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

12.755 Lecture 3/4 Cellular 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

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

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  1. 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 2003 Sunda and Huntsman 2000 Saito et al., 2008 Background: Hudson and Morel 1990, Droop 1973, Sunda and Huntsman 1997 (iron light colimitation study); Anderson and Morel 1982, Droop Biography

  2. (Moore, Doney, and Lindsay, GBC 2004)

  3. Growth models Single 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/m’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

  4. [S] = substrate concentration Monod growth: Droop cellular quota: m/m’m = 1-kQ/Q m = r/Q Q = cellular quota r= uptake rate Km = half saturation constant kQ= saturation constant, value of Q when D (growth rate) = 0 mmax andm’m = maximum growth rate What happens when S is a small number? A large number? Likewise for Q?

  5. Monod • Droop Fe’ Dilution rate = growth rate in chemostat, Note when D=0 Q is not zero

  6. Measurements of cellular uptake and quotasHow to read a Sunda and Huntsman manuscript:(Sunda and Huntsman 1997, Nature) Same equation as: Q=r/m • 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

  7. A curious thing…Uptake is related to Surface Area for many different species and iron concentrations

  8. Diffusion limited uptake: Physical Limits on Uptake and Acclimation through synthesis of transporters r = 4prD[M’] r = 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

  9. Small cell size allows increased nutrient acquisition efficiency through increased surface to volume ratios T. weissfloggii V= 1370 mm3 A= 598 mm2 SA:V = 0.44 T. oceanica V= 171 mm3 A=145 mm2 SA:V = 0.85

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

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

  12. 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, 1978

  13. 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”

  14. 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?

  15. 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 2003 Sunda and Huntsman 2000 Saito et al., 2008 Background: Hudson and Morel 1990, Droop 1973, Sunda and Huntsman 1997 (iron light colimitation study); Anderson and Morel 1982, Droop Biography

  16. Competitive inhibition • Theoretically derived from enzyme kinetics with an “inhibitor” substrate • Kmapp = Km * (1+ [I]/KI) • v = vmax*S/(S + Km*(1+ [I]/KI))

  17. The often forgotten/overlooked • concept of biodilution • Droop cellular quota: • m/m’m = 1-kQ/Q • m = r/Q • = mQ

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

  19. 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. From Shaked, Kustka, and Morel, L&O 2005 A General Kinetic Model for Iron Acquisition by Eukaryotic Phytoplankton

  20. 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 2005 A General Kinetic Model for Iron Acquisition by Eukaryotic Phytoplankton

  21. From Hudson and Morel, 1990 L&O

  22. 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

  23. 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 2003 Sunda and Huntsman 2000 Saito et al., 2008 Background: Hudson and Morel 1990, Droop 1973, Sunda and Huntsman 1997 (iron light colimitation study); Anderson and Morel 1982, Droop Biography

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

  25. To be shamelessly anthropomorphic: what do the bugs really feel? By Quota Closed=Liebig Open=Multiplicative By Dilution

  26. Environmental Bioinorganic Chemistry’s subconscious goal: Finding a crucial pinprick in a biogeochemical cycle Superoxide dismutase Carbonic anhydrase Nitrogenase

  27. Differing cobalt requirements in marine phytoplankton Different biochemistries, biochemical substitution aka cambialism (Data for E. huxleyi from Sunda and Huntsman 1995) Cobalt, cadmium and zinc – A trace metal trio: Biochemical substitution in diatoms but not the cyanobacteria Isn’t this colimitation too?? Saito et al, Limnology and Oceanography, 2002

  28. We’ll call it “Type II Biochemical Substitution” For critics of colimitation, this is a completely different type, removed from Leibig debateHow do you parameterize biochemical substitution as a colimitation?

  29. But perfect and complete substitution is rarely the reality…

  30. 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 chemistry we don’t believe in zero… we can always measure lower ;)

  31. Connecting bioinorganic chemistry to the concept of colimitation Type I. Colimitation Between Two Independent Nutrients Type 0. No Colimitation Type III. Colimitation BiochemicallyDependent Nutrients Type II. Colimitation with Biochemical Substitution

  32. Nutrient co-limitation pairs in the marine environment Nutrient Couple Co-Limitation Type Zinc and Cobalt (Cyanobacteria) 0 or I Only one nutrient/ Independent Nitrogen and Phosphorus I Independent Nitrogen and Light I Independent Nitrogen and Carbon I Independent Iron and Cobalt I Independent Iron and Zinc I Independent Iron and Phosphorus I Independent Iron and Vitamin B12 I Independent Light and Iron I Independent Zinc 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)

  33. Nutrient co-limitation pairs in the marine environment Nutrient Couple Co-Limitation Type Zinc and Cobalt (Cyanobacteria) 0 or I Only one nutrient/ Independent Nitrogen and Phosphorus I Independent Nitrogen and Light I Independent Nitrogen and Carbon I Independent Iron and Cobalt I Independent Iron and Zinc I Independent Iron and Phosphorus I Independent Iron and Vitamin B12 I Independent Light and Iron I Independent Zinc 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)

  34. 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 2003 Sunda and Huntsman 2000 Saito et al., 2008 Background: Hudson and Morel 1990, Droop 1973, Sunda and Huntsman 1997 (iron light colimitation study); Anderson and Morel 1982, Droop Biography

  35. 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

  36. Gedanken experiment Is the inorganic or organic form bioavailable?

  37. Gedanken experiment Is the inorganic or organic form bioavailable? Red = “eukaryotic” phytoplankton Green = non-diazotrophic cyanobacteria Yellow + Green = diazotroph cyanobacteria

  38. 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… Small cell size allows increased nutrient acquisition efficiency through increased surface to volume ratios T. weissfloggii V= 1370 mm3 A= 598 mm2 SA:V = 0.44 T. oceanica V= 171 mm3 A=145 mm2 SA:V = 0.85

  39. 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

  40. 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

  41. Light and Iron Type I or Type III? Moore et al ecosystem model treated a Type I Multiplicative = 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 dark

  42. To understand colimitation we need to understand: • Natural concentrations of nutrients • Bioinorganic chemistry of key biochemistries • 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: • Do you believe Type I multiplicative or Type I Leibig is correct? • What kind of colimitation is iron-light? • How difficult would it be to switch this whole discussion over to a Droop based system instead of a Monod? • What do you think about Droop’s comment about natural communities as “an envelope” which to model collectively?

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