Marine Inorganic Biochemistry: From Photoreactive ... · • Previously isolated from Vibrio parahemol tic sparahemolyticus (enteropathogenic, estuarine bacterium) • Isolation of

Marine Inorganic Biochemistry:From Photoreactive Siderophores

toto Iodide in Kelpp

Frithjof C. Küpper

OverviewOverview

• Introduction: Metals in the oceanIntroduction: Metals in the ocean

Case studies:• Marine siderophores• Marine siderophores• Iodide accumulation in kelpp• Algal genomes

OverviewOverview



Transition metals in terrestrial organisms

• First row series: V, Mn, Fe, Co, Ni, Cu, ZnFirst row series: V, Mn, Fe, Co, Ni, Cu, Zn • Second row series: Mo

• Fe usually very abundant in soil and f h t t !freshwater ecosystems!

• Concentrations of other metals may varyConcentrations of other metals may vary widely.

Metals in the OceanMetals in the OceanMo 100 nMMo 100 nMV 20 - 35 nMNi 2 - 12 nM

Relative abundance of “exotic” biometals

Zn 0.1 – 8 nM

Cu 0.6 - 5.7 nM

Cr 3 - 4.5 nM

F 0 05 0 7 M

Surface concentrationsare often lower than in deeper waters!Fe 0.05 – 0.7 nM

Mn 0 03 0 8 nM

p(Exceptions: Mn, Co)

Mn 0.03 – 0.8 nM

Metals in the OceanMetals in the Ocean

Butler A: Acquisition and Utilization of Transition Metal Ions by Marine Organisms.-Science 281, 207-210Science 281, 207 210

Fundamental differences between the marine and

t t i l bi hterrestrial biosphere• Iron tends to be scarce in the ocean!• In fact, marine primary productivity is

limited in HNLC (high nitrogen lowlimited in HNLC (high nitrogen, low chlorophyll) regions by lack of iron.

• In the sea, Mo, V, Ni, Zn, Cu are much b d t th i !more abundant than iron!

Strategies of marine organismsStrategies of marine organisms to cope with low iron availabilityp y

• Highly efficient uptake and recycling systems

• Use of chemical alternatives to iron

Marine metalloen mes MoMarine metalloenzymes: Mo• Dimethylsulfoxide (DMSO) reductase

(CH ) SO + 2H+ + 2e- (CH ) S + H O(CH3)2SO + 2H + 2e (CH3)2S + H2O

• O atom transfer (sulfoxide / sulfide)

• Membrane-bound or periplasmatic enzyme f i b t iof marine bacteria

Marine metalloenzymes:Marine metalloenzymes: Zn, Co and Cd carbonic

anhydrases from diatoms

CO2 + H2O HCO3- + H+

• No homologies to other carbonic anhydrases

CO2 + H2O HCO3 + H

• Can substitute Co or Cd for Zn! • Efficient Lewis acid-typeEfficient Lewis acid type

catalysts• The first documented caseThe first documented case

of Cd in biology!

F.M.M. Morel et al.

The World OceanThe World Ocean, a halogen-rich environment!a halogen rich environment!

• Marine organisms produce a plethora of halogenated natural productsproducts

• In many cases, metalloenzymes are involved in the biosynthesis

Marine metalloenzymes: V di (V) h l idVanadium (V) haloperoxidases

from marine red and brown algaefrom marine red and brown algae

X- + H2O2 + R-H + H+ R-X + 2 H2O

• Other peroxidases: heme (Fe) peroxidases p ( ) pin most terrestrial organisms, W peroxidases / oxidoreductases inperoxidases / oxidoreductases in hyperthermophilic archaea

Vanadi m halopero idasesVanadium haloperoxidases• Vanadium (V):

vanadatevanadate• Homology to acidic

phosphatases

Butler A, 1998: Science 281, 207-10

Vanadium haloperoxidasesVanadium haloperoxidases• A role in the synthesis of halogenated

marine natural products (esp. red algae: p ( p gLaurencia sp., Plocamium cartilagineum)

Butler A, 1998: Curr. Op. Chemical Biology 2, 279-85

OverviewOverview



Siderophore-mediated metal uptake

Siderophore Greek: “iron carrier”Siderophore, Greek: iron carrier

id hexcretion

Bacterium + Fe3+

apo-siderophore

siderophore – Fe3+

Bacterium + Fe3uptake

siderophore Fe

Marine siderophoresMarine siderophores

R. T. Reid, D. H. Live, D. J. Faulkner, A. Butler, Nature 366, 455 (1993).

Marine siderophoresMarine siderophores

• Some well-known structures (e.g. b ti ) b t tl ltiaerobactin), but mostly many novelties

• Mediate prokaryotic Fe / metal uptake in p y pmarine systems: availability to eukaryotes not well established yet (e g in algal-bacterialwell established yet (e.g. in algal-bacterial symbioses)M k i l h l• Most eukaryotic algae seem to have plasma membrane-bound ferrireductases

• Several marine siderophores are photoreactive!photoreactive!

