will we be able to construct a synthetic bacterium?adanchin/lectures/ecsb_07.pdf · for the...

Genetics of Bacterial Genomeshttp://www.pasteur.fr/recherche/unites/REG/

WILL WE BE ABLE TO CONSTRUCT ASYNTHETIC BACTERIUM?

ECSB, Sant Feliu de Guixols, 26 november 2007


ACKNOWLEDGEMENTS

Génétique in silico Marc Bailly-Béchet Massimo Vergassola

Génoscope AGC Claudine Médigue

Génétique des Génomes Bactériens(in silico)

Gang FangEtienne LarsabalGéraldine PascalEduardo Rocha

The University of Hong KongDpt of Mathematics and HKU-Pasteur Research Centre

Stanislas Noria (collective name, working seminarin « Conceptual Biology »)

The BioSapiens and the Probactys Consortia


THREE REVOLUTIONS

1944 - 1985 MOLECULAR BIOLOGY

1985 - 2005 GENOMICS

2005 - … SYMPLECTIC (SYNTHETIC) BIOLOGY(highly multidisciplinary !)

« Symplectic » is in Greek (συν, together, πλεκτειν, toweave) the same word as « Complex » in Latin; used hereto avoid the unwanted fuzzy connotations associated to« Complexity »; a connotation in Geometry will not interfere


LIFE AND COMPUTATION SOME SIMPLE PHYSICAL CONSTRAINTS TRANSLATION ORGANIZES THE BACTERIAL GENOME DISSYMMETRY OF REPLICATION THE PALEOME: CONSTRUCTOR AND REPLICATOR THE CENOME: THE “PURPOSE” OF THE MACHINE REPRODUCTION vs REPLICATION: THE ESSENTIALITY

OF METABOLISM


Three co-existing processes constitute life:

Metabolism | aCompartmentalization | machine

Information transfer | a “program” (declarative, not| prescriptive)

The cell is the atom of life

WHAT LIFE IS


THE “GENETIC PROGRAM”

Physics: matter, energy, timeStatistical physics: Physics + « information »Biology: Physics + information, coding, control...Arithmetics: sequences of integers, recursivity, coding…Computation: Arithmetics + programs + machine...

The « genetic program » metaphor (or model?) haspractical consequences: we know how to manipulate genesand gene products, do we have the conceptual tools topush the metaphor to its ultimate consequences?


Two entities permit computing:

A machine able to read and write

A program on a physical support, split (in practice, but notconceptually) into two entities:

Program (providing the apparent “goal”)Data (providing the context)

The machine is distinct from the program

WHAT COMPUTING IS


THE TURING MACHINE

the program(data)as a linearsequenceof symbols

the machine(read/write)

is physicallydistinctfrom


CELLS AND COMPUTERSGenetics rests on the description of genomes as texts written with a four

letter alphabet: do cells behave as computers?

Horizontal Gene TransferVirusesGenetic engineeringDirect transplantation of a naked genome into a recipient cell withsubsequent change of the recipient machine into a new one (2007)

all points to separation between

«Machine» (the cell factory) and «Data/Program»

Need: conceptual analysis of biological information (algorithmic complexity,logical depth...)


A GENETIC COMPUTER?

In a computer the machine is distinct from data/program

In the cell, data and program play the same role (theyare ‘ declarations ’ not prescriptions); they can bemodified by the machine (Pol IV, Pol V...)

General reflection (Number Theory) considers theactions of the machine, but not the way it is constructedin practice


AN ALGORITHMIC VIEW OFBIOLOGICAL ACTIONS

Replication, transcription, translation: high parallelism

“Begin, Check Control Points, Repeat, End”

The action is always oriented, with a beginning and an end

The processes of time control (check points) are rarely takeninto account (except for the replication/division processes), buttheir role is essential to allow coordination of multiple actions inparallel

Need: conceptual analysis of check points; experimentalidentification


John von Neumann, trying to understand the brain, suggested thatwere a computer both to behave as a computer and to construct themachine itself, it should harbour an image of the machinesomewhere

That special computer had to be split into a replicator and a constructor,which expresses the program for construction of both the replicatorand the constructor

The metaphor does not appear to apply to the brain, does it apply to thecell?

IS THERE A MAP OF THECELL IN THE CHROMOSOME?


