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Metabolic co-dependence gives rise to collective oscillations within biofilms.

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160118 journal club
論文紹介
バクテリアが集まって作るBaiofilm内でバクテリアが長距離コミュニケーションする話
Metabolic co-dependence gives rise to collective oscillations within biofilms. Nature 523, 550–554 (2015)
Microbiology: Electrical signalling goes bacterial. Nature 527, 44–45 (2015)
Ion channels enable electrical communication in bacterial communities. Nature 527, 59–63 (2015)

Published in: Science
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Metabolic co-dependence gives rise to collective oscillations within biofilms.

  1. 1. Journal Club 2016/01/18 Kazuya Horibe Paper A Paper B
  2. 2. Why did I choose these papers? Swarm Intelligence(= the self-organized system, natural or artificial) Emergence of global behavior How swarm dynamics is organized? Interaction between individuals How individuals in communities interact? Division of labor and production of common goods Competition with each other for limited resources How individuals in communities increase the overall fitness of the population? ADVANCESIN PHYSICS,2000, VOL.49, NO.4, 395 554 PLoS Comput. Biol. 11 (8) (2015) e1004273 Proc. Natl Acad. Sci. USA 112, 4690–4695 (2015)
  3. 3. The life cycle of biofilm Nature Reviews Microbiology 11, 157-168 (2013) A biofilm is a group of microbe which stick to each other on a surface. Cells in a biofilm are embedded within a self-produced matrix of extracellular polymeric substance. Bacteria scale; 1μm Biofilm scale; 100μm~
  4. 4. Oscillations in biofilm growth: an example of Bacillus subtilis Nature 523, 550–554 (2015)
  5. 5. Schematic summary (of the two papers) YugO; potassium ion chanel c. The signal propagation reduces the uptake of glutamate in exterior cells. The cycle is reset when interior cells are not starved. Nature 527, 44–45 (2015) a. When exterior cells take up glutamates, interior cells become starved. Nutrient-stress cases to secrete K+ . b. The release of K+ changes the transmembrane voltage of cells and leads to the subsequent release of K+ from neighbouring cells. Paper A, Fig1a,b Paper A, Fig4a Trade off(Protection and Nutrient access) Metabolic co-dependence Resilience to external attack Paper A, Fig3a,b How B.subtilis biofilms grow in periodic cycles once the colony reaches a threshold size? How the metabolic state of cells is communicated over long distances?
  6. 6. Is the oscillations depend on cell replication or growth? PaperA,Fig1 the average cell replication time=3.4 0.2 h the average period of oscillations= 2.5 0.8 h Oscillations arise during biofilm formation(cell growth). the diameter at which a colony initiates oscillations = 576 85 μm
  7. 7. Media cell membrane Which nutrient conditions cause oscillations in biofilm growth? GDH; glutamate dehydrogenase GS; glutamine synthetase PnasA; the promoter activated upon glu limitation (1) Which of these substrates could be responsible for the observed glutamine limitation? (2) Whether interior or peripheral cells exhibited changes in growth? biofilms under nutrient-limited conditions cell growth is controlled by metabolism →Carbon, Nitrogen? Addition of exogenous glutamine eliminated periodic halting of biofilm growth.
