Unit: Molecular Programming and Genetic Toxicology - CNRS URA 1444 affiliée à l’INSERM
Director: CLÉMENT Jean-Marie
Our activity is devoted to four axes : 1): the study of Escherichia coli chromosome organization (repeated sequences), 2) the study of the maltose transporter and of ABC (ATP Binding Cassette) systems, 3) bacterial super-integrons which associate mobile genes on the chromosome and 4) the study of genotoxic agents responsible for damages to the genetic material of bacteria or transgenic mice.
First theme: BIMEs, Repeated DNA and Insertion sequences.
(Jean-Marie Clément, Caroline Wilde).
An increasing number of completely sequenced bacterial genomes are avalaible in databanks. Functionnal genomics aim to characterize gene functions and regulation, but bacterial chromosome structure is still poorly investigated and understood.
Repeated sequences, usually described in eukaryotes, are also abundant in prokaryotes. BIMEs (Bacterial Interspersed Mosaic Elements) constitute a family of extragenic repeated sequenced we discovered in E.coli and closely related Enterobacteria. We created a specialized databank devoted to intergenic DNA repeats in enterobacteria (http://www.pasteur.fr/recherche/unites/pmtg/repet/index.html). The function of BIMEs remains hypothetical. We suggested that at least some of them could bind proteins and participate to the chromosome structure.
An analysis of intergenic regions from E. coli natural isolates brought two important results:
Each isolate harbors a characteristic pattern of repeated sequences in interenic regions. This finding found an application for the typing of bacterial strains, including Bacillus anthracis, the causative agent of anthrax.
In some isolates, IS1397, a new insertion sequence (IS) is inserted specifically into Palidromic Units (PUs), one of the basic constituents of BIMEs. In Klebsiella pneumoniae, ISKpn1, a second PU-specific IS was discovered. Such a target sequence specificity is unprecedented.
We selected IS1397 transposition events in Enterobacteria (E. coli, Salmonella typhimurium, K. pneumoniae and Yersinia pestis). IS1397 inserts almost exclusively into E. coli PU types, whereas ISKpn1 is specific for K. pneumoniae PUs which are slighly different. A study carried out in Yersinia pestis, a species closely related to E. coli but free of PUs, indicated that DNA circles encompassing the transposable module are probably intermediates of IS1397 transposition and that IS1397 binds directly its PU target.
Sequence comparison of IS1397 and ISKpn1 insertion sites in E. coli and K. pneumoniae together with the use of genetic systems allowing transposition into plasmids allowed us to demonstrate that target specificity of the twi ISs is entirely due to their transposases and is independent from their terminal inverted repeats. The two ISs have thus evolved to be species-specific, being able to transpose safely into intergenic regions. Their dissemination among other enterobacterial species requires a new adaptation which involves mutations enabling recognition of a different type of PU.
Second theme: The ABC maltose transporter and phylogenetic analysis of ABC systems (Elie Dassa, Philippe Bouige, Dorothée Murat, Alireza Pak, Jocelyn Mahaux, Anne Charlery, Olivier Garcia)
The ATP-Binding Cassette (ABC) systems constitute the largest family of paralogues ever found (http://www.pasteur.fr/recherche/unites/pmtg/abc/index.html). They are involved in a variety of biological processes, including not only transport across membranes, but also regulation of translation and transcription or DNA repair. In humans, 15 severe inherited diseases (including cystic fibrosis) are caused by the dysfunction of ABC transporters.
We are pursuing two objectives :
1) Orphan ABC proteins
Number of ABC proteins have no identified membrane associated partners. Several functions have been characterized and deal with transcription or translation regulation and DNA repair. In Staphylococcus aureus, Vga is involved in streptogramin resistance. A collaboration with O. Chesneau was undertaken to understand the resistance mechanism. Four Escherichia coli genes with unknown functions belong to the same family and are investigated (D. Murat, A. Pak, J. Mahaux et E. Dassa). The Programme tranversal de Recherches N°55 co-ordinated by E. Dassa investigates the consequences on inactivation of homologous ORFs in the genomes of S. typhimurium, S.aureus, H. pylori, V. cholerae and Synechocystis.
2) Evolution of ABC systems
We developped a comprehensive database (ABCISSE) on these systems gathering sequences, structures and functions . More than 2650 different ABC ATPases and over 7000 ABC-related genes (taking their partners into account) are registered. An automated annotation tool allows to predict the functions (rarely investigated experimentally, and unknown in half of the cases), the substrate specificities and the ATPases partners (http://www.pasteur.fr/recherche/unites/pmtg/abc/database.iphtml).
