First theme. BIMEs, Repeated Sequences and Genome. (Jean-Marie Clément, Sophie Bachellier, Caroline Wilde, Muguette Jehanno, Patricia Lambert).
Bacterial chromosome structure is still poorly understood, despite the full sequencing of several genomes. Repeated sequences, usually described in Eukaryotes, are also abundantly present in Prokaryotes. Besides prophages, transposons and Insertion Sequences, an increasing number of examples is supporting this observation. We undertook a systematic computer survey of recently sequenced genomes to reveal new families of intergenic repeated sequences. The function of such sequences remains hypothetical. We suggested that at least some of them could bind proteins and participate to the chromosome structure.
We are particularly interested in BIMEs (Bacterial Interspersed Mosaic Elements), a family of extragenic repeated sequenced we discovered in E.coli and closely related Enterobacteria. Mosaic structure of BIMEs follows a very precise organization pattern. We developped a specialized databank devoted to this type of bacterial repeats (freely accessible at the following address : http://www.pasteur.fr/recherche/unites/pmtg/).
We try to elucidate BIME function in E. coli, noteworthy by the identification of BIME binding proteins. Three of them have been discovered (one was identified by our group). To find others, we use yeast one-hybrid technique : BIMEs from various types have been inserted into yeast chromosomes and E. coli proteins able to bind them can be selected by a functionnal assay. This approach will be completed by in vitro interaction studies.
A comparative examination of E. coli natural isolates revealed the presence of a new Insertion Sequence (IS1397) specific for Palindromic Units (PUs), the basic constitutive motif of BIMEs. Such a target sequence specificity is unprecedented. We showed that tranposase overexpression is toxic , leading to filamentation and nucleoid desagregation. Resistant mutants were isolated and are currently under study. The development of appropriate genetic tools allowed us to select IS1397 transposition events in Enterobacteria. This transposition is RecA independent and is almost exclusively specific for PUs in E. coli, Salmonella typhimurium and Klebsiella pneumoniae. IS1397 inserts however preferentially in PUs of the E. coli type which are slighly different from the consensus found for other species. In Klebsiella pneumoniae, a second IS was discovered. ISKpn1 seems this time specific for Klebsiella pneumoniae PUs. The mechanisms accouting for this difference in specificity are under study. In Yersinia pestis, a species closely related to E. coli but free of PUs, IS1397 transposes either downstream of sequences which share homology with E. coli PUs or downstream of a resident IS in which the ends of inverted terminal repeats (IR) are almost identical to IS1397. We also found DNA circles which were probably the result of an excision from the chromosome, which confirms the role of such circular intermediates in the process of.transposion.
We aim to substantiate our initial hypothesis of the role of BIMEs in chromosome structure. Undergoing studies seek to identify proteins which bind IS1397 transposase (Yeast double hybrid method and study of mutants resistant to the toxic effect of transposase overexpression). A systematic deletion of BIMEs from the E. coli chromosome is also on progress.
Second theme. The maltose transporter MalFGK2 complex and the phylogenetic analysis of ABC systems (Elie Dassa, Philippe Bouige, Olivier Pellegrini, David Laurent, Tristan Rossignol)
The ATP-Binding Cassette (ABC) superfamily is composed of systems that are widespread in all living organisms. These systems form the largest family of paralogues ever found. They are involved in a large number of living processes including primarily but not only transport. In humans, 15 severe genetic diseases including cystic fibrosis are caused by the dysfunction of ABC transporters .The most prominent feature of these system is to share a highly conserved domain, displaying conserved Walker motifs A and B common to ATPases and another motif characteristic of ABC transporters, the LSGGQ or signature motif. This domain is able to bind and to hydrolyze ATP, thereby coupling ATP hydrolysis to a large variety of biological processes, including transmembrane transport, translation transcription and their regulation. The ABC maltose import system of Escherichia coli is a model of ABC transporters. Its functional characteristic are investigated in our group since the early 70's. We contributed significantly to the physiological, genetic and more recently to the molecular characterization of this system. Bacterial ABC systems have been studied in details at the functional and the structural points of view and, due to the high conservation of their overall architecture and functional properties, the analysis of their properties will enrich and stimulate research on human ABC systems involved in pathologic manifestations.