Photolysis of aquachelinFe(III)-Aquachelin complex Photolysis products

O O

FeIII

NHO

O

NHO

O

hvHN

N

HN

NO

O O

N

HN

O

NR

-O O-O

OH

N-O

N-O

HN

NH

HN

NH

O

OHO

O

O

O

NH

HN

OH

O

OH

NH

NH OHO O

H2NO

NHO

NH

OH OH

OHO O

H2NO

OOH OH

O

Fe2++O O

D-glnL-serD-serL-threo--OH-asp

D-N-OH,N-Ac-orn

D-ser L-N-OH,N-Ac-orn

+ R

O O

O O

Aquachelin A

Aquachelin B

Aquachelin C

Aquachelin D

R:

KcondFe(III)’ = 1012.2 M-1 Kcond

Fe(III)’ = 1011.5 M-1

Barbeau, Rue, Bruland & Butler, 2001: Nature 413, 409-13

Other cases of photoreactive siderophoresPetrobactin Aerobactin

Common feature:Common feature: -hydroxy carboxylic acid moietymoiety

Barbeau et al., 2002: JACS 413, 409–13Bergeron et al., 2003: Tetrahedron 59, 2007-14

Key questions• Mechanistic aspects of photolysis• Structure of photoproduct

p p y

• How do the physicochemical properties of the photoproduct compare to those ofof the photoproduct compare to those of the original siderophore?

• Is photoproduct-bound Fe(III) as bioavailable as Fe(III) bound to thebioavailable as Fe(III) bound to the original siderophore?

=> Case study: AEROBACTIN

ESI-MS (positive ion mode):ESI MS (positive ion mode): Fe-aerobactin photolysis

decarboxylationm/z = 46 amum/z = 46 amum/z 46 amu

apo-aerobactinapo-photoproduct

Fe3+-photoproduct Fe3+-aerobactin

Quantum mechanical structure optimization of the Ga3+

complexes of aerobactin and its photoproductp p p

Ga-aerobactin Ga-photoproduct

(Spartan 2000 - PM3 level of theory) (C. J. Carrano)

Summary & conclusions: aerobactin photoproductSu a y & co c us o s ae obact p otop oduct

• Photochemistry results in a new ligand of similar physiological properties in bacterial cells

• Photochemistry of Fe-aerobactin yields reactive Fe2+, potentially available for a wide range of other organisms and ligands over a transient time spanF b d t th h t d t b id d t b f li it d

O hi / l i l i li ti (thi i lik l t b l t l

• Fe bound to the photoproduct can be considered to be of limited access to marine biota

• Oceanographic / ecological implications (this is likely to be relevant only in euphotic open-Ocean conditions, yet not for Enterobacteria also

d i b ti !)? I th l ti d t fproducing aerobactin!)? Is there any evolutionary advantage of producing photoreactive siderophores?

Küpper FC, Carrano CJ, Kuhn J-U, Butler A, 2006: Photoreactivity of Iron(III)-Aerobactin Photoprod ct Str ct re and Iron(III) CoordinationAerobactin: Photoproduct Structure and Iron(III) Coordination.-Inorganic Chemistry 45(15), 6028-33

Summary:Sid h h t h i tSiderophore photochemistry

apo-siderophore

+ Fe3+

p pexcretion

siderophore – Fe3+

Bacteriumuptake

hBacterium

Fe3+O2

Fe2+

Uptake!

Fe+ siderophore photoproduct

Fe3+ photoproduct

+ Fe3+Uptake!

Fe3 -photoproduct

Algal bacterial symbiosesAlgal-bacterial symbioses

• Bacterial symbionts of the dinoflagellateG di i t t (D H G )Gymnodinium catenatum (D.H. Green)

Isolation of siderophores from culture pmedia of bacterial symbionts

OOO H

HH

N

OO

HO HHH

H

H NNH

HOO

HHHHH

H

H

OHO

HO

HO

H

OHO

Vibrioferrin

• Vibrioferrin

Vibrioferrin• Previously isolated from Vibrio

parahemol tic s (enteropathogenicparahemolyticus (enteropathogenic, estuarine bacterium))

• Isolation of an unusual derivative – an t d i t tt t d thunexpected isotope pattern suggested the

presence of an element other than the p ese ce o a e e e o e a eusual C, H, N, O or S

=> Boronylated vibrioferrin!y

Boronylated vibrioferriny

(Spartan 2000 - PM3 level of theory)

