|Director : Antoine Danchin (email@example.com)|
The genomic revolution has led to the interpretation of heredity as the reading of a text written in a four-letter alphabet. The text of the genome organises the expression of the genes, but is also directly linked to the architecture of the cell. Two bacterial models are used for these investigations: Escherichia coli and Bacillus subtilis. Our research combines the computer-based study of genomic sequences (analysis in silico) and validation of the predictions obtained, in vitro and in vivo. Our studies focus principally on sulphur metabolism because this atom seems to play a key role in cellular organisation in all living organisms, and in determining the pathogenic nature of bacteria. Studies of sulphur metabolism may therefore provide a means of developing appropriate control methods (e.g. against anthrax, which has recently been used as a bioterrorism agent in the US). The Unit was behind the creation of the HKU Pasteur Research Centre at Hong Kong, where similar studies are carried out on pathogens of local interest.
A Rosetta Stone: comparing the texts of related genomes
In the first year of its activity, the Genetics of Bacterial Genomes Unit carried out a comparative study of two pairs of genomes, each consisting of one reference bacterium genome and one genome of a bacterium of interest as a disease model. The Unit has benefited from the arrival in the laboratory of Jean-François Charles, and entomologist and specialist in the Bacillus cereus/Bacillus thuringiensis complex. Dr Charles brought a large collection of these organisms with him to the laboratory.
Two model bacteria have been used: Escherichia coli, one of the oldest models used by geneticists and the most frequently studied of all bacteria, and Bacillus subtilis, a bacterium from which numerous enzymes used in industry are obtained, and which is often found attached to the surface of leaves and on the ground. E. coli has been compared with Photorhabdus luminescens, which kills insects and propagates in an intermediate nematode host. Bacillus subtilis has been compared with the Bacillus anthracis/Bacillus cereus/Bacillus thuringiensis complex of sporulating bacteria implicated in numerous diseases, including the dangerous anthrax agent, which has recently become infamous in the US. This study focuses on identification of the function of key genes in the overall adaptation of the bacterium to its environment. One of the aims of the study is the identification of possible targets for specific drugs.
The H-NS protein and the response to metabolic transitions (Philippe Bertin)
The capacity to adjust the availability and activity of proteins makes it possible for cells to grow and to adapt to changes in the environment. The H-NS protein is involved in numerous cellular functions and affects the expression of genes regulated by environmental factors (e.g. temperature, pH, osmolarity). It modifies the expression of many genes, affects recombination and transposition and plays a role in bacterial virulence, by controlling motility and the original type of secretion.
Electron microscopy studies of bacteria with mutations in the hns gene have shown that these bacteria have lost their flagella. The synthesis of flagella requires the expression of a large number of genes, in a hierarchical manner. The overexpression in a hns mutant of the flhDC genes the master operon in flagellum biosynthesis restores motility, showing that these genes are a preferential target of H-NS. The mechanism underlying this effect is not entirely understood. The work of the Unit has shown that it affects DNA coiling.
Analysis of all of the products of transcription (the transcriptome), associated with analysis of all the proteins of the bacterium separated by two-dimensional electrophoresis (proteome) was used to compare the wild-type and mutant strains. This study showed that the accumulation of more than 60 proteins, more than half of which have been identified, was affected in the hns mutant. These very powerful methods have brought to light large regions (the H-NS regulon comprises more than 250 genes) regulated in the same manner in the cell. The use of protons is one of the key elements modulated by the presence or absence of H-NS.
The construction of sulphur-containing amino acids (Isabelle Martin-Verstraete)
Sulphur is essential for all cells. However, very little is known about its metabolism in gram-positive bacteria. The work of the Unit, as part of the BACELL programme of detailed functional analysis of the Bacillus subtilis genome funded by the European Commission, has focused on the characterisation of genes involved in the synthesis of methionine and cysteine in this bacterium. In addition to the direct characterisation of the principal genes involved in the synthesis of these amino acids, this study has provided new evidence for the progressive specialisation of enzyme activities during the course of evolution, from an initial primary activity with relatively little specificity. As for the study of H-NS, complementary studies have been carried out involving analysis of the protein synthesis profile (analysis of the proteome by two-dimensional electrophoresis) and of the transcription profile by membrane hybridisation (transcriptome analysis). These analyses showed that sulphur metabolism plays an essential role (fully justifying the choice of this topic as central to the genomic approach) and led to the identification of a certain number of co-regulated blocks of genes. This was used as the basis for the implementation of an original statistical treatment of the results. We identified several genes of unknown function that are probably involved in sulphur metabolism. The work of the Unit has also focused on the identification of essential regulatory genes involved in sulphur metabolism. Three of these genes have been characterised. They differ markedly from what has been found in gram-negative bacteria such as Escherichia coli, rendering the findings of this study particularly interesting.