GENE ORDER AND CELL SHAPE

Tamames J, Gonzalez-Moreno M, Mingorance J, Valencia A, Vicente MBringing gene order into bacterial shapeTrends in Genetics (2001) 17: 124-126

The mur-fts cluster


CELL SHAPE

Constructing a synthetic cell would best take thisconjecture into practice, and implement the relevantgene order in the synthetic genome



OF METABOLISM


PHYSICS OF REPLICATION

DNA forms a long folded thread: how do thedaughter molecules separate?

Are physical constraints reflected in thesequence?

[Replication is oriented: the physics of a strandcannot be that of its complement]

A correct use of physics helps!


A TEXTBOOK VIEW OF ENTROPY

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.

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.. .... .Benjamin Crowell, licensed under the CreativeCommons Attribution-ShareAlike license

S = k log Ω


AN INCREASE IN ENTROPY IS ENOUGH TOSEPARATE CHROMOSOMES

0

tJun S, Mulder B. Entropy-driven spatial organization of highly confined polymers: lessonsfor the bacterial chromosome .Proc Natl Acad Sci U S A. 2006 103:12388-12393

Danchin A, Guerdoux-Jamet P, Moszer I, Nitschke P. Mapping the bacterial cell architectureinto the chromosome. Philos Trans R Soc Lond B Biol Sci 2000, 355:179-190


BREAKING SYMMETRY

Optimal use of the driving force of entropy requiressymmetry breakage: this needs to be implemented inthe structure of the synthetic cell


CONCEPTS AND PATCHES

The processes constituting life can be analyzedconceptually. They need however to be implementedwith concrete objects, having idiosynchraticproperties. The DNA sequence cannot be a smoothlinear double helix, simply because of the chemicalnature of its nucleotides; it winds, turns and bends.However it needs to be recognized by control orstructural elements. How can these divergentconstraints be reconciled?


RECURSIVE MODELLING

Realistic Model 1 <=> Real sequence

Prediction 1


Prediction 2


Prediction 3

…..


Evolution optimises replication, while DNA needs alsoto support gene sequences

This is witnessed by: A period 3, signature of the codon succession in

genes (constrained by the genetic code rule)

A period 10-11.5 of yet unknown function...

CONSTRAINTS IN THE DNA SEQUENCE


PERIODS IN GENOMES

One observes a correlationbetween base pairs withperiod three

After deconvolution of thisperiod there remains asomewhat fuzzy period of 10to 11.5 base pairs

Eskesen et coll. BMC Molecular Biology Volume 5, 12, 2004


A UNIVERSAL FEATURE OF THE GENOME TEXT: 10-11.5

motifs

0 10 20 30 40 50 60 70 80 90 100bp

0 10 20 30 40 50 60 70 80 90 100bp

ffss(G(G--))-0.01

0

0.01

0 10 20 30 40 50 60 70 80 90 100bp

real

model

validation

Helicobacter pylori

Discrepancy


TYPE A FLEXIBLE MOTIFS

methods

1- 1-xxAAxxxxxxxxTTxxxxxxxxAAxxxxxxxxTTTTxxxxxxxxxxAAxxxxxxxxTTxxxxxxxxAAxxxxxx: : All domainsAll domains 2-2-xxxxxxxxxxxxxxxxxxxxxxGGxxxxxxxxTTTTxxxxxxCCxxxxxxxxxxTTxxxxxxxxxxxxxxxxxx:: ProteobacteriaProteobacteria 4- 4-xxxxxxxxxxxxTTxxxxxxxxAGAGxxxxxxTTTTxxxxxxxxxxxxxxxxTTxxxxxxxxxxxxxxxxxxxx:: ArchaeaArchaea 55''--xxxxxx-10xxxxxxxxx0xxxxxxxx10xxxxxx-10xxxxxxxxx0xxxxxxxx10xxxxxxbp-3bp-3''

TTTTxxxxxxGGxxxxxxTTxxxxxxxxxxxxxxxxxxxxTTTT

Nucleotides forming this class of motifsare fully accessible on this side of thehelix and the dinucleotides are locatedin the small groove

Nucleotides forming this class of motifsare fully accessible on this side of thehelix and the dinucleotides are locatedin the large grooveTTTT

GG TT TTTT

AAAACC

AA AAAA


FLEXIBLE MOTIFS ACCOMMODATE LOCALVARIATIONS OF THE DNA STRUCTURE

The flexiblility of these motifs allow DNA to take intoaccount superturns and bends

Larsabal, E, Danchin, AGenomes are covered with ubiquitous 11 bp periodic patterns, the "class A flexible patterns »BMC Bioinformatics. 2005 6:206


OPEN QUESTIONS

The constraints resulting from the presence offlexible motifs is so large that it should be visiblein gene products

