View
2
Download
0
Category
Preview:
Citation preview
Tight regulation of electrically-charged
substrate transport in bacteria
Emory University
Minsu Kim
Three famous quotes
• An anonymous philosopher once said
– “Life is nothing but a dream”
Three famous quotes
• An anonymous philosopher once said
– “Life is nothing but a dream”
• Nobel prize winner Albert Szent-Györgyi said
– "Life is nothing but an electron looking for a place
to rest".
Three famous quotes
• An anonymous philosopher once said
– “Life is nothing but a dream”
• Nobel prize winner Albert Szent-Györgyi said
– "Life is nothing but an electron looking for a place
to rest".
• Nobel prize winner François Jacob said
– “the dream of every cell is to become two cells.”
Three famous quotes
• An anonymous philosopher once said
– “Life is nothing but a dream”
• Nobel prize winner Albert Szent-Györgyi said
– "Life is nothing but an electron looking for a place
to rest".
• Nobel prize winner François Jacob said
– “the dream of every cell is to become two cells.”
I will talk about one problem related to electric
charge when a cell strives to achieve its dream.
What does it take for one cell to become
two cells?
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
Carbon and nitrogen uptake
C N
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
Carbon and nitrogen uptake
C N
amino
acidprotein
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
Carbon and nitrogen uptake
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
C N
amino
acidprotein Biomass
Carbon and nitrogen uptake is important
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
C N
amino
acidprotein Biomass
Carbon and nitrogen uptake is important
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
C N
amino
acidprotein Biomass
Carbon uptake and metabolism have been extensively characterized.
However, much less is known about nitrogen uptake
C N
amino
acidprotein Biomass
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
However, much less is known about nitrogen uptake
C N
amino
acidprotein Biomass
Nitrogen uptake is peculiar.
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
Nitrogen metabolism and regulation
NH4+/NH3
C N
amino
acid
NH4+ H+ +NH3, [NH4
+]/[NH3]~180 at pH 7
protein Biomass
Biomass of a bacterial cell: ~50% Carbon, ~15% Nitrogen . .
NH3 passive diffusion can limit growth
NH3 NH4+
NH3 is very permeable to membrane (~30 times more than H2O)
NH3 passive diffusion can limit growth
NH3 NH4+
NH3 is very permeable to membrane (~30 times more than H2O)
NH3
NH3 passive diffusion can limit growth
NH3 NH4+
NH3 is very permeable to membrane (~30 times more than H2O)
NH3
NH3 passive diffusion can limit growth
NH3 NH4+
NH3 is very permeable to membrane (~30 times more than H2O)
BiomassNH3 NH4+
NH3 passive diffusion can limit growth
NH3 NH4+
NH3 is very permeable to membrane (~30 times more than H2O)
BiomassNH3 NH4+
NH3 passive diffusion can limit growth
AmtB transports NH4+
NH3 NH4+
BiomassNH3 NH4+
AmtB
NH3 is very permeable to membrane (~30 times more than H2O)
AmtB transports and concentrates NH4+ internally
Concentrate?
NH4+
NH4+
AmtB
NH3 is very permeable to membrane.
Concentrate?
NH4+
NH4+NH3H+ +
AmtB
NH3 is very permeable to membrane.
Concentrate?
NH3
NH4+
NH4+NH3H+ +
AmtB
NH3 is very permeable to membrane.
Concentrate?
NH3
NH4+
NH4+NH3H+ +
AmtB
NH3 is very permeable to membrane.
AmtB may be harmful
NH3 is very permeable to membrane.
NH4+
NH3 leaky diffusion
Acidification
or
ATP drainage
Challenge
NH3
NH4+
NH4+NH3H+ +
AmtB
AmtB may be harmful
NH3 is very permeable to membrane.
NH4+
NH3 leaky diffusion
ATP
NH3
NH4+
NH4+NH3H+ +
AmtB
How much NH4+ should AmtB transport?