The degradation of aromatic compounds, iron and oxygen (Francis Biville)
The degradation of aromatic compounds plays a key role in many organisms. This process is of particular interest because it uses oxygen (in the gaseous, molecular form) and is therefore directly involved in control of the intracellular concentration of this highly reactive gas. With the aid of computer-based, genetic, physiological and global analysis (proteome, transcriptome) analytical methods, the work of the Unit, in collaboration with a Japanese laboratory, has made it possible to identify the targets of two structurally similar regulators (the hcaR and yhaJ activators), the level of expression of which increases on entry into the stationary phase. The inactivation of hcaR has a major effect on the expression of seven genes involved in the response to oxygen. This effect manifests itself as a large decrease in resistance to certain oxygen derivatives in stationary phase. The product of the hcaR gene is also a regulator involved in control of the initial steps in the catabolism of 3-phenylpropionic acid. We are currently studying the effects of hcaR inactivation on metabolism on entry into the stationary phase. The results obtained with the yhaJ regulator show that the overexpression of this gene leads to a change in the process of cell division. Identification of the targets of this regulator, involved in this phenotype, is underway. A third, previously unknown regulator of the LysR type (ygiP) was identified as a positive regulator of the neighbouring genes encoding proteins with an activity required for growth on glycerol and anaerobiosis. The comparative analysis of transcriptomes led us to suggest that this activator modulates the expression of genes involved in motility and its regulation, the transport of iron and acid stress. The results of physiological experiments have shown that the inactivation of ygiP leads to an increase in the speed of biofilm formation, thus confirming the results of transcriptome analysis concerning the role of this regulator in the regulation of flagellum biosynthesis.
Annotation, data management and analysis in silico (Ivan Moszer, Eduardo Rocha, Paris; Claudine Médigue, Evry)
Due to the very large body of data generated by genome sequencing and the enigmatic nature of many of the genes discovered, experimental studies carried out with molecular genetic techniques are supplemented by a series of studies using the techniques and concepts of information technology, statistics and mathematics. A specialist Bacillus subtilis database, which has served as the model for several other databases, is accessible via the Internet at http://genolist.pasteur.fr/SubtiList/). This database is currently being updated and improved, as much in conceptual terms as in terms of data, by addition of the transcriptome analysis results generated by the BACELL programme. A genetic annotation platform, Imagene, is being used to establish a general model for the identification of errors in genomic sequences, resulting in a major update of the sequences and annotations of the genome of B. subtilis. This platform has been used to reannotate all of the complete bacterial genome sequences currently available and to identify numerous sources of errors, which multiply in databases. The future of this platform is at the heart of a consortium (Geno*) combining the Unit, a laboratory of the INRIA and the companies Hybrigenics and GenomExpress. The more biological and naturalist aspects of this research aim to determine the link between the major functions of the living being and cellular architecture. Genome analysis has shown that the order of the genes on the chromosome is not random, but is instead linked to the architecture of the cell. The results of our sulphur metabolism studies suggest that the selection pressure responsible for this unexpected link is the diffusion of reactive molecules, gases and free radicals. These reactions are due to the product of numerous genes, the function of which remains unknown. Thus, cellular compartmentalisation may be at the origin of a characteristic of genomes that caused much surprise on its discovery: the presence of numerous genes of unknown function.
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|Office staff||Researchers||Scientific trainees||Other personnel|
DANCHIN Antoine (Professor, Head of Unit at Pasteur, Research Director (DR1 CNRS) firstname.lastname@example.org
BERTIN Philippe (C.R. Pasteur) email@example.com
BIVILLE Francis (C.R. Pasteur) firstname.lastname@example.org
CHARLES Jean-François (C.L. Pasteur) email@example.com
DERZELLE Sylvianne (Postdoc, CDD Industrial contract) firstname.lastname@example.org
MOSZER Ivan (C.R. Pasteur) email@example.com
PIMENTEL CACHAPUZ ROCHA Eduardo (C.R. CNRS) firstname.lastname@example.org
AUGER Sandrine (3rd year PhD student, Univ. Paris 7) email@example.com
MOREIRA Sandrine (2nd year student: DESS Informatique) firstname.lastname@example.org
MARISA Laetitia (1st year student: DESS Informatique)
SOUTOURINA Olga (3rd year PhD student, Univ. Versailles) email@example.com
HOMMAIS Florence (3rd year PhD student, Univ. Paris 7) firstname.lastname@example.org
KRIN Evelyne (I. CNRS) email@example.com
FABRY Cécilia (I.E. INRA) firstname.lastname@example.org
TURLIN Evelyne (T.S. Pasteur) email@example.com
NGO Saravuth (A.I., CDD) firstname.lastname@example.org
BRITO Emma (Laboratory technician) Ebrito@pasteur.fr
MARANGHI Laurence (Laboratory assistant)