Boron• Discovered by Joseph-Louis Gay-Lussac and Louis-

Jaques Thénard, and independently by Sir Humphry Davy in 1808 (isolation of boron by combing boricDavy, in 1808 (isolation of boron by combing boric acid (H3BO3) with potassium)

• Typical oxidation state: +3, stable isotopes: 10

5B 115B

• In seawater: fairly abundant - about 0.4 mM (but

stable isotopes: 5B, 5B

sea a e a y abu da abou 0 (buvariable) (22nd most abundant element in Earth’s crust after N and Cu)

• Well-studied in the context of seawater

crust, after N and Cu)

• Well-studied in the context of seawater desalination

BoronBoron• Applications: enamels, borosilicate glass,

pyrotechnics, nuclear reactors, boron fibers in py , ,engineering

• Very few (< 5) boron-containing natural products known so far including a quorumproducts known so far, including a quorum sensing molecule

• Boron is an essential trace elements for algae and terrestrial plants!and terrestrial plants!

• Unknown function in eukaryotesUnknown function in eukaryotes

The Boron StoryThe Boron Story

Harris WR, Amin S, Küpper FC, Green DH, Carrano CJ, 2007: Borate-binding to siderophores: Structure and stability.-g p yJournal of the American Chemical Society 129, 12263-71

A i S Kü FC G DH H i WR C CJ 2007Amin S, Küpper FC, Green DH, Harris WR, Carrano CJ, 2007: Boron binding by a siderophore isolated from marine bacteria "associated" with the toxic dinoflagellate G. catenatum.-a o a d o d o ag a G a a uJournal of the American Chemical Society 129, 478-479

The Boron Story

Fe-vibrioferrin is highly g yphotoreactive

• Unlike for aerobactin and all other photoreactive siderophores known so far, photochemistry destroys the Fe-bindingphotochemistry destroys the Fe binding backbone of VF

• VF photochemistry generates free reactive iron intermediates which enhance iron uptake of theintermediates which enhance iron uptake of the algal symbiont

Boron, iron and vibrioferrin (VF)Boron, iron and vibrioferrin (VF)VF

+ Fe3+Excretion + BO33-

B t iVF–Fe3+Uptake VF–B

hBacteriumO

Uptake?Fe3+O2

Fe2+Biol. effects?

“phycosphere”

+ VF photoproduct

( F 3+ bi di ) phycosphere(no Fe3+ binding)

Uptake!!

Alga

Vibrioferrin photochemistryVibrioferrin photochemistry – a key factor in an algal-bacterial symbiosis

Amin SA, Green DH, Hart MC, Küpper FC, Sunda WG, Carrano CJ, 2009: Photolysis of iron–siderophore chelates promotes bacterial–algal mutualism.-Proceedings of the National Academy of Sciences of th USA 106(40) 17071 6the USA 106(40), 17071–6

A i SA G DH Kü FC C CJ 2009Amin SA, Green DH, Küpper FC, Carrano CJ, 2009: Vibrioferrin, an unusual marine siderophore: Iron binding photochemistry and biological implicationsbinding, photochemistry, and biological implications.-Inorganic Chemistry 48, 11451-8

OverviewOverview



Iodine in seaweeds: A bit of historic background

• Goiter-preventing effects of seaweeds: p gKnown to the Chinese emperor Shen-Nung (third millennium B C !)(third millennium B.C.!)

U f b t d d di t• Use of burnt seaweeds and sponges as diet supplements for the same purpose: Common in ancient Greece at the time of physician Hippocrates [460-370 B.C.] pp [ ]

Biological model: gLaminaria digitata(Phaeophyceae)

Iodine as a novel chemical element was discovered in the ashes of Laminaria

Courtois, B. (lu par / read by N.

Clément), 1813:),

Découverte d’une substance

nouvelle dans le Vareck.

Annales de Chimie 88, 304-310

2 I- + H2SO4 → I2 + SO32- + H2O 2 4 2 3 2

•Strong corrosion of copper flasks•Strong corrosion of copper flasks•Emission of violet vapor=> Fr IODE / En IODINE=> Fr. IODE / En. IODINEGreek ιώδης, i.e. violet

Kelp as the world’s first raw material for iodine productionfor iodine production

Re-enactment of traditional kelppharvest, Breton folk festival in Roscoff, Brittany, France

(Gouel Rosko, 1997)


Re-enactment of traditional kelppburning, Breton folk festival in Roscoff, Brittany, France

(Gouel Rosko, 1997)


Historic kelp incinerator inPorspoder, Finistère, Brittany, France (June 2004)

Iodine production from seaweeds became a major economic activity in coastal regions of Europe - in particular, in parts of Brittany, Normandy Ireland and ScotlandNormandy, Ireland and Scotland

Current-day harvesting of kelp l t i l f i di d ti– no longer a raw material for iodine production

Kelp harvesters (goëmoniers) near Porspoder, Finistère, Brittany,Kelp harvesters (goëmoniers) near Porspoder, Finistère, Brittany, France – use of kelp for alginate extraction (June 2004)

The Breton coast – where the seaweeds during the pioneering erawhere the seaweeds during the pioneering era

of iodine research came from

Near Porspoder, Finistère, Brittany, France (June 2004)

The Breton coast – where the seaweeds during the pioneering erawhere the seaweeds during the pioneering era

of iodine research came from

Near Porspoder, Finistère, Brittany, France (June 2004)

Iodine accumulation in Laminaria

• Laminariales (kelps) are a major ( p ) jbiogeochemical pump of iodine!

