Causes and consequences of phenotypic variability: a preliminary study of life & death of individual E. coli.

Causes and consequences of phenotypic variability: a preliminary study of life & death of individual E. coli

The paradigm of genetics

Phenotype = Genotype Environment

… but is there any phenotypic variability when genotype and environment remain constant ?

In theory phenotypic variability could favour

Bet-hedging strategies in face of an uncertain future (Do not put all your genomes in one phenotypic basket, Balaban Science 2004)

Rapid epigenetic changes

(e.g. inherited through autocatalytic feedback loop)

Division of labour (including altruistic behavior)

(as the cells with identical genome maximize their inclusive fitness)

Classical sources of phenotypic variability

Environmental differencesGeographical

Temporal

Differences in the life cycle stages e.g new-born vs reproducing

Genetic differences caused by mutation recombination (Horizontal Transfer)

Is there other sources of variability of individual lifehistory when genotype and environment are constant ?

Measurement errors (minimized by repeated measures)

Epigenetic (non genetic heritability ?)

Aging (in a symetrically dividing organism?)

Stochastic sources quantitative (small numbers of big molecules) qualitative (error rates > 0)

Noise in gene expression is affected by genotype and environment

2 different fluorescent proteins controlled by identical promoters

Elowitz Science 2002

Life with small number of big molecules

DNA

RNA

Proteins

Mutations

aberrant RNA

aberrant proteins

10-9

10-4

10-5

Error rates

Functions

Cells

Functional degeneracy

cell death

Genes involved

mutS, mutT

mutT

gidA, mnmE

Maintenance ?

Functional fidelity ?

Strategies to maintain DNA integrity

Eliminate source of lesions

Physical protection

Template maintenance

Pool sanitization

Polymerase fidelity

Quality control

Strategies to maintain DNA integrity

• Eliminate source of lesions

• Physical protection

• Template maintenance

• Pool sanitization

• Polymerase fidelity

• Quality control

R

Preventing RNA infidelity• Transcription coupled repair (preferential repair of transcribed DNA strand)

• RNA polymerase fidelity (Blank Biochemistry 1986)

• alkB repair of alkylated mRNA, Aas Nature (2003)

• Release of ribosome facing truncated/damaged mRNA (tmRNA encoded by ssrA) Keller Science (1996)

• MutT sanitizes the ribonucleotide poolTaddei Science (1997)

DNA

mRNA

tRNA

relativeβ - galactivity

- T A G -- A T C -

- U A G -

S T O P

10-5

- G A G -- C T C -

- G A G -

- C U C -

1

. . .

Glu

- T A G -- A T C -

- C U C -. . .

Glu

- G A G -°

rGTP° rGTPOHRNA

polymerase

MutT

MutT controls transcription fidelityScience (1997) 278 128-130

lacZ-lacZ+Genotype

lacZ-

10-3

mutT+ mutT-

°

MutT hydrolyses dG°TP & rG°TP

Taddei Science 1997

RNA polymerase incorporate 8-oxoG

GTP

8-oxo-GTP

UTP

8-oxo-GTP

GTP

Matrice ADN Matrice dA-dTGenomic DNA template

Poly dAdT template

Taddei Science 1997

Errors during transcription lead to protein oxidation

Dukan PNAS 2000

Error in translation increase

misfolding & protein oxidation

Dukan PNAS 2000

Translation error as a limiting step for protein oxidation

Dukan PNAS 2000

8-oxo-G concentration increase in the brain during neuro-degeneration

Nunomura J Neuroscience 1999

8-oxo-G-ARN binding protein

Degradation?