The primary concern of our research team is to understand the dynamic interplay of protein interactions in multicomponent transporters, taking the maltose transporter in E. coli as a model. We use genetic and biochemical methods to detect and measure the strength of such interactions in order to characterize the different structural states of the transporter during its functional cycle. We also make functional and evolutionary studies on the superfamily of ABC systems.
a) Interactions between cytoplasmic membrane components in the maltose transporter (with T Rossignol and O Pellegrini)
Integral membrane proteins of bacterial import ABC systems display a short hydrophilic conserved motif, the EAA motif located at about 100 residues from the C-terminus. The motif is hydrophilic and was found to reside in a cytoplasmic loop. Our previous work suggested that EAA motifs could be directly interact with the ATPase MalK. We have investigated the proximity of residues in the conserved EAA sequence of MalF and MalG the MalK subunits by means of site-directed cysteine chemical cross-linking. We demonstrated that critical residues in MalK participate in constitution of asymmetric interaction sites with the EAA loops of MalF and MalG. These interactions are strongly modulated by ATP, indicating a structural rearrangement of the subunits during the transport cycle. Thus, it is tempting to speculate that the observed changes in subunit-subunit interactions may reflect structural rearrangements that are required to initiate ATP hydrolysis and transport. Recently, we demonstrate that EAA regions could be cross-linked by the same technique, suggesting a close proximity of theses loops in the transporter.
b) Evolution of ABC systems (with D Laurent and P Bouige).Our recent analyses show that ABC systems cluster in three phylogenetic classes: importers (exclusively prokaryotic), exporters and ABC systems apparently devoid of transmembrane domains probably involved in regulatory processes. This fits remarkably well with the three functional classes of ABC systems described above. The analysis of the distribution of ABC systems in 5 eukaryotic genomes led to two remarkable observations. First, the ABC protein content of yeast (29 systems), the fruit-fly (55), worm (60) and Arabidopsis thaliana (116) and man (48) is conserved in number and in nature. The higher number of Arabidopsis ABC systems is due to extensive multiple duplications. This observation suggests that eukaryote use a relatively limited set of essential ABC systems, irrespectively of very large differences in global gene number. Second, we identified 6 typical prokaryote systems in the nuclear genome of Arabidopsis thaliana. This supports our hypothesis that almost all eukaryote ABC systems were acquired from the genomes of the symbiotic bacteria which were the ancestors of mitochondria and chloroplasts.
Our phylogenetic studies on the components of ABC systems led to the first comprehensive phylogenetic and functional classification of ABC systems. The sequences of more than 2650 different ABC ATpases are presently deposited in the Genbank database. The total number of ABC-related genes is about 7000 when the partners of these ATPases are taken in account. About the half is comprised of systems discovered during genome sequencing projects and their annotation is very limited. Our goal is to establish a knowledge database of these systems, that will include functional, sequence and structural information. This database will be helpful to annotate accurately ABC systems and to identify the partners of the ATPases. We will provide a quasi automatic annotation tool that perform in a single step the prediction of the function, the substrate specificity and the putative partners of any newly described ABC ATPase. A working version of this database is available on the following Internet site:
The third research axis deals with super-integrons: i) specifically their phenotypic impact, ii) the functions of the genes which are found in their cassettes, and iii) the mechanisms of gene recruitment and cassette assembly. (Didier Mazel, Anne-Marie Guérout, Dean Rowe-Magnus, Latefa Biskri, Chiho Mashimo, Gaëlle Demarre)
Horizontal gene tranfers play a fundamental role in bacterial evolution. One can find many examples, from the expression of virulence factors in pathogens to the development of new metabolic traits, to illustrate this statement. Yet, the most striking, and perhaps the most worrysome, example of the impact of gene transfer has been the development of multi-drug resistance over the last half of the 20th century. In this global phenomenon, the contribution of a peculiar class of genetic elements, the integrons, in the evolution of resistance in Gram-negative bacteria has been substantiated. Integrons constitute a natural genetic engineering device that captures, expresses and disseminates exogenous DNA. We have shown the existence of another type of integrons with a broader role - the chromosomal super-integrons. We have identified such integron super-structures in 9 distinct genera throughout the g-proteobacterial radiation (Vibrio, Shewanella, Xanthomonas, Pseudomonas, ...). These super-integrons gather hundreds of cassettes on the chromosome in these bacterial species. Phylogenetic analysis of the associated integrases revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures that may have evolved from an integron carried by the genome of the common ancestor of these bacterial groups. Thus this system plays and played the role of an evolutionary device in bacteria for millions of years. These structures maintain a considerable reservoir of adaptive functions (metabolism, detoxification, resistance, virulence...). Furthermore, this system appears to be able to capture genes from remote and diverse origin, as cassettes carrying genes only described in eukaryotes, archaebacteria or viruses have been identified. Our recent studies point the super-integrons as the source of both the multi-resistant integrons platforms and their resistance cassettes. Our current projects deal with several aspects: the inventory of these elements and of the functions that they could encode, the expression profile of the gene cassettes, the recombination parameters and the cassette assembly process.