• Laminaria is the strongest iodine accumulator in life

Still l

in life

Still unclear:

Ch i l f f l t d i di ?• Chemical form of accumulated iodine? • Biological significance?Biological significance?

The Global Iodine Cycleiodine oxide (IO)iodine oxide (IO),

and: I-, IO3-, HOI

IO provides condensation Thyroidlactoperoxidase-catalysed

I2, iodocarbons (R-I)(CH I CHI CHI etc )

photolysisnuclei for cloud formation

precipitation

diet

lactoperoxidase catalysedincorporation in thyroxine

(CH2I2, CHI3, CHI3 etc.)

“Iodovolatilisation”

dietCH3I

kelp forests dietaryfertilizers

euphotic zone

kelp forestsand planktonic algae(accumulation of iodine, iodinated natural products)

Nitrate reductase(phytoplankton, bacteria?)

ysupple-ments

I2, HOI

+ organicmatter

iodinated natural products)IO3

-(p y p , )

terrestrial runoff

deep waters IO -

+ O2, O3 or H2O2

I-

fossil iodide / iodate deposits

deep waters

S

IO3-

(predominantly) I-(release of small amounts

from POC)Millions of years ago

sedimentsR-I R + I-

H2S

Bacterial decomposition

)

Adapted / modified from Feiters, Küpper & Meyer-Klaucke, 2005: J. Synchrotr. Radiation 12, 85-93


• Requirement of an intact cell wall (apoplast)cell wall (apoplast)

• Role of hydrogen peroxide and haloperoxidases in iodine uptake

• Iodine efflux upon oxidative stress

Küpper, F.C.; Schweigert, N.; ArGall E ; Legendre J M ; VilterArGall, E.; Legendre, J.M.; Vilter, H.; Kloareg, B., 1998: Iodine uptake in Laminariales involves

t ll l h l idextracellular, haloperoxidase-mediated oxidation of iodide. Planta 207, 163-171


• Strong seasonality!3

DW

)te

nt (%

2

ine

cont

1

Iod

00Winter Spring Summer Autumn

ArGall, E.; Küpper, F.C.; Kloareg, B., 2004: A survey of iodine contents in Laminaria digitata. Botanica Marina 47, 30-37.


• Accumulation in cortical tissues

P ti l i d d X i i(stipe section)

Particle-induced X-ray emissionElemental map of the meristoderm and outer cortes in a L. digitata stipe section

25 m

Verhaeghe, E.F.; Fraysse, A.; Guerquin-Kern, J.-L.; Wu, T.-D.; Devès, G.; Mioskowski, C.; Leblanc, C.; Ortega, R.; Ambroise, Y.; Potin, P., 2008:Mi h i l i i f i di di t ib ti i th b l L i iMicrochemical imaging of iodine distribution in the brown alga Laminaria digitata suggests a new mechanism for its accumulation. Journal of Biological Inorganic Chemistry 13, 257-269.


Techniques used in this study:

• GC/MS• Gas-phase particle counters

C th di t i i• Cathodic stripping square wave voltammetry (CSSWV)

• X-ray absorption spectroscopy using synchrotron radiation (XAS)synchrotron radiation (XAS)

etc.

Biological XAS at the EMBL O t t ti H bEMBL Outstation, Hamburg

lit 2

13 elementfluorescence

detectormonochromatic

beam

whitebeam

Al foilslits 1

slits 2

I0 It

detector

beam

focussingmirrororder sorting

sample

ionization

calibrator crystal(Si 220) withscintillation

e+

synchrotron

double crystalmonochromator

(Si 111)

ionizationchambers

scintillationcounters

synchrotron

Biological XAS at the EMBL O t t ti H bEMBL Outstation, Hamburg

Iodine XAS of Laminaria tissuesod e S o a a a t ssues

Iodide (I-) is the accumulated form(Iodine K-edge)

Iodide (I ) is the accumulated form of iodine in Laminaria!

