12.755 lecture 3/4 cellular uptake of trace elements by microbes

12.755 Lecture 3/4Cellular Uptake of Trace Elements by Microbes

Major Concepts• Growth eqns for phytoplankton: Monod• Growth eqns for phytoplankton: Droop/Cellular Quota

• Principles of Uptake: Diffusion limited uptake• Principles of Uptake: High affinity and low affinity transport• Principles of Uptake: Free ion model• Principles of Uptake: Kinetic versus thermodynamic control

• Dual metal effects: Competitive Inhibition• Dual metal effects: Biodilution• Dual metal effects: Colimitation

• A real (complicated) example: Bioavailability• Another real (complicated) example: Fe acquisition systems• Another real (complicated) example: Fe – light colimitation

Related Readings:Kustka, Shaked, Morel L&O 2003Sunda and Huntsman 2000Saito et al., 2008Background: Hudson and Morel 1990, Droop 1973, Sunda and Huntsman 1997 (iron light colimitation study);

Anderson and Morel 1982, Droop Biography 1

(Moore, Doney, and Lindsay, GBC 2004)

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Growth modelsSingle substrate models:• Michaelis-Menten enzyme kinetics

• Monod 1942, empirical hyperbolic relationship between growth rate and substrate based on Michaelis-Menten equation:

• Droop 1968, cell quota model (growth related to intracellular concentration not extracellular):

/’m = 1-kQ/Q

Under steady-state conditions the Monod equation and Droop equations are equivalent (Burmaster, 1979)

- chemostat cultures are needed for steady-state- trace metal buffered batch cultures act as chemostats 3

Q = cellular quota

Monod growth:

Droop cellular quota:

’m = 1-kQ/Q

Q

= uptake rate

[S] = substrate concentration

Km = half saturation constantkQ= saturation constant, value of Q when D (growth rate) = 0max and’m = maximum growth rate

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What happens when S is a small number? A large number?Likewise for Q?

• Monod

• Droop

Dilution rate = growth rate in chemostat,Note when D=0 Q is not zero

Fe’

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Measurements of cellular uptake and quotasHow to read a Sunda and Huntsman manuscript:

(Sunda and Huntsman 1997, Nature)

Same equation as: Q=/

• Derived from Droop’s cellular quota ideas

• Possible because all experiments in steady-state (Burmaster showed equivalence between these equations at steady-state)

• Key concept: Biodilution of metal (for multiple metals), if growth rate changes and uptake stays maximal, quota must change

A curious thing…

Uptake is related to Surface Area for many different species and iron concentrations

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Diffusion limited uptake: Physical Limits on Uptake and

Acclimation through synthesis of transporters= 4rD[M’]

= Maximal diffusion rate

r = cellular radius

D = diffusion rate constant (2 x 10-6 cm2 s-1)

Acclimated at low Zn

Acclimated at high Zn

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Small cell size allows increased nutrient acquisition efficiency through increased surface to volume ratios

T. weissfloggiiV= 1370 m3

A= 598 m2

SA:V = 0.44

T. oceanicaV= 171 m3

A=145 m2

SA:V = 0.85

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Diffusion limitation and Cell Size (from Chisholm, 1992) (equivalent cellular nitrogen quota)

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High affinity and low affinity transporters even have isotope effectsSeth John et al., L&O 2007

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The basis for the free-ion or M’ model (Late 1970’s early 80’s)log K’s:

FeNTA= 17.9 FeEDTA =27.7

FeDTPA = 32.6

Figures from Classic Paper Anderson and Morel 1982, also see Sunda and Guillard, 197812

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Cell transporters are ligands too, are they in equilibrium?

Transporters are in equilibrium. Back reaction (k-L ) is possible

Transporters are under kinetic control. Back reaction (k-L ) is too slow to matter

So many cells present that dissociation of metal-buffer (M-EDTA) MY is too slow to supply cells M , the Buffer is “Blown”

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Using the Kinetic Model for Phytoplankton Uptake: How do the chemical properties of the metals affect uptake?