  8. 8. (1)Which substrates could be responsible for the glutamine limitation? (2)Which cells exhibited changes in growth? Paper A, Fig2 Paper A, FigS4 How peripheral cells could experience periodic ammonium limitation despite a constant supply of glutamate in the media? (1) Critical substrate is ammonium. (2) periodic reduction in the growth of peripheral cells Media cell membrane
  9. 9. Mathematical model for metabolic co-dependence Paper A, Fig2a,d Media Flow Ammonium limitation for peripheral cells may arise due to glutamate limitation for interior cells. Main assumptions (1) Consumption of glutamate during growth of peripheral cells deprives interior cells of this nutrient and thus inhibits ammonium production in the biofilm interior. (2) The growth of peripheral cells depends predominantly on ammonium that is produced by metabolically stressed interior cells. modeling Ammonium ion can cross the cell membrane and be lost to the extracellular media.(Arch. Microbiol. 139, 245–247 (1984)) ? Separation cells(interior and peripheral) Two subpopulations depends on nutrient availability. Paper A, Fig3a,b
  10. 10. A; the concentration of ammonium G; the concentration of glutamate in the biofilm interior H; the concentration of active glutamate dehydrogenase r; the rate of biomass production (Metabolic condition) ρ; the cell density μ; the growth rate of biofilm i→interior, p→peripheral
  11. 11. A; the concentration of ammonium G; the concentration of glutamate in the biofilm interior H; the concentration of active glutamate dehydrogenase r; the rate of biomass production (Metabolic condition) ρ; the cell density μ; the growth rate of biofilm
  12. 12. A; the concentration of ammonium G; the concentration of glutamate in the biofilm interior H; the concentration of active glutamate dehydrogenase r; the rate of biomass production (Metabolic condition) ρ; the cell density μ; the growth rate of biofilm
  13. 13. A; the concentration of ammonium G; the concentration of glutamate in the biofilm interior H; the concentration of active glutamate dehydrogenase r; the rate of biomass production (Metabolic condition) ρ; the cell density μ; the growth rate of biofilm
  14. 14. A; the concentration of ammonium G; the concentration of glutamate in the biofilm interior H; the concentration of active glutamate dehydrogenase r; the rate of biomass production (Metabolic condition) ρ; the cell density μ; the growth rate of biofilm
  15. 15. The simple model accounted for experimental observations. Paper A Fig3c-h(model) Paper A, Fig2(observation) Growth rate of Interior cells oscillates, not periphery Critical substrate is ammonium.
  16. 16. Why peripheral cells do not increase intracellular production? GDH overexpression - stopped growth oscillations. - resulted in high levels of cell death in the colony interior. The Biofilm can regenerate itself in an external attack by metabolic co-dependence.
  17. 17. Schematic summary (of the two papers) YugO; potassium ion chanel c. The signal propagation reduces the uptake of glutamate in exterior cells. The cycle is reset when interior cells are not starved. Nature 527, 44–45 (2015) a. When exterior cells take up glutamates, interior cells become starved. Nutrient-stress cases to secrete K+ . b. The release of K+ changes the transmembrane voltage of cells and leads to the subsequent release of K+ from neighbouring cells. Paper A, Fig1a,b Paper A, Fig4a Trade off(Protection and Nutrient access) Metabolic co-dependence Resilience to external attack Paper A, Fig3a,b How B.subtilis biofilms grow in periodic cycles once the colony reaches a threshold size? How the metabolic state of cells is communicated over long distances?
  18. 18. Communication through electrical signaling (Intro Paper B) Glutamate (Glu−) and ammonium (NH4+) are both charged metabolites, whose respective uptake and retention is known to depend on the transmembrane electrical potential and proton motive force.(J. Bacteriol. 177, 2863–2869 (1995)) FEMS Microbiol. Rev. 29, 961–985 (2005) The role of ion channels in bacteria has remained unclear. Proc. Natl Acad. Sci. USA 109, 18891–18896 (2012) Biofilms can exhibit fascinating macroscopic spatial coordination.
  19. 19. Membrane potential oscillates in the biofilm growth. Paper B Fig1 ThT; Membrane potential PaperB FigS1d We focus on potassium because this ion is the most abundant cation in all living cells and has been implicated to have a role in biofilm formation.