Iodine metabolism d id ti tand oxidative stress

• Iodine uptake requires low H2O2 levels (< 25 M)

Hi h t ti f H O lt i i di• Higher concentrations of H2O2 result in iodine efflux

O id ti t i L i iOxidative stress in Laminaria:

• Oxidative (respiratory) burst – a defense reaction( p y)

• Desiccation, high temperatures, high irradiance d t t h i id t t l tidand exposure to atmospheric oxidants at low tide

The oxidative burst in Laminaria• A key element in eukaryotic innate immunity• Triggers in Laminaria: bacterial endotoxins (LPS), gg ( ),

oligoguluronates (oligoalginates)

123

123

120 s

3

420 s

3

120 s 420 s

1 123

23

DCFH-DA : dichlorohydro-fl i

660 s 840 sfluorescein diacetate

Küpper et al., 2001: Plant Physiology 125, 278-91

Monitoring the iodine pool d i th id ti b tduring the oxidative burst

in Laminaria with XASin Laminaria with XAS

• EXAFS: Oxidative stress results in a change gof the solution environment of accumulated iodide (towards an aqueous hydrated form)iodide (towards an aqueous, hydrated form)

• XANES: No changes in the redox state of giodine – only iodide is detectable

Cathodic stripping square lt t (CSSWV)wave voltammetry (CSSWV)

Oligoguluronate treatmenttreatment

Strong iodide efflux upon oxidative stress

• No increased levels of oxidized or organic iodine species

Scavenging of ozone (O3)Scavenging of ozone (O3) by Laminariay

Flow reactor scheme

(Palmer et al., 2005: Env. Chem. 2, 282-90)

Scavenging of ozone (O3) g g ( 3)by Laminaria

Laminariathalli effectively scavenge ozoneg

When light is present:present: ultrafine particle pa t c eformation

Kelp forests contribute to aerosol formationcontribute to aerosol formation

in the coastal environment• Iodine oxides as condensation nuclei• “Particle bursts” in the coastal atmosphere

above kelp beds at low tide and high p girradiance

O’Dowd et al., 2002: Nature 417, 632-6

Kelp forests contribute to aerosol formationcontribute to aerosol formation

in the coastal environment

O’D d t l 2002O’Dowd et al., 2002:Nature 417, 632-6

Kelp iodine emissions into the coastal atmosphere

• “Iodovolatilisation” discovered in 1920s by Kylin and Dangeard: I2 detected with starch paperDangeard: I2 detected with starch paper

• J. Lovelock, 1973: Discovery of methyl iodide emissions from seaweedsemissions from seaweeds

• B. Alicke et al., 1999: High IO levels above kelp beds at low tideat low tide

• L.J. Carpenter et al., 2000: CH2I2 main species itt d b L i iemitted by Laminaria

(total iodine emissions: 0.09 – 0.5 pmol g FW-1 min-1)• This study: High I2 fluxes due to reaction of O3 with I-

on seaweed surface (130 pmol g FW-1 min-1)

You can smell it!You can smell it!

I2 I2 I2

Taynish National Nature Reserve, Argyll, Scotland, June 8, 2008

Sea water: High tide Atmosphere: Low tideParticle formation

XASIO•

I-

XASh

I•I-

I2

hO3

II- I-

I2

Iodine

I-I-I

I-Iodine uptake

V-haloperoxidaseH2O2 / ROS2 2

I-

Biological significance: Iodine accumulation in Laminaria

• Laminaria accumulates iodide as an inorganic antioxidant: the first case in a living system!a living system!

Oth i h th h i l• Other, previous hypotheses: chemical defense (grazers, pathogens)(g p g )

• Implications for atmospheric & marineImplications for atmospheric & marine chemistry

Biological significance: Iodine accumulation in Laminaria

• Iodo(hydro)carbons: quantitatively insignificant as H2O2 scavenging products

• Must have another function – defense?!

• Iodine is a better leaving group than bromine or hl ichlorine:

I > Br > Cl

=> High reactivity / strong alkylating potential of iodinated compoundsiodinated compounds

What makes iodide a suitable antioxidant?

• Reactions with major oxidants (H2O2, O2-, OH.,

1O2, O3) are very favourable kinetically and

thermodynamicallythermodynamically

• Iodide compares well with established, organic

biological antioxidants

• Iodide is the only halide with potential antioxidantIodide is the only halide with potential antioxidant

properties

• Confirmed in a heterologous system: iodide

quenches respiratory burst in human blood (IC50 =quenches respiratory burst in human blood (IC50 =

2.9 mM)

Complementarity to other p yantioxidant systems

• Iodide is an apoplastic antioxidant (i.e. limited to the

cell wall space)

• Ascorbate and glutathione are strictly intracellular (not

excreted / effluxed)

• EST data suggest that Laminaria has fewer and lower• EST data suggest that Laminaria has fewer and lower

expression levels of typical antioxidant enzymes than

other eukaryotes

Cell wall (apoplast)

Cell / cytosolSea water

Cell wall (apoplast)

