Transcript Phenotypic variations in a monoclonal bacterial population

Phenotypic variations in a monoclonal
bacterial population
Oleg Krichevsky, Itzhak Fishov, Dina Raveh,
Ben-Gurion University, Beer-Sheva
J. Wong, D. Chatenay, M. Poirier, S. Ghozzi, J. Robert
Laboratoire Jean Perrin, FRE 3132 CNRS-UPMC
24 rue Lhomond, 75005 Paris
Escherichia Coli Bacteria
4 µm
Electronic microscopy
1 colony in phase contrast microscopy
Schematic bacterium
Membrane (glycolipide)
Cytoplasme (H2O+ions monovalents et divalents)
• Acides nucléiques (ADN, ARN)
• protéines (enzymes dont polymérases)
• small molecules(ribosome)
•Small numbers of molecules (par ex. 1 chromosome, 10-10000 ARNs; protéines)
•Dynamic enzymatic reaction: production, transformation,
degradation of the species with time.
Bacterium Biochemistry (simplified!)
1) Central dogma:
translation
ADN chromosome transcription
ARNm
ARNpolymérase
ribosome
2) DNA replication
ADNpolymérase, gyrase…
Protéine: un gène
Bacterial culture
Growth by division: 1 bacterium→2 daughter bacteria genetically identical (clone)
Duplication, repartition of the constituants (in particular of the chromosome)
t
Densité optique (600 nm)
1
0.1
0.01
0
50
100
temps(min)
150
200
Division time: 30’à 37°C in nutritive medium(pH~7, protéines, glucides)
Population/individual
•
Culture of a single colony in homogenous medium, obtain a monoclonal population
(typically: 1ml de medium grown 12 hours~108 bacteria).
•
J. Spudich et D. Koshland revealed the individual character of chemotactism. (Nature
262 1976)
•
Mutations don’t explain this individuality→ non geneticorigin.
(mutation rate: 10-10/pb/génération)
•
The authors invoked fluctuations of the small number of particle, of chemical rates
to explain those non genetic variability.
•
This process is more efficient than mutation to allow species adaptation to rapidly
fluctutating environnment.
Genetic expression network:
• ADNARNProtéine (fluorescente)
Example with a negative feedback loop:
Gène
ADN
Promoteur
Taux de transcription kR
ARN
Dégradation gR
Taux de traduction kP
Protéine
Dégradation gP
• Fluctuations. Network noise. Variability.
• Ozbudak et al.: origin of the protein noise expression:
transcription/translation Nature genetics 31 (2002).
• Elowitz et al.: Intrinsic noise(Fluctuations des éléments du
réseau)/extrinsic noise(fluctuations des autres composants de la cellule) Science 297
(2002).
• Influence of the regulation mechanism
DNA in bacterium
1 chromosome (4 Mpb)
N (1<n<300) plasmid copy number (entre 2 kpb et 100 kpb)
Plasmid
•
•
•
•
•
extrachromosomal DNA fragment
Code for its copy number (replicon sequence: ori, regulation)
Uses the host to replicate
Adds an advantage against otherwise toxic medium (Antibiotic resistance.)
Symbiotic plasmid/bacterium association
Partition system
Without partition system
With partition system
Plasmid copy number (PCN) inE. Coli
PCN=phenotype choice
Measured individual PCN on population scale(~104 individus)


Distribution : variability
Antibiotic resistance: adaptability
<n>
0.3
Nombre de cellules
0.25
0.2
s Standard deviation
0.15
0.1
0.05
0
-0.05
0
20
40
60
80
100
Nombre de Copie de Plasmide
120
Direct Visualisation directe of plasmids in bacteria:
G. Scott Gordon, Dmitry Sitnikov, Chris D. WebbOgden Aurelio Teleman, Aaron Straight, Richard
Losick, and Schaechter, Schaech Andrew W. Murray, and Andrew Wright, Cell 1997.
Fluorescent protein bounds to the plasmid sequence
Disadvantage: homologous recombinaison
Indirect Method
Fluorescent protein mOrange coded by the plasmid
[protein] plasmid copy number
Fluorescent intensity bacteriaPCN
•Expression copie unique sur le chromosome protéine verte.
•Fluorescent gene expression under IPTG inducible tac promoter.
Promoter choice
Promoter tac
fluorescent gene
Termination seq
RNA-polymerase
RNA-polymerase
LacI repressor→no transcription, no gene expression
IPTG LacI repressor titration→transcription, gene expression
Strong induced promoter: minimise expression noise (Elowitz et al.)
Measure the fluorescent intensity
Phase contrast
Fluorescent image
Measurement over a population~104, every individual at the
same developpment
Low level fluorescence
→Flow cytometry+fluorescent microscopy set up
Set up:
cell
optic
detection
Soft lithography microchannel
UV exposure
Mask
photosensitive resin
glass
develop, fix
Ready to use channel
Spread PDMS, bake at 90° C
Unmold, fix on a cover glass
Optical differentiel Interferometry profilometry image of the channel
(z=2µm)
Field of view: 10µm
Bacterial speed: ~0.1-1 mm/s
Optical elements detail
Time series of fluorescent intensity
FV
FO
FV = PV + AV
FO = aPO + AO + bPV
Fi: i channel measure of fluorescent intensity
Pi: i protein fluorescent intensity
Ai: i channel autofluorescent intensity
a: normalization constant between green and orange fluorescence
b: leak of green toward orange channel
Rq: a posteriori, no orange to green
Bacteria preparation
(E. Coli TOP10 strain)
1.
2.
3.
4.
5.
6.
Culture 37°C 12h of a clone picked on a petri dish
Dilution 500X, re culture→DO=0.2
Re dilution 100X, re culture →DO=0.2
Induction 1h 1mM IPTG →protein fluo. production
Bloque chloramphénicol →stop protein production
Wash phosphate buffer, 12h. Protein maturation
Bacteria in exponential phase
→reproductibility
No protein production
Fluorescence level
Limit autofluorescence
Calibration
1.
"Green" bacteria no plasmid
Induced: leak gren→orange, b=0.17
Non induced :autofluorescence
2.
"Orange" bacteria, no plasmid (b=0)
Coefficient a=0.58
3.
Fluorescent gene in 1 et 2 copies on a plasmid
Linearity between gene copy number and protein expression
Study as a function of the replicon
(ampicillin resistance)
•
F: single copy, partition system
R1+:partition system
•
R1: low copy number
•
ColE1: medium copy number, no partition system
R1-:without partition system
Hypothesis: On average gene expression does not depend on the copy nor its origin
F
R1-
R1+
ColE1
<PV> (a.u.)
27,1
28,5
26,5
25,7
<PO> (a.u.)
27,0
244
173
2167
<n>=<PO>/<PV>=<nP>/<nC>
1,0
7,8
6,5
95
qPCR
0,5
3,2
3,8
23,4
We take <nC>=1,7 (E. Coli and Salomonella, p.1553, ASM Press, 1996)
≈constant
Mean plasmid copy number per
chromosome
Variance et variability
Hypothesis on correlation and autoforrelation of fluorescent
protein expression [ <PaPb>, (a,b=O,V)]
n P2 =
n P nC  2
nP
P

