PROBACTYS
Life has always seemed to me like a plant that lives on its rhizome. Its true life is invisible, hidden in the rhizome. The part that appears above ground lasts only a single summer
Carl Gustav Jung
Programmable bacterial catalysts
The European Union supports research via grants which permit development of research activities associating several european partners, according to the principle of subsidiarity. The Probactys programme has been meant to identify properties of a bacterial chassis to permit metabolism of aromatic compounds. The effort presented here corresponds to our contribution via grant CT-2006-029104.
This page aims at informing the general public (in particular members of european countries which steer the European Union) about the ultimate developments of this research.
The central discovery in the programme, after definition of the two large domains of genome organisation in bacteria (Pseudomonas putida in particular), the minimal (or universal) paleome (core gene functions for life) and the cenome (genes for life in context, see preceding report), has been that the minimal paleome comprises twice as many genes as the number of genes deemed essential for life. In silico analysis of the structure of the paleome has shown that, beside about 250 genes deemed essential, it comprises usually non-essential genes which code for degradation functions that use energy (proteases and RNases in particular) and genes which code for metabolic incompatibilities (metabolic "frustration"). Understanding this split is necessary to be able to construct relevant synthetic bacteria.
Comparison between P. putida and E. coli permitted us to define an intermediary region (tentatively named the "mixome") where genes are slowly (in the time course of evolution) recruited from the cenome to belong to the species-specific paleome.
The majority of the genes in this domain are genes coding for metabolic functions. Degradation of aromatics appears to play a central role there, in a way which is not yet understood. Because of want of enough personnel to study this question we extended our study to the independent project on E. coli (Coliscope). The central observation, which needs to be further documented, is that the presence of functional operons in a genome correlates with avirulence. This is visible in Pseudomonadales where P. aeruginosa (which can become pathogenic) lacks the operons while P. putida (which is a GRAS organism) has them.
To help in retrieving information fast on P. putida we used our database PutidaList, which has useful facilities to recover motifs in DNA and proteins.
Analysis of the minimal paleome, formed of about 500 genes, showed us that it comprised two sets of genes. The first set is easy to understand as it corresponds to genes that are deemed “essential” as they are necessary for growth in rich media under laboratory conditions. Their number is about 250. Many non-essential genes in the paleome coded for degradation functions in enzymes using energy (ATP-dependent proteases, ATP-dependent helicases, in particular, but also other enzymes involved in maintenance, repair and degradation, using for example S-adenosyl-methionine). This led us to develop a conjecture based on the novel development of theories of information. Briefly, we see living organisms as information traps. The way natural selection works is by making room, using energy, not to degrade, but to prevent degradation of functional entities.
If this conjecture is right, then it has enormous consequences for Synthetic Biology, and for all processes aiming at constructing functional minimal genomes, with the aim of achieving cell factories. Based on the conceptual separation between reproduction and replication, analysis of the degradosome in Bacteria led us to discover that a ubiquitous source of energy which could be used for degradation is the mineral polyphosphate. This opens a large number of constraints for the construction of a perennial synthetic bacterium. In fact we think that, unless we can harness the genes which are required to make a young progeny from an old culture (using polyphosphates as the ultimate energy source), a minimal genome will end up with cells which will be endowed of only a limited number of generations. This means that the problem of scaling up will be central to Synthetic Biology. In short, either we construct cells which will be limited in their multiplication power, but will faithfully develop following the goal expected from their construction, or we will have an endless multiplication capacity, but with a trade off, the systematic emergence of unexpected and unplanned properties. This will correspond to the capacity of trapping information using functions that degrade unfunctional products (in particular RNA and proteins) using energy to prevent degradation of what is functional, whatever its origin, a feature antagonistic to the engineering view.
This work led us to explore and develop novel ideas about information as an authentic principle of Nature, and we proposed a rationale to identify those genes which permit accumulation of information in the cell's progeny. To this aim we devised an experimental set up, constructing "intelligent" bacteria, which can produce, after onset of an ageing process, a progeny which is able to use nutrients that the parent cells could not use. We have now obtained hundreds of mutants from these constructs, and several have been sequenced using the Solexa / Illumina technology. The initial work has been performed on E. coli MG1655 using the recombineering technology. This work needs to be completed.
The second set of non-essential minimal paleome genes contains many genes of unknown function. In particular, surprisingly, it contains at least one catabolic system. This system codes for serine dehydratase, a complex iron-sulfur dependent enzyme that degrades serine to pyruvate, permitting it to enter the standard core intermediary metabolism. As stated in the preceding report, this led us to explore again an old observation, that of serine toxicity, well-known in industry as it interferes dramatically with methods for serine overproduction.