I-Ascorbate, glutathione, etc.

ywater


Küpper FC, Carpenter LJ, McFiggans GB, Palmer CJ, Waite T, Boneberg E-M, Woitsch S, Weiller M, Abela R, Grolimund D, Potin P, Butler A, Luther III GW, Kroneck PMH, Meyer-Klaucke W, Feiters MC, 2008:

Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistryantioxidant impacting atmospheric chemistry.-

Proceedings of the National Academy of Sciences of the USA oceed gs o t e at o a cade y o Sc e ces o t e US105 (19), 5954-58

A place where you can smell the tide level…

Roscoff, Brittany, France

OverviewOverview



Algal genome projectsAlgal genome projectsDiatomsDiatoms:

• Thalassiosira (Armbrust EV et al 2003: Science 306: 79 86)al., 2003: Science 306: 79-86)

• Phaeodactylum (Bowler et al., 2008: Nature 456: 239-244 )

Algal genome projectsAlgal genome projects

• Ostreococcus tauriOstreococcus tauri• Ostreococcus lucimarinus

Emiliania h le i• Emiliania huxleyi• Micromonas pusillap• Bathyococcus sp.• etc• etc.

All l l i d l fAll algal genomics models so farare unicellular!are unicellular!

Ectocarpus siliculosusctoca pus s cu osus

• Filamentous, cosmopolitan brown alga – mostly from temperate seaso te pe ate seas

• One of the best-studied seaweeds• The first fully sequenced multicellular alga!• The first fully-sequenced multicellular alga!• > 300 fully-characterized strains in public-domain

c lt re collections (CCAP KU MACC)culture collections (CCAP, KU-MACC)

Cock JM, Sterck L, Rouzé P, Scornet D, Allen AE, Amoutzias G, Anthouard V, Artiguenave F, Aury J-M, Badger JH, Beszteri B, Billiau K, Bonnet E, Bothwell JHF, Bowler C, Boyen C, Brownlee C, Carrano CJ, Charrier B, Cho GY, Coelho SM, Collén J, Corre E, Da Silva C, Delage , , , , , , gL, Delaroque N, Dittami SM, Doulbeau S, Elias M, Farnham G, Gachon CMM, Gschloessl B, Heesch S, Jabbari K, Jubin C, Kawai H, Kimura K, Kloareg B Küpper FC Lang D Le Bail A Leblanc C Lerouge P LohrKloareg B, Küpper FC, Lang D, Le Bail A, Leblanc C, Lerouge P, Lohr M, Lopez PJ, Martens C, Maumus F, Michel G, Miranda-Saavedra D, Morales J, Moreau H, Motomura T, Nagasato C, Napoli CA, Nelson DR Nyvall Collén P Peters AF Pommier C Potin P Poulain PDR, Nyvall-Collén P, Peters AF, Pommier C, Potin P, Poulain P, Quesneville H, Read B, Rensing SA, Ritter A, Rousvoal S, Samanta M, Samson G, Schroeder DC, Ségurens B, Strittmatter M, Tonon T, T J V l ti K D P Y i hi T V d P YTregear J, Valentin K, von Dassow P, Yamagishi T, Van de Peer Y, Wincker P, 2010:

The Ectocarpus genome and the independent evolution of multicellularity in the brown algae.- Nature 465, 617-21

June 3, 2010June 3, 2010

Features of the Ectocarpus genome

• 214 Mbpp• Very high number of introns • Repeated sequences (Transposons, p q ( p ,retrotransposons, helitrons) make up > 22% of the genome! • No ferritins!!• Halogen metabolism (1 V haloperoxidase, 21 putative dehalogenases and 2 haloalkane dehalogenases)• Many bacterial genes• … and a high percentage of genes of still unknown f ti !function!

Iodine in Ectocarpus?Iodine in Ectocarpus?

• Ectocarpus accumulates iodine, but at much lower levels than Laminaria (data from Roscoff & Vernaison)(data from Roscoff & Vernaison)

• Genome annotation has revealed the presence of one vanadium haloperoxidase genehaloperoxidase gene

• X-ray absorption spectroscopy using y p p py gsynchrotron radiation (XAS) on EctocarpusEctocarpus

Iodine K-edge XAS ofEctocarpus siliculosus

5.0E-02

6.0E-02

ce u

nits

4.0E-02

ores

cenc Ectocarpus in regular Provasoli-enriched sea

water, without supplementsEctocarpus in Provasoli-enriched sea water,with 50 uM iodide supplement

3.0E-02

X-ra

y flu

o with 50 uM iodide supplementEctocarpus in Provasoli-enriched sea water,with 50 uM iodate supplement

1 0E 02

2.0E-02

Rel

ativ

e X

0.0E+00

1.0E-02

3 29E 04 3 30E 04 3 31E 04 3 32E 04 3 33E 04 3 34E 04 3 35E 04 3 36E 04 3 37E 04

R

3.29E+04 3.30E+04 3.31E+04 3.32E+04 3.33E+04 3.34E+04 3.35E+04 3.36E+04 3.37E+04

Energy (eV)

Lik L i i E t l t Like Laminaria, Ectocarpus accumulates iodine as iodide!