  20. 20. Extracellular potassium has a role in the synchronized oscillations in membrane potential. ThT; Membrane potential APG-4; Extracellular potassium ANG-2; Extracellular sodium Paper B FigS2
  21. 21. Oscillations in membrane potential were driven by flow of potassium across the cell membrane. ThT; Membrane potential APG-4; Extracellular potassium ANG-2; Extracellular sodium Paper B Fig2 300mM KCl; matching the intracellular potassium concentration
  22. 22. Active and extracellular propagation of potassium signal: speed and intensity The signal travels at a constant rate of propagation. The amplitude of the signal does not decay with distance travelled. paper B Fig2 f,g,h paper B FigS3 c,d L = v*t L; distance v; velocity t; time
  23. 23. The molecular mechanism of signal propagation Since glutamate limitation is known to drive the underlying metabolic oscillations, we anticipated that transient removal of glutamate could initiate potassium release. (1)Glutamate limitation can trigger the potassium signal via the YugO potassium channel. YugO; potassium channel in B. subtilis TrkA; gate domain of YugO kCl shock; transient bursts of external potassium (300 mM KCl) paper B Fig S4a paper B Fig 3a paper B Fig 3c (2)YugO appears to have a role in propagating the extracellular potassium signal within the biofilm. paper B Fig 3b (1) (2) (3) (3)Glutamate transport capacity ∝ The proton motive force J. Bacteriol. 177, 2863–2869 (1995) FEBS Lett. 585, 23–28 (2011) Nature Rev. Microbiol. 9, 330–343 (2011)
  24. 24. Mathematical modeling of electric signaling V; the membrane potential n; channel open during a fraction of time S; the concentration of stress-related metabolic products E; the excess extracellular potassium concentration T; the ThT concentration Nature 527, 59–63 (2015)Supplementary Information
  25. 25. Mathematical modeling of electric signaling V; the membrane potential(Hoggkin-Huxley model) n; channel open during a fraction of time S; the concentration of stress-related metabolic products E; the excess extracellular potassium concentration T; the ThT concentration Nature 527, 59–63 (2015)Supplementary Information
  26. 26. Mathematical modeling of electric signaling V; the membrane potential n; channel open during a fraction of time S; the concentration of stress-related metabolic products E; the excess extracellular potassium concentration T; the ThT concentration Nature 527, 59–63 (2015)Supplementary Information
  27. 27. Mathematical modeling of electric signaling V; the membrane potential n; channel open during a fraction of time S; the concentration of stress-related metabolic products E; the excess extracellular potassium concentration T; the ThT concentration Nature 527, 59–63 (2015)Supplementary Information
  28. 28. Mathematical modeling of electric signaling V; the membrane potential n; channel open during a fraction of time S; the concentration of stress-related metabolic products E; the excess extracellular potassium concentration T; the ThT concentration Nature 527, 59–63 (2015)Supplementary Information
  29. 29. Mathematical modeling of electric signaling V; the membrane potential n; channel open during a fraction of time S; the concentration of stress-related metabolic products E; the excess extracellular potassium concentration T; the ThT concentration Nature 527, 59–63 (2015)Supplementary Information
  30. 30. Mathematical modeling of electric signaling V; the membrane potential n; channel open during a fraction of time S; the concentration of stress-related metabolic products E; the excess extracellular potassium concentration T; the ThT concentration Nature 527, 59–63 (2015)Supplementary Information
  31. 31. Schematic summary (of the two papers) YugO; potassium ion chanel c. The signal propagation reduces the uptake of glutamate in exterior cells. The cycle is reset when interior cells are not starved. Nature 527, 44–45 (2015) a. When exterior cells take up glutamates, interior cells become starved. Nutrient-stress cases to secrete K+ . b. The release of K+ changes the transmembrane voltage of cells and leads to the subsequent release of K+ from neighbouring cells. Paper A, Fig1a,b Paper A, Fig4a Trade off(Protection and Nutrient access) Metabolic co-dependence Resilience to external attack Paper A, Fig3a,b How B.subtilis biofilms grow in periodic cycles once the colony reaches a threshold size? How the metabolic state of cells is communicated over long distances?