= kL [M’] LT

kL = rate of reaction with uptake ligands

[M’] = abundance of bioavailable metal

LT = number of transporters (per cell surface)

All three are crucial… Which ones can biology change?



• Principles of Uptake: Diffusion limited uptake• Principles of Uptake: High affinity and low affinity transport• Principles of Uptake: Free ion model • Principles of Uptake: Kinetic versus thermodynamic control





Competitive inhibition

• Theoretically derived from enzyme kinetics with an “inhibitor” substrate

• Kmapp = Km * (1+ [I]/KI)

• v = vmax*S/(S + Km*(1+ [I]/KI))

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The often forgotten/overlookedconcept of biodilution

Droop cellular quota:

’m = 1-kQ/Q

Q

Q

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Box model for cellular uptake – Hudson and Morel 1990 (see newer papers for updated versions)

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Fe’ model for bioavailability in doubt? Earlier data is consistent when recast as a Fe(II) model

Despite the prevalence of experimental data supporting it, the Fe’ model is now in doubt. Electrochemical measurements have shown that most of the iron in seawater is bound by strong organic ligands that buffer such low concentrations of Fe’ that they should not support phytoplankton growth. Laboratory studies have shown that phytoplankton cultures can obtain Fe and grow in the presence of model siderophores … which like the oceanic ligands, maintain very low Fe’ concentrations.

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From Shaked, Kustka, and Morel, L&O 2005A General Kinetic Model for Iron

Acquisition by Eukaryotic Phytoplankton

d

And this brings us back to bioavailability:• Problem reconciling Sunda’s Droop style experiments

with new evidence for a iron reductase system• Iron is a great example:

– Fe3+ (Fe’) uptake– FeL reduction (eukaryotic phytoplankton)– Fe-siderophore uptake (DFB)– Other mechanisms?

– EDTA = metal buffer in culture– DFB = siderophore– L = natural ligands measured

by electrochemistry

Shaked, Kustka and Morel 2005 L&O

From Shaked, Kustka, and Morel, L&O 2005A General Kinetic Model for Iron

Acquisition by Eukaryotic Phytoplankton 22

• From Hudson and Morel, 1990 L&O24

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Intracellular Storage of Metals – Metal Quotas are very “plastic”Monod growth does not include storage, Droop equation does

Most of our approach is Monod, Droop is better

How do you parameterize colimitation?Type I: Independent Nutrient Colimitation

-14-13

-12-11

-10

-14

-13

-12

-11

-100

0.1

0.2

0.3

0.4

0.5

S1 (mol L-1)S2 (mol L-1)

Gro

wth

Rat

e (d

-1)

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To be shamelessly anthropomorphic: what do the bugs really feel?

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By Quota

By Dilution

Closed=LiebigOpen=Multiplicative

Environmental Bioinorganic Chemistry’s subconscious goal: Finding a crucial pinprick in a biogeochemical cycle

Superoxide dismutase Carbonic anhydrase Nitrogenase

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Saito et al, Limnology and Oceanography, 2002

(Data for E. huxleyi from Sunda and Huntsman 1995)

0.00

0.25

0.50

0.75

1.00

1.25

-8-9

-10-11

-12-13

-14

-10-11

-12-13

-14

Gro

wth

Rat

e (d

-1)

log

Zn' (M

)

log Co' (M)

Emiliania huxleyi

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

-11

-12

-13-11-12

-13

Gro

wth

Rat

e (d

-1)

log

Zn'

(M)

log Co' (M)

Prochlorococcus

Differing cobalt requirements in marine phytoplanktonDifferent biochemistries, biochemical substitution aka cambialism

Cobalt, cadmium and zinc – A trace metal trio: Biochemical substitution in diatoms but not the cyanobacteria

Isn’t this colimitation too??