Ectocarpus and iron: paxenic cultures are CAS active!

CAS = Chrome azurol S (a colorimetric assay for Fe3+-binding activity)• CAS activity is caused by secretion of strong Fe(III)-binding ligands– or acidification

Siderophores in Ectocarpus?p p

• Siderophore production is known to occur in• Siderophore production is known to occur in bacteria, fungi and monocotyledons – but no eukaryotic algae so far!eukaryotic algae so far! (Only phytosiderophores from monocots / higher plants)

• CAS activity in axenic Ectocarpus cultures• CAS activity in axenic Ectocarpus cultures seemed like an exciting finding

• Still, no siderophores could be isolated so far

• CAS activity might be caused by external acidification

Features of the E tEctocarpus genome

from the bioinorganic perspective:from the bioinorganic perspective:Iron uptake

• homologs of fro2, a cell surface Fe(III) reductase

• several divalent metal ABC transporters (ZIP type)

• NRAMP (M2+ H+ symporter with preference for Fe(II))• NRAMP (M2+-H+ symporter with preference for Fe(II))

consistent with simple reductase/permease pathway

• absence of multicopper oxidases

f id h bi th ti th i iti ll• presence of siderophore biosynthetic pathway initially

hypothesized, but not corroborated

Iron uptake in EctocarpusIron uptake in Ectocarpus• Short term iron uptake studies: Fe is taken up in a time and concentration dependent manner => Consistent with an active transport process. • Derived kinetic parameters: similar to those reported in the few available studies in other red, green and brown algae

Macrocystis Gracilaria Laminaria Undaria Ectocarpus

Vm 1.6 pmol/cm2/hr 0.26l/ /h

2.7l/ 2/h

6.4l/ 2/h

0.25l/ /hpmol/mg/hr pmol/cm2/hr pmol/cm2/hr pmol/mg/hr

Km 3.5 µM 0.6 µM 0.54 µM 6.4 µM 1.5 µM

Ref Manley 1981 Liu 2000 Matsunaga1991

Matsunaga1991

This work

Fe(III) chelate reductase activity in Ectocarpus

A) iron replete (30 µM) and B) iron starved (4 nM) cultures ofA) iron replete (30 µM) and B) iron starved (4 nM) cultures of Ectocarpus siliculosus.Negative controls: C) dead cells and D) live cells minus FZ.g ) )Error bars = ± 1 S.D. from triplicate measurements

Iron uptake from 55FeEDTA in Ectocarpus siliculosus cultures over 800 hrsin Ectocarpus siliculosus cultures over 800 hrs

Error bars represent ± 1 S.D. from three separate experiments with replicate time points for each.

Features of the E tEctocarpus genome

from the bioinorganic perspective:from the bioinorganic perspective:Iron storageg

• no ferritins!!

typical feature of heterokont organisms

• No other iron store obvious from the Ectocarpusgenome

need for physical techniques need for physical techniques

Ectocarpus pand iron:

How to store iron withoutiron without

ferritin?

• probing the intracellular Fe pool by MössbauerMössbauer spectroscopy

…in search of a novel mode of i t ll l iintracellular iron storage…

Ectocarpus and iron: pHow to store iron without ferritin?

• probing the intracellular Fe pool by XAS

Extracted EXAFS spectrum of 52 merged spectra transformed in k-space. Black squares:experimental data; red line: fit. Green line: setting of the range.

Ectocarpus and iron: pHow to store iron without ferritin?

Mössbauer and X-ray absorption spectroscopy show y p p py2 main components of the cellular Fe pool:

(1) an iron sulfur cluster (approx 26% of the total(1) an iron-sulfur cluster (approx. 26% of the total intracellular iron pool)

(2) a second component with spectra typical of a (Fe3+O6) system with parameters similar to the

h h h i h i l famorphous phosphorus-rich mineral core of bacterial and plant ferritins (approx. 74% of the

ll l i l)cellular iron pool)

=> suggests that Ectocarpus contains a non-gg pferritin but mineral-based iron storage pool

Exotic bioelements in Ectocarpus?