G°

Translation

Erroneous Protein

8-oxo-G-RNA G°

TP

Oxydative Stress

RNA polymerase

8-oxo-G-ARN

direct oxidation of RNA

MutT

GTP Oxidation

G°

G°MP + PPiG°

RNA

RNA

Cause & consequences of 8-oxo-G in RNA

Consequences of RNA infidelity

• from a mutant gene may come transient function, leakiness

• from a wild-type gene may come a transient function loss

1 erroneous mRNA --> 40 erroneous protein

Non uniform distribution of erroneous proteins

Can transient transcription errors lead to phenotypic change that have long lasting consequences

> Transient mutators: wild-type bacteria that exhibit a mutator phenotype due to transcription/translation errors

Ninio suggests that a 1% subpopulation of cells is

transiently deficient for a protein involved in DNA fidelity

>How to capture and quantify transient events (via heritable consequences, epigenetic switch)

lac operon

• set of coordinately expressed genes under the negative control of lac repressor

• classical induction system: the active inducer is a product of one of the controlled enzymes

• lac repressor is a rare protein (~10-20)• transient depletion of repressor will lead to a

transient derepression of operon and to a burst of lacZYA gene expression

growth in low inducer level

high inducer

uninduced culture

Fully induced

Monod, ‘preinduction effect’ 1956

Fully induced

dilute single cells into maintenance inducer level

growth in maintenance inducer level

high inducer

β-galactosidase assays on ‘single-cell’ cultures

growth in maintenance inducer level

uninduced

induced

Novick & Weiner, 1957; maintenance

dilute single cells into maintenance inducer level

growth in maintenance inducer level

high inducer intermediate inducer

β-galactosidase assays on ‘single-cell’ cultures

growth in maintenance inducer level

uninduced cultures

induced mixed

Novick & Weiner, 1957; ‘all or none’

dilute single cells into maintenance inducer level

growth in maintenance inducer level

high inducer intermediate inducer

β-galactosidase assays on ‘single-cell’ cultures

growth in maintenance inducer level

uninduced cultures

induced mixed

Novick & Weiner, 1957; ‘all or none’

Ozbudak et al., Nature 427, 737 (2004)

Ozbudak et al., Nature 427, 737 (2004)

Ozbudak et al., Nature 427, 737 (2004)

Monitoring phenotypic variability in cell lineages

Development of molecular tools, microfluidic, databases, image analysis, statistical tools, tweezers, microscopes

Time-lapse of a bacterial lineage

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cette image.

Manually corrected mask

Automaticallygenerated mask

Data available after image analysis

• >100 movies (E. Stewart)• > 100000 divisions

(R. Madden)• Morphometry :

– Length– Positions

• Exhaustive genealogies > 10 generations

Individual sizes grow exponentially within a lineage

Distributions of individual phenotypes

Biomasse(µm)

Growth rate(µm/min)

Time to division(min)

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For phenotype to depend only genotype and environment

One must take into account DNA extended environment

(intracellular environment is dynamic, ~ heritable & local)

Why change ?Population geneticsGodelle Gouyon Brown Maynard-Smith

Change where ?Microbial ecology Fons

Who changes ?Molecular epidemiologyBinguen Denamur Picard Brisabois Berche

A network approach of bacterial variability

GiraudLechatBambou

B. ToupanceO. TenaillonJ-B André

Duriez

Change what?Bio-informaticsRocha

Who has changed ?Molecular PhylogenyLecointre Darlu

How to change ? Molecular biology Matic Radman Vulic Dionisio BjedovBregeon Leroy Hayakawa Sekiguchi Dukan

Change when ?transcriptome analysis Knudsen Cerf

Phenotypic variabilityLife History Stewart Madden Lindner Paul Gabriel Fontaine Depaepe Bredèche Mosser

Why change ?Population geneticsGodelle Gouyon Brown Maynard-Smith

Change where ?Microbial ecology Fons

Who changes ?Molecular epidemiologyBinguen Denamur Picard Brisabois Berche

A network approach of bacterial variability

GiraudLechatBambou

B. ToupanceO. TenaillonJ-B André

Duriez

Change what?Bio-informaticsRocha

Who has changed ?Molecular PhylogenyLecointre Darlu

How to change ? Molecular biology Matic Radman Vulic Dionisio BjedovBregeon Leroy Hayakawa Sekiguchi Dukan

Change when ?transcriptome analysis Knudsen Cerf

Phenotypic variabilityLife History Stewart Madden Lindner Paul Gabriel Fontaine Depaepe Bredèche Mosser

www.necker.fr/tamara/

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