How does E. coli regulate it?
Microfluidic chemostat
Microfluidic chamberE. coli NCM3722, in Rich Defined Medium +0.5% glycerol
Channel (Flow of fresh medium)
1 m
1000 m
100 m
Capillary0.6m
Nutrient exchange rate: ~40s
Time
[NH4+ /NH3]
Channel (Flow of fresh medium)
101
102
103
104
0.0
0.2
0.4
0.6
NH4
+ (M)
Gro
wth
rat
e h
r
Growth rate with and without AmtB
E. coli K-12 NCM3722 in Neidhardt’s
MOPS medium with 0.4% of glyc with
various ammonium concentrations.
: WT
: amtB
101
102
103
104
0.0
0.2
0.4
0.6
NH4
+ (M)
Gro
wth
rat
e h
r
AmtB promoter-GFP
E. coli K-12 NCM3722 in Neidhardt’s
MOPS medium with 0.4% of glyc with
various ammonium concentrations.
: WT
: amtB
101
102
103
104
1
10
100
NH4
+ (M)
Am
tB P
rom
ote
r-G
FP
(a.
u.)
: WT
: amtB
101
102
103
104
1
10
100
NH4
+ (M)
Am
tB P
rom
ote
r-G
FP
(a.
u.) 10
110
210
310
40.0
0.2
0.4
0.6
NH4
+ (M)
Gro
wth
rat
e h
r
AmtB promoter-GFP
E. coli K-12 NCM3722 in Neidhardt’s
MOPS medium with 0.4% of glyc with
various ammonium concentrations.
: WT
: amtB
AmtB promoter activities deviate for the two strains.
: WT
: amtB
101
102
103
104
1
10
100
NH4
+ (M)
Am
tB P
rom
ote
r-G
FP
(a.
u.) 10
110
210
310
40.0
0.2
0.4
0.6
NH4
+ (M)
Gro
wth
rat
e h
r
AmtB promoter-GFP
E. coli K-12 NCM3722 in Neidhardt’s
MOPS medium with 0.4% of glyc with
various ammonium concentrations.
: WT
: amtB
AmtB promoter activities deviate for the two strains.
AmtB promoter
Internal NH4+ (via Gln)
: WT
: amtB
Quantitative analysis
+ ext + int
0 4 Δ 4 Δ ([NH ] -[NH ] ) (1)N C P
0N )][NH]([NH int
3
ext
3 P
C: conversion factor
N0 = # of Nitrogen per cell
= Growth rateP: membrane permeability
N assimilation into biomass = NH3 diffusion
NH3amtB
Quantitative analysis
+ ext + int
0 Δ 4 Δ 4 Δ ([NH ] -[NH ] ) (1)N C P
0N )][NH]([NHP int
3
ext
3
C: conversion factor
P: membrane permeability
N assimilation into biomass = NH3 diffusion
NH3amtB
+ ext + int
0 4 wt 4 wt AmtB ([NH ] -[NH ] ) (2)wtN C P J
WT
N assimilation into biomass = NH3 diffusion + NH4+ transport
N0 = # of Nitrogen per cell
= Growth rate
Quantitative analysis
+ ext + int
0 Δ 4 Δ 4 Δ ([NH ] -[NH ] ) (1)N C P
amtB
+ ext + int
0 4 wt 4 wt AmtB ([NH ] -[NH ] ) (2)wtN C P J
WTWalter and Gutknecht(1986)
P=0.13cm/sec
[NH4+]int and JAmtB
For 65min DT growth
1
10
100
[NH
+
4]in
t (
M)
10 100-10
0
10
[NH+
4]
ext (M)
N f
lux (
M/h
r)
: WT
: amtB
NH3 leaky flux
NH4+ flux through AmtB
1
10
100
[NH
+
4]in
t (
M)
10 1000.0
0.2
0.4
0.6
[NH+
4]
int (M)
h
r
10 100-10
0
10
[NH+
4]
ext (M)
N f
lux (
M/h
r)
[NH4+]int and JAmtB
: WT
: amtB
N*
N*
NH3 leaky flux
NH4+ flux through AmtB
1
10
100
[NH
+
4]in
t (
M)
10 1000.0
0.2
0.4
0.6
[NH+
4]
int (M)
h
r
[NH4+]int and JAmtB
: WT
: amtB
N*
Precise
10 1000
5
10
N f
lux (
M/h
r) [NH
+
4]
int (M)
N*
10 100-10
0
10
[NH+
4]
ext (M)
N f
lux (
M/h
r)
NH3 leaky flux
NH4+ flux through
AmtB
Sensitive
AmtB is regulated very sensitively so that internal ammonium is maintained
precisely at the minimal level needed for the optimal growth.