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We’ll call it “Type II Biochemical Substitution” For critics of colimitation, this is a completely different type, removed from Leibig debate

How do you parameterize biochemical substitution as a colimitation?

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But perfect and complete substitution is rarely the reality…

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• What if one of those metalloenzymes affects the ability to take up another nutrient?

• Lots of examples: carbonic anhydrases (Zn, Co, Cd) are involved in C acquisition, Urease contains nickel, nitrogenase contains Fe and Mo (or V)

• (Metals are really useful in enzymes)

We’ll call that Type III Biochemically Dependent Colimitation

The equation doesn’t work if S=0, but in trace metal analytical chemistrywe don’t believe in zero… we can always measure lower ;)

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Connecting bioinorganic chemistry to the concept of colimitation

Type I. Colimitation Between Two Independent Nutrients

Type II. Colimitation with Biochemical Substitution

Type III. Colimitation BiochemicallyDependent Nutrients

Type 0.

No Colimitation

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Nutrient co-limitation pairs in the marine environment

Nutrient Couple Co-Limitation Type Zinc and Cobalt (Cyanobacteria) 0 or I Only one nutrient/ IndependentNitrogen and Phosphorus I IndependentNitrogen and Light I IndependentNitrogen and Carbon I IndependentIron and Cobalt I IndependentIron and Zinc I IndependentIron and Phosphorus I IndependentIron and Vitamin B12 I IndependentLight and Iron I IndependentZinc and Cobalt (Eukaryotic Phytoplankton) II Biochemical substitution (CA)*Zinc and Cadmium (Diatoms) II Biochemical substitution (CA)*Copper and Zinc II Biochemical substitution (SOD)*Zinc and Cobalt (hypothesized) II Biochemical substitution (AP)*Zinc and Phosphorus III Dependent (AP)* Cobalt and Phosphorus III Dependent (AP)*Zinc and Carbon III Dependent (CA)*Cobalt and Carbon III Dependent (CA)*Cadmium and Carbon III Dependent (CA)*Iron and Copper III Dependent (FRE and MCO)*Iron and Nitrogen (N2 fixation) III Dependent (NIF)*Molybdenum and Nitrogen (N2 fixation) III Dependent (NIF)*Nickel and Urea (Nitrogen) III Dependent (Urease)

(Saito, Goepfert and Ritt, 2008)

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-14-13

-12-11

-10

-14

-13

-12

-11

-100

0.1

0.2

0.3

0.4

0.5

S1 (mol L-1)S2 (mol L-1)

Gro

wth

Rat

e (d

-1)

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Nutrient co-limitation pairs in the marine environment

Nutrient Couple Co-Limitation Type Zinc and Cobalt (Cyanobacteria) 0 or I Only one nutrient/ IndependentNitrogen and Phosphorus I IndependentNitrogen and Light I IndependentNitrogen and Carbon I IndependentIron and Cobalt I IndependentIron and Zinc I IndependentIron and Phosphorus I IndependentIron and Vitamin B12 I IndependentLight and Iron I IndependentZinc and Cobalt (Eukaryotic Phytoplankton) II Biochemical substitution (CA)*Zinc and Cadmium (Diatoms) II Biochemical substitution (CA)*Copper and Zinc II Biochemical substitution (SOD)*Zinc and Cobalt (hypothesized) II Biochemical substitution (AP)*Zinc and Phosphorus III Dependent (AP)* Cobalt and Phosphorus III Dependent (AP)*Zinc and Carbon III Dependent (CA)*Cobalt and Carbon III Dependent (CA)*Cadmium and Carbon III Dependent (CA)*Iron and Copper III Dependent (FRE and MCO)*Iron and Nitrogen (N2 fixation) III Dependent (NIF)*Molybdenum and Nitrogen (N2 fixation) III Dependent (NIF)*Nickel and Urea (Nitrogen) III Dependent (Urease)

(Saito, Goepfert and Ritt, 2008)

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• A real (complicated) example: Bioavailability• Another real (complicated) example: Fe – light colimitation• Another real (complicated) example: Fe acquisition systems



Are the oceans are poised for colimitation ?

