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)

1. Journal Club
2016/01/18
Kazuya Horibe
Paper A
Paper B

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. 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. Oscillations in biofilm growth:
an example of Bacillus subtilis
Nature 523, 550–554 (2015)

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. 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. 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. (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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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?

33. 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)

34. 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

35. 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.

36. 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.

37. 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

38. 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.

39. 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

40. 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)

41. 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.

42. 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)