NH4+ flux through AmtB
[NH4+]int and JAmtB
NH4+
NH3 leaky diffusion
1
10
100
[NH
+
4]in
t (
M)
N*
Precise
10 100-10
0
10
[NH+
4]
ext (M)
N f
lux (
M/h
r)
NH3 leaky flux
: WT
: amtB
NH4+ flux through AmtB
[NH4+]int and JAmtB
N*
NH4+
NH3 leaky diffusion
1
10
100
[NH
+
4]in
t (
M)
N*
Precise
10 100-10
0
10
[NH+
4]
ext (M)
N f
lux (
M/h
r)
NH3 leaky flux
: WT
: amtB
NH4+ flux through AmtB
[NH4+]int and JAmtB
N*
NH4+
1
10
100
[NH
+
4]in
t (
M)
N*
Precise
10 100-10
0
10
[NH+
4]
ext (M)
N f
lux (
M/h
r)
NH3 leaky flux
: WT
: amtB
NH4+ flux through AmtB
NH4+
NH3 leaky diffusion
Putting together known interactions
aKG : alpha-ketoglutarate.
M. Merrick(2010)
aKG: a nitrogen carrier
biomass N assimilation[aKG]d
J Jdt
biomass N assimilation[aKG] J J dt
c
aKG: a nitrogen carrier
biomass N assimilation[aKG]d
J Jdt
biomass N assimilation[aKG] J J dt
External ammonium Leaky diffusion
N*
NH4+
NH3 leaky diffusion
N*
NH4+
NH3 leaky diffusion
External ammonium Leaky diffusion
Leaky diffusion [NH4+]int
N*
NH4+
NH3 leaky diffusion
[aKG] AmtB
N*
[aKG] J biomass - JN assimilation dt NH4
+
NH3 leaky diffusion
AmtB [NH4+]int
N*
[aKG] J biomass - JN assimilation dt NH4
+
NH3 leaky diffusion
AmtB [NH4+]int
N*
[aKG] J biomass - dt NH4+
NH3 leaky diffusion
JN assimilation
AmtB [NH4+]int
N*
[aKG] J biomass - dt NH4+
NH3 leaky diffusion
JN assimilation
1
10
100
[NH
+
4]in
t (
M) : WT
: amtB
N*
[NH4+]ext (M)
Negative integral feedback
N*
[aKG] J biomass - dt NH4+
NH3 leaky diffusion
Amplify signal
JN assimilation
Negative integral feedback
N*
[aKG] J biomass - dt NH4+
NH3 leaky diffusion
Amplify signal
JN assimilation
10 1000
5
10
N f
lux (
M/h
r)
[NH+
4]
int (M)
NH4+ flux through
AmtB
Negative integral feedback
Negative integral feedback
Integral feedback control
• Integral feedback control : Common engineering scheme that
allows a system to track a desired set-point robustly.
– e.g. thermostat in the room, cruse control in a car.
• AmtB is regulated very sensitively so that internal ammonium
is maintained precisely at the minimal level needed for the
optimal growth.
Kim, M. et al. (2012) Mol. Syst. Biol. 8:616
Tribute to Sydney Kustu
Recommended