PAPPE:

Potential autotrophic production per element

SW concentration (mol X/L) x Extended Redfield (mol C/mol X) = mol POC/L from X

What seawater concentration should be used?

What chemical form (or “species”) should be used? Most nutrients are now known to have interactions with organic (carbon-based) molecules.

Extended Redfield Ratio – Warning: be very wary of many bad papers on this topic– A Quota DOES NOT EQUAL a biochemical requirement

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Gedanken experiment

Is the inorganic or organic form bioavailable?

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Gedanken experiment

Is the inorganic or organic form bioavailable?Red = “eukaryotic” phytoplanktonGreen = non-diazotrophic cyanobacteria Yellow + Green = diazotroph cyanobacteria

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Small cell size allows increased nutrient acquisition efficiency through increased surface to volume ratios

T. weissfloggiiV= 1370 m3

A= 598 m2

SA:V = 0.44

T. oceanicaV= 171 m3

A=145 m2

SA:V = 0.85

But if you can grow on ML instead of M’ that’s a game changer (ML>>M’), Remember the limits of diffusion can control M’ uptake eventually…

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What does “bioavailable” really mean?

• Chemical species dependent• Biological species dependent

• What are the energetic costs associated with utilization of organic forms of nutrients (higher, but how significant is that on fitness relative to the community?)

• Perhaps best considered as essentially a kinetic term (Morel, Allen, Saito, 2003)– ZnL dissociation is going to be much faster than CoL dissociation with conditional

stability constants of 109-1011 for Zn and >1016.8 for Co

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The problem of studying colimitation

• To understand colimitation we need to understand

– Natural concentrations of nutrients– Bioinorganic chemistry of key

biochemistries– Bioavailability of all nutrients

Field incubation experiments are like sledgehammers, powerful enough to detect primary limitation, but likely not nimble enough to detect colimitation?

- need molecular diagnostics

- carbonic anhydrase as an example

- iron bioavailability as an example

Station 4 Bottle Incubation #2

Days

0 1 2 3 4 5

Chl

orop

hyll

g/L

0

1

2

3

4

5

6

7

Control +500pM Cobalt+500pM Co, +2nM Fe+2nM Fe+500pM Cobalt Rep

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Light and Iron Type I or Type III?

Moore et al ecosystem model treated a Type IMultiplicative = independent

But Fe is directly involved in light acquisition (PS I is very iron intensive) = Type III biochemically dependent (reducing Km of other nutrient)

And worse, light is involved in iron uptake, through supply for energy for the iron reductase system = Type III biochemically dependent

Can you have a two-way Type III dependence?

Have to look at the available data carefully. Even the best experiments have woefully low resolution for Type I/III interpretations.

Maldonado 1999 Colimitation of iron and light in the North Pacific only 6 points on the 3D plot.

Iron uptake occurs in dark48

To understand colimitation we need to understand:1. Natural concentrations of nutrients2. Bioinorganic chemistry of key biochemistries3. Bioavailability of all nutrients

“One is driven to the conclusion that the biochemical details of uptake and utilization of the various nutrients have very little bearing on the appearance of the kinetic relationship between substrate concentration and growth.” He continues that: “The burden of this argument holds some comfort for ecologists, for it suggests that they may be spared the necessity of becoming biochemists in addition to being mathematicians (Droop 1974).”

Discussion questions:1. Do you believe Type I multiplicative or Type I Leibig is correct?2. What kind of colimitation is iron-light?3. How difficult would it be to switch this whole discussion over to a Droop

based system instead of a Monod?4. What do you think about Droop’s comment about natural communities as

“an envelope” which to model collectively?

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12.755 lecture 3/4 cellular uptake of trace elements by microbes

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