It may result in non random distribution of genesif some functions are associated to regularities inproteins (alpha helices, beta sheets, beta turnsetc)



OF METABOLISM


MULTIVARIATE ANALYSESMultivariate analyses try to extract information by reducing as much aspossible the number of descriptors of the objects of interest

Laplace-Gauss statistics

Principal Component Analysis uses the centered average and a simpledistance (identity); it is the reference method

Correspondence Analysis belongs to the same family, but it uses the χ2

measure as a distance (Benzécri, 1965)

Absence of normality (or log-normality)

Independent Component Analysis uses the non gaussian character ofthe values associated to descriptors; it characterizes objects belongingto common independent clusters (the « cocktail party » theorem),(Hérault, 1984)

Further methods need to be developed


LOCAL BIASES OF CODON USAGE

Correspondence Analysis shows that genes with similarbiases are functionnally related. How is this reflected in thechromosome?

A clustering method (Vergassola et al.) based on informationtheory groups the genes into homogeneous families, whichappear not to be randomly spread in the chromosome. Themethod identifies 4 classes in E. coli and 5 in B. subtilis.Genes sharing similar codon bias tend to be close to eachother on the chromosome, in coherent patches extended onaverage ten times the extent of transcriptional units


Genes with similar bias areorganized into groups longer thanoperons, showing some translation-driven organization of thechromosome

A major part of this effect comes fromthe recycling or rare transfer RNAmolecules. It is essential tounderstand that individual molecules(not concetration!) are important inthe cell

GENOMIC TRANSLATION ISLANDS

M. Bailly-Béchet


TRANSLATION ISLANDS

One groups is associatedto high expression (blue).

The other groups arealso functionnallyconsistent: horizontallytransferred genes (red),motility (yellow) andintermediary metabolism(green). M. Bailly-Béchet

M Bailly-Bechet, A Danchin, M Iqbal, M Marsili, M VergassolaCodon usage domains over bacterial chromosomesPLoS Computational Biology (2006) 2: e37


SEQUENCES ANDARCHITECTURES

The non-random distribution of genes in the genome suggestsstrong constraints of the 3D distribution of molecules in thecell. Escherichia coli has to accomodate in less than one cubicmicrometer 20,000 ribosomes, 150,000 tRNAs, 1,000 mRNAs(each 3 times longer than the cell), and a DNA molecule 1,000longer than the length of the cell, together with a huge numberof proteins. Occupation of space is therefore a major questioncombining constraints related to the physics of diffusion andthe physics of polymers. Furthermore, the « concentration » ofmany small molecules is meaningless (1 µM = 600 moleculesin E. coli)…



OF METABOLISM


LOOKING FOR THE REPLICATORAND THE CONSTRUCTOR

Are genes grouped randomly in the chromosomes?

Do we find different gene categories, in terms of the waythey are organized?

At first sight, consistent with different DNA managementprocesses in different organisms not much is conserved,while genes transferred from other organisms are distributedthroughout genomes

However, groups of genes such as operons or pathogenicityislands tend to cluster in specific places, and they code forproteins with common functions. « Persistent » genes areclustered together


Genes are preferentially located in theleading replication strand in Bacteria. Thereis however much variation, depending on theorganism, with a considerable bias in A+T-rich Gram-positive organisms


180

90

0

270

55% leading

Escherichia coli

Ori

Ter

90270

65% leading

Treponema pallidum

Ori

Ter

180

9027075% leading

Bacillus subtilis

Ori

Ter

90270

87% leading

Thermoanaerobactertengcongensis

Ori

Ter

Gene densityGene density inthe leading strand


DISTRIBUTION OF HIGHLY-EXPRESSED GENES

C. c

resc

entu

s

M. t

uber

culo

sis

E. c

oli

B. su

bitli

s

rRNA Highly-expressed genes are

clustered near the origin ofreplication in fast growingbacteria

Origin

Terminus

Middle

Ori

Ter

10%

20%

30%

40%

50%

60%

0%

70%

10%

20%

30%

40%

50%

0%


TO LEAD OR TO LAG...

Is it possible to see whether there is a difference inthe nucleotide composition, between the leadingand the lagging strand? Does that have aconsequence on the codon biases? Does that havea consequence for the protein amino acidsequence?


TO LEAD HAS A COST: BIASVISIBLE IN PROTEINS…

GT in the leading strand, CA in the lagging strand....