While in our hands most bacteria are sensitive to serine, this is not so, at least under standard conditions, with P. putida. This led us to analyze more in depth serine sensitivity in Enterobacteria, first, then in other gamma-proteobacteria. The relative insensivity of P. putida is due to the fact that the genome harbours three serine dehydratase genes which immediately scavenge serine when it is in excess. This is important to note when constructing a minimal genome of P. putida. Briefly, our phylogenetic analyses and experiments demonstrate that the source of serine toxicity is one of its derivatives. This observation corresponds to the chemical fact that reactive compounds result in a phenomenon of "frustration" which implies channelling or processes to lower the internal concentration of the molecule at possibly reactive sites. Looking for mutants, we found that it was impossible to obtain mutants resistant to some derivatives, suggesting that their targets are numerous. In fact, even in the presence of isoleucine and valine (aceto-hydroxyacid synthases were found to be targets of serine several decades ago) it was still impossible to get mutants except for some mutants (sequenced using Solexa / Illlumina) in transport system, thus demonstrating that there is at least three targets of the molecule in the cell, involving major pathways.
To uncover the chemical steps involved using a variety of constructs from diverse bacteria we performed whole genome transcriptome analyses both in E. coli and in P. putida after serine and other additions to the medium. The experimental data are being explored. Beside standard stress induced conditions as well as the expected involvement of serine dehydratase, we found in E. coli that a novel organisation of the metabolic pathway connected to the TCA cycle was created. Unexpectedly, we also observed a huge involvement of part of the aromatic biosynthesis pathway. By contrast in P. putida, beside the expected effect on serine dehydratases, a large number of catabolic pathways is triggered. It is likely that this is highly related to the general degradative capacity of the organism. This work, and in particular statistical analysis, needs to be pursued.
Comparison of Pseudomonas species indicate that there exists some significant differences between different species, with P. putida comprising more aromatic degradative potential (perhaps associated to the process described in the preceding paragraph). Browsing bacterial genomes showed us that the situation is similar in Enterobacteria, where we looked for an organism with unusual aromatic biosynthesis / degradation potential. Photorhabdus luminescens is a rare example of organisms synthesizing cinnamic acid (gene stlA (plu2234), coding for phenylalanine ammonia-lyase). This protein is similar to HutH, histidine ammonia-lyase in P. putida (E-score 3 e-62). We explored the role of cinnamic acid in P. luminescens, showing that it may have a role in quorum-sensing). We also unravelled some of the features of the toxicity of this metabolite, using intrinsic bioluminescence as a probe: there seems to be a clear impact on ATP production, a fact that will need to be incorporated in our knowledge of the action of aromatics in Gamma-proteobacteria.
Finally, the project ColiScope (cited above), meant to compare several tens of E. coli strains substantiated this observation, showing us that aromatic operons may have other unsuspected roles. The corresponding work (involving analysis of pathogenic effects) needs to be pursued. This is particularly important for the minimisation of the P. putida genome if we aim at answering concerns about Synthetic Biology raised in the public.
Our effort in terms of spreading the concepts of Synthetic Biology is developed via our journal, Symplectic Biology.
All publications (stars indicate experimental work), with the
Probactys logo 
are
displayed at our bibliography
pages
V Barbe, S Cruveiller, F Kunst, P Lenoble, G Meurice, A Sekowska, D Vallenet, TZ Wang, I Moszer, C Médigue, A Danchin From a consortium sequence to a unified sequence: The Bacillus subtilis 168 reference genome a decade later Microbiology (2009) 155: 1758-1775
S Chalabaev, E Turlin, S Bay, C Ganneau, E Brito-Fravallo, J-F Charles, A Danchin, F Biville Cinnamic acid, an autoinducer of its own biosynthesis, is processed via Hca enzymes in Photorhabdus luminescens Appl Environ Microbiol (2008) 74:1717-1725
A Danchin Natural selection and immortality Biogerontology (2009) 10: 503-516
A Danchin Bacteria as computers making computers FEMS Microbiol Rev (2009) 33: 3-26
A Danchin A phylogenetic view of bacterial ribonucleases Prog Nucleic Acid Res Mol Biol (2009) 85: 1-41
A Danchin Sélection naturelle et immortalité in: Les mondes darwiniens (T Heams, P Huneman, G Lecointre, M Silberstein, eds), Syllepse (2009) pp 445-470
A Danchin, A Sekowska Frustration: Physico-chemical prerequisites for the construction of a synthetic cell in: Synthetic Chemistry, Beilstein Institut (2009) 1-13
V de Lorenzo, A Danchin Synthetic biology: discovering new worlds and new words EMBO Rep (2008) 9: 822-827
A Danchin
Saurons-nous construire une bactérie synthétique ?
Médecine/Sciences 24: 533-540
A Danchin
Fabriquer une bactérie comme un ordinateur
Pour la Science
60: 66-72
A Danchin
Les organismes vivants comme pièges à information
Ludus
vitalis (2008) 16: 30
A Danchin
Quelles cellules saurons-nous construire ?