• No Cd carboanhydrases (homologous to those in centric diatoms) identified in genome

• However, Ectocarpus cell walls accumulate high levels of strontium (Sr)! (Detected by X-ray

)microprobe)

S i th lk li t l f th 2nd i• Sr is an earth alkali metal – from the 2nd main group (beneath calcium)

• Hypothesis: Probably bound to alginate

• No biological functions of Sr are known

Acknowledgements • Carl J. Carrano (San Diego State University)

• Alison Butler (UC S t University)(UC Santa Barbara)

• David H. Green (SAMS)

• Kathy Barbeau (formerly UCSB,

• Shady Amin (SDSU)( o e y UCS ,now Scripps Institution of Oceanography)

•• Martina StrittmatterMartina StrittmatterWes Harris (University of •• Martina Strittmatter Martina Strittmatter (SDSU & SAMS)(SDSU & SAMS)

• Wes Harris (University of Missouri, St. Louis)

AcknowledgementsP t M H K k (U i it f K t G )-- Peter M.H. Kroneck (University of Konstanz, Germany)

-- Sonja Woitsch, Markus Weiller (dto.)

-- Lucy J. Carpenter and Carl Palmer (University of York, UK)

-- Gordon McFiggans (University of Manchester, UK)

-- Bernard Kloareg and Philippe Potin (CNRS, Roscoff, France)

-- Martin Feiters (University of Nijmegen, The Netherlands)

-- Wolfram Meyer-Klaucke and Gerd Wellenreuther (European Molecular Biology Laboratory, Hamburg, Germany)

-- Eva M. Boneberg (Biotechnologie Institut Thurgau, Switzerland, formerly at University of Konstanz)

-- Alison Butler (University of California, Santa Barbara, USA)

-- George Luther & Tim Waite (University of Delaware, Lewes, USA)g ( y )

-- Rafael Abela & Daniel Grolimund (Swiss Light Source, Paul ScherrerInstitute, Villigen, Switzerland)

Thank you.y

Dunstaffnage Marine Laboratory, Oban, Argyll / West Highlands, Scotland

The following is reserve material gfor self study

Reserve slides for the Laminaria-iodine ti f llsection follow


Cl- Br- I-Thermodynamics

Cl Br IOne-electron transfer per X- GR (kJ mol-1) X- + 3O2 [X] + O2

• - 247.97 200.7 143.76

X- + OH• (g) [X] + OH- 49.21 1.93 -55.0(g)

X- + OH•(aq) [X] + OH 67.54 20.26 -36.66

X- + 1O [X] + O • - 152 45 105 17 48 24X + 1O2 [X] + O2 152.45 105.17 48.24

X- + H2O2 [X] + OH• + OH- 235.42 188.15 131.22

X- + H+ + HO2 • [X] + H2O2 93.59 46.31 -10.61

X- + O3 [X] + O3• - 135.08 87.80 30.87


Cl- Br- I-Thermodynamics

Cl Br ITwo-electron transfer per X- GR (kJ mol-1) 2 X- + H+ + O3 X2 + O2 + OH- - 57.9 -112.5 - 217.3

X- + H+ + O3 HOX + O2 -111.8 -141.4 -210.8

X- + H+ + 1O2 + H2O HOX + 62.48 32.88 -36.43H2O2

X- + 2H+ + 1O2 X2 + H2O2 96.66 42.06 -62.76

X- + H2O2 HOX + OH- 28.21 -1.39 -70.81

X- + 2 H+ + H2O2 X2 + 2 H2O -77.66 -132.26 -237.082 2 2 2


Kinetics

Compound k (M-1 s-1)Compound k12 (M-1 s-1)

O3 reactions with9I- 1.2 x 109

Br- 2.48 x 102

Cl- < 3 x 10-3

Ascorbate 4.8 x 107

Glutathione 2.5 x 106

Singlet oxygen (1O2) reactions withI- 1 x 108 (aprotic solvents)

8 7 x 105 (pH ~ 7)8.7 x 105 (pH ~ 7)Br- 1.0 x 103

Cl- 1.0 x 103

A b t 8 3 106Ascorbate 8.3 x 106

Glutathione 2.4 x 106 (in D2O, 310 K, pD = 7.4)


Kinetics


OH radical (OH) reactions with10I- 1.2 x 1010

Ascorbate 1.1 x 1010

Glutathione 1.3 x 1010 (pH = 5.5)( )Dimethyl sulfoniopropionate 3 x 108

Dimethyl sulfide 1.9 x 1010

Dimethyl sulfoxide 6.6 x 109Dimethyl sulfoxide 6.6 x 10

Superoxide (O2-) reactions with

I - 1 x 108 (no data available for I-)I3 1 x 108 (no data available for I )Ascorbate 2.7 x 105 (pH= 7.4) Glutathione 2.4 x 105 (pH= 7.8)


Kinetics


Hydrogen peroxide (H2O2) reactions withI- 0.69Br- 2.3 x 10-5

Cl- 1.1 x 10-7

Ascorbate 2 x 100

Glutathione 2 – 20 x 100

Glutathione peroxidase 6 x 107Glutathione peroxidase 6 x 10

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