0

5

10

15

0 5 10 15

% V

% T

Borreliaburgdorferi

0

5

10

15

0 5 10 15

% V

% T

Chlamydiatrachomatis


Proteins are made of 20 amino acid types, amongwhich Valine and Threonine, and one observes thatValine-rich protein are on the leading strand whileThreonine-rich proteins are on the lagging strand!Isologous proteins replace preferentially one residue forthe other when their gene change strand

This should be taken into account in models ofevolution


ESSENTIAL GENES LOCATE INTHE LEADING STRAND

hi ghlyexpressed

0%

25%

50%

75%

100%

non-highlyexpressed

essential genes non-essential genes

lagging

leading

non-highlyexpressed

highlyexpressed


PHYSICAL CAUSALITY: AVOIDING COLLISIONSBETWEEN RNA AND DNA POLYMERASES

DNAPdeceleration

End oftranscription

Stop of RNAP & DNAP

Transcriptionaborts

Co-oriented FrontalConsequences:1. Slowing down of

replication

2. Loss of transcripts

Consequences:1. Truncated

transcripts

2. Truncated essentialproteins


REPLICATION DISSYMMETRY

The genes required to construct the cell are betterplaced in the leading DNA strand. The physics ofreplication has to be taken into account, possibly byusing two DNA polymerase genes (note that threecomplexes are involved in replication, one for theleading strand and two for the lagging strand)

McInerney P, Johnson A, Katz F, O'Donnell M.Characterization of a triple DNA polymerase replisome Mol Cell 2007 27:527-538



OF METABOLISM


PERSISTENT GENES

Laboratory essential genes are located in the DNAleading strand. They are conserved in a majority ofgenomes. By contrast the genes that are conserved andlocated in the leading strand make a particular category,which doubles the number of « essential » genes.

These genes make a universal category; 400-500genes persist in a majority of bacterial genomes; theyare not only involved in the three processes needed forlife, but in maintenance and in adaptation to transientphenomena; a fraction manages the evolution of theorganism.


TWO CATEGORIES OF GENEPERSISTENCE

Which functional category?

Information transfer

Compartmentalization

Anabolism

Stress, maintenance and repair

Highly non random!

Persistent genes


The contribution of gene divergence correlatesbetween some orthologous pairs and 16S rRNA

(A) Approximately half of B. subtilis (resp. E. coli)persistent genes show a correlation coefficient>0.9 for sequence similarity of the pair of orthologsand 16S RNA

Some genes (B) evolve in an erratic way. Thismay be due to horizontal gene transfer, localadaptations leading to change in evolutionarypace, or simply wrong assignments of orthology.The latter is a significant problem, especially inlarge protein families

GENE PERSISTENCE

G Fang, EPC Rocha, A DanchinHow essential are non-essential genes?Mol Biol Evol (2005) 22: 2147-2156


PERSISTENT GENES ARE CLUSTEREDTOGETHER

Persistent genes are functionally defined. They arelocated in the DNA replication leading strand

The way they group along chromosomes in morethan 250 bacteria (genome length > 1,500) displaysthree clusters that reflect a scenario of the origin oflife. This is why it is proposed to name paleome(from παλαιος, ancient) this group of core genes


EXISTENCE IMPLIES CLUSTEREDPERSISTENCE

Why are persistent genes clustered? A simple model showsthat if, in addition to horizontal gene transfer, there is aprocess deleting genes in groups in genomes, then anygene contributing to fitness frequently enough overgenerations will tend to cluster with other genes with similarproperties. This accounts for clustering of essential genes,but most probably also for clustering of antibiotic resistancegenes in bacteria found in hospitals....

As a consequence gene clustering will precede, not derivefrom co-transcription or protein-protein interaction (nointelligent design!)

Note: the model needs to be refined. It may yield interestingchaotic behaviours


EXISTENCE IMPLIES CLUSTERING


PERSISTENT GENESCONNECTIVITY

Using 228 genomes with more than 1500genes and « correct » annotations, we haveidentified genes that tend to remain close toone another; this « mutual attraction »constructs a remarkable network made ofthree layers


PERSISTENT GENES RECAPITULATETHE ORIGIN OF LIFE

G. Fang

The external network, made ofgenes of intermediarymetabolism (nucleotides andcoenzymes, lipids), is highlyfragmented; the middle networkis built around class I tRNAsynthetases, and the innernetwork, almost continuous,organized around the ribosome,transcription and replicationmanages information transfers

A Danchin, G Fang, S NoriaThe extant core bacterial proteome is an archive of the origin of lifeProteomics. (2007) 7:875-889


