Marine Inorganic Biochemistry: From Photoreactive...
Transcript of Marine Inorganic Biochemistry: From Photoreactive...
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
• Introduction: Metals in the oceanIntroduction: Metals in the ocean
Case studies:• Marine siderophores• Marine siderophores• Iodide accumulation in kelpp• Algal genomes
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
• Introduction: Metals in the oceanIntroduction: Metals in the ocean
Case studies:• Marine siderophores• Marine siderophores• Iodide accumulation in kelpp• Algal genomes
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
• Introduction: Metals in the oceanIntroduction: Metals in the ocean
Case studies:• Marine siderophores• Marine siderophores• Iodide accumulation in kelpp• Algal genomes
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)
Kelp as the world’s first raw material for iodine productionfor iodine production
Re-enactment of traditional kelppburning, Breton folk festival in Roscoff, Brittany, France
(Gouel Rosko, 1997)
Kelp as the world’s first raw material for iodine productionfor iodine production
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
Iodine accumulation in Laminaria
• 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
Iodine accumulation in Laminaria
• 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.
Iodine accumulation in Laminaria
• 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.
Iodine accumulation in Laminaria
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
Iodine accumulation in Laminaria
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
• Introduction: Metals in the oceanIntroduction: Metals in the ocean
Case studies:• Marine siderophores• Marine siderophores• Iodide accumulation in kelpp• Algal genomes
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
What makes iodide a suitable antioxidant?
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
What makes iodide a suitable antioxidant?
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
What makes iodide a suitable antioxidant?
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)
What makes iodide a suitable antioxidant?
Kinetics
Compound k (M-1 s-1)Compound k12 (M-1 s-1)
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)
What makes iodide a suitable antioxidant?
Kinetics
Compound k (M-1 s-1)Compound k12 (M-1 s-1)
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