Our phylogenetic analysis clusters ABC systems in three classes which match perfectly with the three functional classes : importers (exclusively prokaryotic), exporters and ABC systems apparently devoid of transmembrane domains probably involved in regulatory processes. The analysis of ABC systems in fully sequenced eukaryotic genomes suggests that eukaryotes use a relatively limited set of essential ABC systems, irrespectively of very large differences in genome sizes. We identified 6 typical prokaryote systems in the nuclear genome of Arabidopsis : most of eukaryotic ABC systems were probably acquired from the symbiotic bacterial ancestors of mitochondria and chloroplasts (E. Dassa, A. Charlery, O. Garcia, P. Bouige).
Third theme: Super-integrons
(Didier Mazel, Latefa Biskri, Gaëlle Demarre, Anne-Marie Guérout, Chiho Mashimo, Dean Rowe-Magnus)
Horizontal gene tranfers play a fundamental role in bacterial evolution. Many examples can be found, from the expression of virulence factors in pathogens to the development of new metabolic traits. The development of multi-drug resistance over the last decades is presently a serious challenge in public health. This is probably one of the most striking and worrysome example of the impact of gene transfer.
The contribution of integrons, a peculiar class of genetic elements in the evolution of resistance in Gram-negative bacteria has been substantiated. Integrons constitute a natural genetic engineering device. Due to the activity of a cassette-associated site-specific recombinase , they capture, express and disseminate exogenous DNA.
We have discovered another type of integrons with a broader role, the super-integrons, which assemble hundreds of cassettes on the chromosome of several γ-proteobacteria (Vibrio, Shewanella, Xanthomonas, Pseudomonas...). Phylogenetic analysis of super-integron integrases indicates that they may have evolved from an integron carried by the common ancestor of these bacteria. Thus this system may have played the role of an evolutionary device in bacteria for millions of years. They presently maintain a considerable reservoir of adaptive functions (metabolism, detoxification, resistance, virulence...). They seem to be able to capture genes from remote and diverse origins, since some of them have only identified homologs in eukaryotes, archaebacteria or viruses. Our recent studies point out the super-integrons as the source of multi-resistant integrons. Our current projects deal with the inventory of these elements and of the functions that they could encode, the expression profile of the cassettes, the recombination parameters and the cassette assembly process (http://www.pasteur.fr/recherche/unites/pmtg/integ/index.html).
Fourth theme: Genetic Toxicology
(Philippe Quillardet, Marie-Ange Rouffaud)
We are involved in a preventive approach consisting in screening environmental compounds (genotoxics) able to generate mutations at the origin of human cancers. We developped SOS-chromotest, a short-term bacterial colorimetric test for the screening of genotoxic agents. It has been validated by the use of a large number of compounds (http://www.pasteur.fr/recherche/unites/pmtg/toxic/index.html).
1) Nitrofuran mutagenesis in Escherichia coli
Nitrofurantoin and nifuroxazide, two mutagenic compounds in bacteria, are widely used against urinary tract infections and acute diarrhoea from bacterial origin. R7000, another member of the nitrofuran family, is one of the most potent mutagens in bacteria. We analysed R7000-induced DNA adducts (mostly on guanines) and resulting mutations (mainly G:C-T:A base pair substitutions and G:C base pair deletions). A SoxRS-dependant metabolic activation by nitroreductases is necessary and generates an oxidative stress which could enhance nitrofuran genotoxicity.
2) Nitrofuran mutagenesis in mice
Genotoxic effects of R7000 have also been studied in an animal model ("Big-Blue"transgenic mice carrying a bacterial reporter gene) in order to evaluate the possible consequences of nitrofurans exposition in humans. Intraperitoneal injection of R7000 induces mutations in small intestine, caecum and colon, the actual targets of the therapeutic action of most nitrofurans. The mutation spectrum, and thus the probable mode of action of R7000 are very similar in mice and E.coli. Oral administration of nitrofurantoin induced a mutagenic effect in kidney and R7000 in stomach. On the contrary, no mutagenic effect was observed with by nifuroxazide.
3) Genotoxics and DNA arrays
E.coli DNA arrays allows to identify genes induced or repressed by a broad spectrum of genotoxic agents (particularly in pollutants from the natural environment) and to define a "signature" for each of them. Their action on metabolism, on activation and on stress response will be depicted and unidentified functions of some ORFs will be uncovered. On the other hand, the construction of bacterial strains carrying fusions between diagnostic genes and a reporter gene could constitute the basis for a test allowing the rapid screening environmental genotoxic agents.
Keywords: Chromosome structure, ABC Systems, Superintegrons, Genome plasticity, Genetic Toxicology, Carcinogenesis