Deliciouspaper
(2009) 3: 6
1. Scientific conferences and seminars
• February 2008, Seminar at the Institut de Microbiologie, Orsay: "Ce que nous apprend la structure des génomes bactériens de l’origine de la vie à la biologie synthétique"
• February 2008, Seminar at the Swiss Institute of Bioinformatics, Geneva, Switzerland: "Natural selection as a new law of physics: an illustration with bacterial genomes"
• March 2008, European Forum for Biotechnology in China, Conference EFBIC-Red, Shanghai, China: "The program and the machine. Where can we find drug targets?"
• April 2008, Conference on the Origin of Life, Origins Institute, McMaster University, Hamilton, Canada "To live and to perpetuate life: prerequisites for the construction of a synthetic cell"
• May 2008, Conference of the Beilstein Institut, Synthetic Chemistry, Bolzano, Italy: "Frustration: physico-chemical prerequisites for the construction of a synthetic cell"
• June 2008, EMBO Workshop, Microbial diversity and metagenomics, Chalkidiki, Greece: "A future for (meta)genomics: constructing a synthetic cell factory"
• September 2008, Tenth Anniversary of the creation of the Swiss Institute of Bioinformatics, Bern, Switzerland: "in vivo, in vitro, in silico: the future of biology, from genomes to synthetic cells"
• October 2008, Seminar at the Faculty of Medicine of the University of Hong Kong, Hong Kong, China: "in vivo, in vitro, in silico: the future of biology, from genomes to synthetic cells"
• November 2008, EMBO Workshop, Science and Society, EMBL, Heidelberg, Germany: "Life and perpetuation of life of a synthetic bacterium"
Audience 80 persons, Scientists, journalists, politician and students, mainly European (including Switzerland, but also, see EMBL site from all over the world)
• March 2009, European Science Foundation, University of Barcelona, Sant Feliu de Guixols, Spain: "Maxwell's demon's genes: Towards a cell factory or towards a living synthetic cell?"
• April 2009, TARPOL Meeting (overlap between the targets of Probactys and TARPOL, and some of the work of our team overlaps, with relevant acknowledgements), Ecole Normale Supérieure, Paris, France: "Maxwell's demon's genes: Towards a cell factory or towards a living synthetic cell?"
• April 2009, 4th Semmering Vaccine Symposium, Baden, Austria: "Natural selection and immortality, or, Maxwell's demon's genes"
• May 2009, International conférence on Synthetic Biology, Collège de France, Paris, France: "Maxwell's demon's genes: Is it possible to construct a synthetic living cell?"
• May 2009, Conférences de l'Institut Cochin de Biologie Moléculaire, Faculté de Médecine Paris V, Paris, France: "Living organisms as information traps"
• June 2009, Annual Conference of the Swiss Society of Microbiology, Lausanne, Switzerland: "Maxwell's demon's genes: Towards a cell factory or towards a living synthetic cell?"2. Courses
• January 2008 Course at the Ecole Normale Supérieure, Paris, France: "Ce que nous apprend la structure des génomes bactériens de l’origine de la vie à la biologie synthétique"
• January 2009 Course at the Ecole Normale Supérieure, Paris, France: "Saurons-nous construire une cellule synthétique?"
• February 2009, Course at the Ecole Centrale, Châtenay-Malabry, France: "Les organismes vivants comme pièges à information"
Specific audience of students from Ecole Centrale des Arts et Manufactures
• June 2009, Course at the Ecole Nationale Supérieure des Mines de Paris, Paris, France: "L'usine cellulaire synthétique : réalité ou fiction ?"3. General public conference
• May 2008, Ecole Normale Supérieure, Paris: "Saurons nous construire une bactérie synthétique ?"
• July 2008, Université de tous les Savoirs, Faculté de Médecine Paris V, Paris, France: "Peut-on concevoir la cellule comme un ordinateur qui ferait des ordinateurs ?"
• 8 January 2009, Université de tous les Savoirs, Cité des Sciences, La Villette, Paris, France: "Vie et calcul : y trouvons-nous des points communs ?"
• 15 January 2009, Université de tous les Savoirs, Cité des Sciences, La Villette, Paris, France: "La vie comme piège à information"
• 22 January 2009, Université de tous les Savoirs, Cité des Sciences, La Villette, Paris, France: "L'usine cellulaire synthétique : réalité ou fiction ?"
• 26 January 2009, Séminaire d'orientation de l'Institut National de Recherche en Informatique et Automatique (INRIA), Lyon, France: "Les organismes vivants comme pièges à information"
• February 2009, European Group on Ethics in Science and New Technologies, Brussels: "Will we be able to construct a synthetic cell?"