Fourth theme: Genetic Toxicology (Philippe quillardet, Eliette Touati, Xavier Arrault, Valérie Michel, Marie-Ange Rouffaud)
The group of "Genetic Toxicology" is involved in a preventive approach consisting in screening environmental compounds (genotoxic) able to damage the genome of our cells (genotoxic effects) and to generate mutations at the origin of human cancers .
We have used the genotoxic response of the bacterial cell to devise a screening test for potential mutagenic and/or carcinogenic agents: the SOS chromotest. The test allows to rapidly evaluate, by a simple colorimetric assay, the capacity of an agent to dammage DNA. It has been evaluated with a large number of compounds (see data base: www.pasteur.fr/units/pmtg/).
In order to study the functions induced by a broader spectrum of toxic agents (potentially carcinogenic genotoxic agents, non-genotoxic carcinogens, various pollutants of the natural environment), we use high density DNA arrays containing all PCR-amplified open reading frame (ORFs) from E.coli K12. The objective is to define classes of toxic agents on the basis of genes for which the expression is modified, and to define genes, or groups of genes, diagnostic for each classes of agents. One considers the spectrum of genes for which the expression is modified by a chemical or physical agent as "signature" of the action of the agent on the cell.
The knowledge of diagnostic genes for classes of toxic agents may bring informations on various aspects of their action (metabolism, activation, stress response) and to reveal the properties of ORF unidentified to day. On an other hand, we hope that this work leads to the construction of bacterial strains carrying fusions between these diagnostic genes and a reporter gene to allow the screening of these agents in the environment.
We are also involved in studying the mechanism of action of genotoxic agents. Particularly, we have studied in details the mechanism of action of an very powerful bacterial mutagen, R7000, a nitrofuran. Nitrofuran derivatives are widely used as antibacterial agent in medecine.
Our objective is to explain, in terms of molecular interactions, why R7000 reaches such a powerful mutgenic potency. We have located the R7000 induced DNA damages and the resulting mutations in Escherichia coli. We have found that R7000 induced DNA adducts with a very high efficiency. These R7000 induced DNA adducts are essentially on modified guanines and the resulting mutations are mainly G:CT:A base pair substitutions and G:C base pair deletions. The genotoxic activity of nitrofurans requires their metabolic activation catalyzed by nitroreductases. During this metabolism a soxRS-dependant oxidative stress is also generated. Although it is likely a side effect it could contribute to the nitrofuran genotoxicity.
The genotoxic effects of R7000 has also been studied in an animal model in order to establish a relationship between results obtained in bacteria and results obtained in animal and to to try to evaluate the possible consequences of nitrofurans exposition in human. Transgenic mice, so called "Big-Blue" have been used. These mice present inserted in their genomic DNA a lambda bacteriophage containing the E.coli lacI gene used as a reporter gene for frequencies of mutations. Thus it is possible to determine the mutagenic potential of a chemical in any mice organs. We found that, when administrated by intraperitoneal route, R7000 induces mutations in small intestine, caecum and colon, organs belonging to the digestive apparatus, the target of the therapeutic action of most nitrofurans. The mutation spectrum induced by R7000 in mice is very similar to what had been found in E.coli suggesting that the mechanism of its genotoxic action is similar in both organisms.
Nitrofurantoin and nifuroxazide are two nitrofuran derivatives widely used in human medecine for therapy of urinary tract infections and acute diarrhoea from bacterial origin, respectively. The two compounds are mutagenic in bacteria and consequently they could have long term adverse effects on human health. The mutagenic action of these two compounds and of R7000 have been evaluated in "Big-Blue" transgenic mice after oral administration. We found that nitrofurantoin induced a mutagenic effect in kidney and R7000 in stomach. No mutagenic effect in any of the organs tested was induced by nifuroxazide.
The aim of another study in our laboratory is to analyze why infection from bacterial origin are susceptible to induce genotoxic events and lead to cancer development in the population. The same transgenic mice cited above constitute an appropriate tool to analyze if the infection results in an increase of the mutant frequency at the infected site, which could be responsible for events at the origin of precancerous lesions. The model choosen is the infection of the gastric mucosa by Helicobacter pylori (work in collaboration with Dr A. Labigne's team and Dr M. Huerre's team at the Institut Pasteur). This bacteria is now recognized as the aetiological factor of chronic gastritis. Its presence in gastric mucosa is associated with the development of peptic ulcer, and should be responsible for the development of gastric cancer. Bacterial, host as well as environmental factors may contribute either directly or indirectly to the promotion of genotoxic events.
Our purpose is to examine in what conditions, chronic infection of mice with Helicobacter pylori, lead to an increase of the frequency of mutational events at the gastric epithelium level which could promote gastric tumors. This work implies the elaboration of an animal model for the study of this infectious disease.