 O
PO PV 
nC

PV2   n P nC

100
ColE1
Poisson
s
10
R1's
1
F
0.1
1
10
<n >
100
1000
P
F
R1-
R1+
ColE1
<nP>
1,71
13,3
11
161
s
0,7
4,2
3,1
40
h (%)
46
34
29,2
25
h=s/<nP>
R1- plasmid loss
Bacteria are cultivated without antibiotic for many generations
with
without, 54 générations
without, 99 générations
Diminution de la Population N+ with plasmid diminishes
Population N- without plasmid increases
Loss rate
• We measure N+(54)=60%, N+(99)=16%
N ( g )
N ( g )
= cte  2
g ( g 1)
T
,g =
T
• We deduce: population + division time est higher than 2
min. compared to population –
• Loss rate/bacterium/generation: 0,5%


 N  ( g )  1  2g 1 


 = 2
g (g 1) 

 N  ( g )  1  2
Boe et Rassmussen, plasmid, 36,p.153 (1996)
Numerical simulations
ADN chromosome transcription
ARNpolymérase
traduction
ARNm ribosome
Protein
10 réactions biochimiques Rµ:
* X0 -> X1
* X1 -> X0
* X1 -> X2 + X0
* X2 -> X3
* X3 -> Ø
* X3 -> X4
* X4 -> X3
* X4 -> X5 + X3
* X5 -> X6
* X6 -> Ø
R0 : free promoter -> RNAP bound promoter
R1 : unbinding of RNAP freeing promoter
R2 : transcription initiation
R3 : transcription, X3 = mRNA
R4 : mRNA deggradation
R5 : reversible mRNA/ribosome complex formation
R6 : reversible mRNA/ribosome complex dissociation
R7 : Translation start freeing RBS
R8 : production of protein X6
R9 : protein degradation
Siggia et al., PNAS October 1, 2002 vol. 99 no. 20 12795
Stochastic simulations
We have M reactions Rµ (m=1,2,…,M) involving N species.
We define P(t,µ)dt as the probability that the next reaction in [t+t, t+t+dt] is
reaction Rµ.
M
One can show that:
P(t , m) = hm c m exp(t  h c )
 =1
cµdt = average probability, to first order in dt, that a particular combination of
Rµ reactant molecules will react accordingly in the next time interval dt.
hµ = number of distinct molecular reactant combinations for reaction Rµ found to

be present in V at time t.
(Daniel T. Gillespie, JOURNAL OF COMPUTATIONAL PHYSICS 2, 403-434 (1976))
Example:
X1 + X2 -> X3
2X -> Y
h = X1X2
h = X (X-1)/2
Implementation: one has to generate (t,µ) according to P(t,µ)
in order to update at each step the number of reactant molecules implied in
reaction m.
Daniel T Gillespie, J. Phys. Chem., 1977, 81 (25), 2340-2361
1 gene which duplicates, binomial repartition
of protein
Ages and division time distributions
Conclusion
• Build up tools in molecular biology, optic and microfluidic to measure
variability in bacterial population
• Application: plasmid copy number measurement
 F: single copy, strictly regulated
 R1: partition System1) lowers PCN and 2) lowers variability
 ColE1: high pcn but low variability
• Plasmid loss rate in absence of partition system
• Plasmid metabolic cost: increase in division time
Perspectives
• Synchronisation of bacterial population
• Antibiotic concentration effect
Poubelle
Réservoir 2
• Sorting:
• Other toxic gene to test variability
Thank you for your attention