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!) 2) DNA replication ADNpolymérase, gyrase… transcription translation ADN chromosome ARNm Protéine: un gène 1) Central dogma: ARNpolymérase ribosome

Bacterial culture Growth by division: 1 bacterium→2 daughter bacteria genetically identical (clone) Duplication, repartition of the constituants (in particular of the chromosome) t 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 Dégradation gR ARN 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)

résistance antibiotique Plasmid Replicon résistance antibiotique 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

Without partition system With partition system

<n> s Standard deviation Plasmid copy number (PCN) inE. Coli PCN=phenotype choice Measured individual PCN on population scale(~104 individus) Distribution : variability Antibiotic resistance: adaptability <n> s Standard deviation

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 Promoter 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

Field of view: 10µm Bacterial speed: ~0.1-1 mm/s Optical differentiel Interferometry profilometry image of the channel (z=2µm)

Optical elements detail

Time series of fluorescent intensity FO FV 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) 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 "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: low copy number ColE1: medium copy number, no partition system R1+: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 ≈constant Mean plasmid copy number per chromosome We take <nC>=1,7 (E. Coli and Salomonella, p.1553, ASM Press, 1996)

Variance et variability Hypothesis on correlation and autoforrelation of fluorescent protein expression [ <PaPb>, (a,b=O,V)] Poisson F R1's ColE1 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% We deduce: population + division time est higher than 2 min. compared to population – Loss rate/bacterium/generation: 0,5% Boe et Rassmussen, plasmid, 36,p.153 (1996)

Numerical simulations transcription traduction ADN chromosome ARNm Protein ARNpolymérase ribosome 10 réactions biochimiques Rµ: * X0 -> X1 R0 : free promoter -> RNAP bound promoter * X1 -> X0 R1 : unbinding of RNAP freeing promoter * X1 -> X2 + X0 R2 : transcription initiation * X2 -> X3 R3 : transcription, X3 = mRNA * X3 -> Ø R4 : mRNA deggradation * X3 -> X4 R5 : reversible mRNA/ribosome complex formation * X4 -> X3 R6 : reversible mRNA/ribosome complex dissociation * X4 -> X5 + X3 R7 : Translation start freeing RBS * X5 -> X6 R8 : production of protein X6 * X6 -> Ø 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. One can show that: 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 h = X1X2 2X -> Y 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 Thank you for your attention Synchronisation of bacterial population Antibiotic concentration effect Sorting: Other toxic gene to test variability Poubelle Réservoir 2 Thank you for your attention