+ in 100 mM phosphate buffer, 1.5

715

mM ascorbate, pH 7; (C) Mo6+ in 100 mM Tris-HCl, pH 8.5. Inset: Control experiment

716

injecting Fe2+ into 100 mM phosphate, pH 7. Upper panels show the thermograms

717

(thermal power as a function of time) and the lower panels show the binding isotherm

718

(normalized heat as a function of the molar ratio).

719 720

Figure 3: AFM topography images of microcystin-LR samples diluted in 20 %

721

methanol (A) and presence of EDC (D). A majority of monomers can be observed in

722

(A), whereas the ratio of dimers increases in (D). (B) 2D image of a monomeric feature

723

from (A) and height profile (corresponding to the blue line) (C). (E) 2D image of a

724

representative dimer from (D) and height profile for the two protomers of the dimer (F).

725

Heights in the Z axis for each protomer are numbered. Monomers and dimers are

726

highlighted in white and red circles, respectively.

727 728

Figure 4: AFM topography images of crosslinked microcystin-LR samples upon

729

incubation with different metals in 20 % methanol: image showing different species

730

(A), a zoomed image showing a trimer in detail (B) and its corresponding height profile

731

highlighting the different units (C), in the presence of Fe2+; image showing different

732

species (D), a zoomed image showing a tetramer in detail (E) and its corresponding

733

height profile highlighting the different units (F), in the presence of Cu2+; image

734

showing different species (G), a zoomed image showing a hexameric chain in detail (H)


24


Page 26 of 32

735

and its corresponding height profile highlighting the different units (I), in the presence

736

of Fe3+. Monomers are rounded in white, dimers in red, trimers in green, tetramers in

737

yellow and hexamers in pink.

738 739


25

Page 27 of 32


740

Table 1. Binding parameters for microcystin-LR interacting with different metals at

741

25°C. The buffers are 150 mM sodium phosphate, 100 mM HEPES or 50 mM Tris-

742

HCl. Buffer

pH

Kd (µM)

∆H (kcal/mol)

Fe2+

Phosphate

7.0

2.4

-5.6

Fe3+

Phosphate

7.0

2.4

-2.8

Mo6+

Phosphate

7.0

37

-8.1

Hepes

7.6

15

-3.2

Tris-HCl

8.0

3.3

-14.3

Tris-HCl

8.5

1.3

-21.8

Hepes

7.6

1.2

-2.3

Tris-HCl

8.0

4.6

-8.5

Tris-HCl

8.5

10

-7.0

Hepes

7.6

1.3

-1.2

Tris-HCl

8.0

31

-8.0

Tris-HCl

8.5

44

-11.9

Cu2+

Mn2+

743

Relative error in Kd is 15%.

744

Absolute error in ∆H is 0.5 kcal/mol.

745


26


Page 28 of 32

746 747

Table 2. Percentages of microcystin-LR oligomeric species detected by AFM. All

748

measurements were performed in 20 % methanol except indicated. The samples were

749

cross-linked (EDC) in the presence of indicated metals. Features correspond to image

750

units, while molecules refer to the amount of individual peptide monomers in the

751

corresponding image units. Error associated to percentage determination was ± 5–15%.

752 CHAIN ASSOCIATION Conditions

Units

Microcystin- Features

Monomers Dimers Trimers Tetramers Hexamers (%)

(%)

(%)

%

%

95

5

----

----

----

LR

Molecules 90

10

----

----

----

+Milli-Q

Features

5

---

----

----

water

Molecules 90

10

---

----

----

+PBS

Features

98

2

----

----

----

pH 7.4

Molecules 96

4

----

----

----

Features

72

27

1

----

----

Molecules 56

42

2

----

----

+EDC

Features

97

3

----

----

----

Mn (II)

Molecules 95

5

----

----

----

+EDC

Features

97

3

----

----

----

Zn (II)

Molecules 95

5

----

----

----

+EDC

Features

65

25

10

----

----

Fe (II)

Molecules 45

34

21

----

----

+EDC

Features

67

20

3

10

----

Cu (II)

Molecules 43

25

6

26

----

+EDC

Features

42

30

----

----

28

Fe (III)

Molecules 16

22

----

----

62

+EDC

95

753 754


27

Page 29 of 32


Figure 1 M. aeruginosa PCC 7806 Control M. aeruginosa PCC 7806 -‐Fe M. aeruginosa PCC 7005 Control M. aeruginosa PCC 7005 -‐Fe

Number of cells (10-6/mL)

50 40

A

30 20 10 0 0

2

4

6

8

9

12

Total protein/cell (µg/cell)

Time (days)

60 50

M. aeruginosa PCC 7806 Control M. aeruginosa PCC 7806 -‐Fe M. aeruginosa PCC 7005 Control M. aeruginosa PCC 7005 -‐Fe

B

40 30 20 10 0 0

2

4

6

8

12

Chlorophyll a (µg/ml)

Time (days)

6

M. aeruginosa PCC 7806 Control M. aeruginosa PCC 7806 -‐Fe M. aeruginosa PCC 7005 Control M. aeruginosa PCC 7005 -‐Fe

C

4 2 0 0

2

4

6

8

9

Time (days)


12


Page 30 of 32

Figure 2!

[Fe(II)]T/[microcystin-LR]T!

dQ/dt (µcal/s)!

B

Time (min)!

C

Q (kcal/mol of injectant)!

dQ/dt (µcal/s)!

Time (min)!


A


dQ/dt (µcal/s)!

Time (min)!

[Fe(III)]T/[microcystin-LR]T!


[Mo(VI)]T/[microcystin-LR]T!

Page 31 of 32


Figure 3

A

B

C

D

E

F



Page 32 of 32

Figure 4

A

B

C

D

E

F

G

H

I

E ACS Paragon Plus Environment

Microcystin-LR binds iron and iron promotes self-assembly

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