12.755 lecture 3/4 cellular uptake of trace elements by microbes major concepts growth eqns for...
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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
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?
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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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?
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 15
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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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
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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
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 26
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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• 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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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 – light colimitation• Another real (complicated) example: Fe acquisition systems
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 41
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?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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