  32. 32. Biofilm growth (Intro Paper A) Proc. Natl Acad. Sci. USA 98, 11621–11626 (2001) Proc. Natl Acad. Sci. USA 109, 18891–18896 (2012) Natl Acad. Sci. USA 110, 848–852 (2013) Proc. Natl Acad. Sci. USA 111, 18013–18018 (2014)
  33. 33. A; the concentration of ammonium G; the concentration of glutamate in the biofilm interior H; the concentration of active glutamate dehydrogenase r; the rate of biomass production (the concentrations of housekeeping proteins) ρ; the cell density μ; the growth rate of biofilm Assumptions
  34. 34. Schematic summary (of the two papers) YugO; potassium ion chanel Paper A, Fig1a,b Paper A, Fig3a,b Paper B, Fig1d Paper B, Fig2c c. The signal propagation reduces the uptake of glutamate in exterior cells. The cycle is reset when interior cells are not starved. Nature 527, 44–45 (2015) a. When exterior cells take up glutamates, interior cells become starved. Nutrient-stress cases to secrete K+ . b. The release of K+ changes the transmembrane voltage of cells and leads to the subsequent release of K+ from neighbouring cells.
  35. 35. Question and Result How the metabolic state of cells is communicated over long distances? Maintenance of the proper intracellular concentrations of glutamate and ammonium depends on the electrical potential across the cell membrane. Membrane potential depends on sodium or potassium ion. (Paper B) How Bacillus subtilis biofilms grow in periodic cycles once the colony reaches a threshold size? These oscillations arise when the cells in the biofilm‘s interior become deprived of glutamate, owing to high consumption of the amino acid by peripheral cells. (Paper A) →metabolic states(biofilm oscillation) depend on sodium or potassium ion. (Paper B) →The first example of a bacterial potassium channel that functions in a signaling role, through long-range coordination of metabolic oscillations.
  36. 36. Question and Result Maintenance of the proper intracellular concentrations of glutamate and ammonium depends on the electrical potential across the cell membrane.(J. Bacteriol. 177, 2863–2869 (1995)) Membrane potential depends on potassium ion. (Paper B) How Bacillus subtilis biofilms grow in periodic cycles once the colony reaches a threshold size? →Metabolic states(biofilm oscillation) depend on potassium ion. (Paper A, Paper B) These oscillations arise when the cells in the biofilm’s interior become deprived of glutamate, owing to high consumption of the amino acid by peripheral cells. (Paper A) How the metabolic state of cells is communicated over long distances(about 200μm)? Question Result Paper A, Fig3a,b Paper B, Fig1d
  37. 37. Media cell membrane Which nutrient conditions cause oscillations in biofilm growth? GDH; glutamate dehydrogenase GS; glutamine synthetase PnasA; the promoter activated upon glu limitation (1) Which of these substrates could be responsible for the observed glutamine limitation? (2) Whether interior or peripheral cells exhibited changes in growth? biofilms under nutrient-limited conditions cell growth is controlled by metabolism →Carbon, Nitrogen? PnasA is the index of glutamine limitation. Addition of exogenous glutamine eliminated periodic halting of biofilm growth.
  38. 38. Oscillations in membrane potential were driven by flow of potassium across the cell membrane. ThT; Membrane potential APG-4; Extracellular potassium ANG-2; Extracellular sodium Paper B FigS3Paper B Fig2 300mM KCl; matching the intracellular potassium concentration Valinomycin; an antibiotic that creates potassium-specific carriers in the cellular membrane
  39. 39. Potassium channel is necessary for long range membrane potential propagation. paper B Fig3 e,f,g YugO channel gating appears to promote efficient electrical communication between long distant cells. model(show later)
  40. 40. Conclusion (of the two papers) YugO; potassium ion chanel Nature 527, 44–45 (2015) a. When peripheral cells take up most of the available glutamate, the interior cells become starved. Nutrient-stressed interior cells secrete potassium ions (K+ ) through the YugO K+ channel. b. The release of K+ ions then changes the trans membrane voltage of cells and leads to the subsequent release of K+ ions from neighboring cells, propagating the starvation signal. c. The signal propagation ultimately reduces the uptake of glutamate in peripheral cells. Glutamate becomes available for interior cells to consume and the cycle is reset.
  41. 41. refarence Metabolic co-dependence gives rise to collective oscillations within biofilms. Nature 523, 550– 554 (2015) Microbiology: Electrical signalling goes bacterial. Nature 527, 44–45 (2015) Ion channels enable electrical communication in bacterial communities. Nature 527, 59–63 (2015)

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