WHAT FUNCTIONS FOR LIFE?SCENARIO FOR THE ORIGIN OF LIFE

To be — to persist in time — can be proposed as the rootfunction of living organisms

Fighting weathering implies chemical turnover(metabolism) on solid surfaces and immobility requiresprotection (compartmentalisation)Compartementalised metabolism creates surfacesubstitutes (RNA)Exploration, associated to sensing and memorizing(information transfer) is the discovery that made life aswe know it

A Danchin Homeotopic transformation and the origin of translation Progress in Biophysics and Molecular Biology (1989) 54: 81-86


METABOLISM AND REPLICATION

This scenario emphasizes the separationbetween metabolism and replication, thelatter being a secondary invention ofprebiotic systems:

Building blocks => nucleotides => tRNA =>ribosome => DNA



OF METABOLISM


THE COMPOSITE GENOME

Expecting two genome components, coding for themachine and for the “purpose” of the machine, we need toseparate between the replicator/constructor andsecondary functions.

Extant genomes should comprize ubiquitous functions (notgenes!) which would correspond to the former (here thepaleome) and functions specific to the environment of theorganism (named the cenome — as in “biocenose” — toexpress the fact that these genes correspond to a specificniche)


CONSERVATION OFGENE CLUSTERING

Clusteringfrequency

Frequencyin genomes

+frequent + rare

Known Unknown

Antibiotics Virulence

PenicillinVancomycin

Isoniazide

Rifampicin

Streptomycin

Tetracycline

Genome core<2000 genes

Variable genesalready > 50000 genes

The cenome(from the Greekκοινος, common)

The paleome(from the Greek

παλαιος, ancient)


A SPLIT PALEOME

Paleome 1 (essential genes) Constructor: DNA specifies proteins which form

the machine that constructs the cell (reproduction)

Replicator: DNA specifies proteins that replicateDNA (replication)

Paleome 2 (persistent non essential genes)Perennisation of life (maintenance), requiresidentification of functional from non functional objects


SYNTHESIS: A TALE OF TWO GENOMES

Life manifests first bygrowth and repair ofweathering: thecorresponding genomeexists since the origin, it isthe paleome.. Explorationof the environment is aninevitable consequence ofexistence, it results fromcontinuous creation andexchange of the geneswhich form the cenome.

To liveTo escape death

To live in contextA. Danchin. Archives or Palimpsests? Bacterial Genomes Unveil a Scenario for the Origin of LifeBiological Theory (MIT Press) (2007) 2: 52-61.


THREE PARTS IN THE GENOME’SORGANIZATION

Anabolism and Replication

Maintenance and Repair: coping witherrors

Life in context (the cenome)



OF METABOLISM


SURVIVAL

Rules for constructing the contructorand the replicator are understood

This substantiates that constructing aliving bacterium will be possible

But will it produce a stable progeny?


METABOLISM AND REPLICATION

Replication accumulates errors (Muller’sratchet and Orgel’s error catastrophe); thereplicator cannot, by itself lead to perennity

Reproduction: can metabolism reproduce inan error-prone context, and improve onunperfect components? The answer is « yes »

Freeman Dyson’s « origins of life » revisited


INFORMATION AGAIN

Metabolism improvement can be conceptuallytolerated as creation of information is reversible(Landauer, 1961; Bennett, 1988); a consistentorganized pathway may slowly improve andbecome robust

Open question: « Room » is needed toaccomodate innovation; how is it obtained?Experiments (including in silico) are needed toidentify the corresponding processes


GENOME EXPLORATION

Is the structure of the paleome homogeneous?

How do we see the dialogue between the youngand the old (creation of information, in practice)?Note that this is « built in » biological systems,and may be at the room of cancer cells’immortality


ESSENTIAL METABOLISM

Recovery from an aged state requires:Stepwise improvement of the « quality » of biologicalobjects

Energy-dependent selection of what needs to bedestroyed (ratchet mechanism)

Persistent genes of unknown function evolvefollowing a tree that differs from that of theanabolic pathways…


CAVEAT: PATCHES FORANECDOTES

Each component of the cell has idiosynchraticproperties, some are incompatible with othercomponentsThe paleome codes for anabolic and maintenancefeatures, except for a few purely catabolic stepsOne example: serine catabolism (accounts for serinetoxicity)

This results in the « anecdotal » appearance of biologicalsystems


THANK YOU

will we be able to construct a synthetic bacterium?adanchin/lectures/ecsb_07.pdf · for the...

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