Les travaux scientifiques ont abouti à la publication de huit articles scientifiques dont sept en anglais avec de bons facteurs d'impact:
- un Nature Chemical Biology: impact factor de 14.6
- un Journal of the American Chemical Society; impact factor de 8.6
- un PLos Genetics: impact factor de 7.7
- un Analytical Chemystery; impact factor de 5.7
- deux Molecular Microbiology: impact factor de 5.6
- un Faraday Discussions: impact factor de 3.7.

Liste des publications:

Baffert, C., Bertini, L., Lautier, T., Greco, C., Sybirna, K., Ezanno, P., Etienne, E., Soucaille, P., Bertrand, P., Bottin, H., Meynial-Salles, I., De Gioia, L., Léger, C. (2011)
 Exogenous carbon monoxide disrupts the reduced H-cluster of FeFe-hydrogenase. A combined DFT and PFV study.
Journal of the American Chemical Society epub
L'article complet en PDF: Baffert et al 2011.pdf
Summary
Hydrogenases (H2ases), the enzymes that catalyze the biological production and oxidation of H2, are classified according to the metal content of their active site. In FeFe-H2ases, H2 activation occurs at the H-cluster, which consists of a [Fe2(CO)3(CN)2(dtma)] subsite (dtma=dithiomethyl amine) covalently bound to a [4Fe4S] cluster. During catalysis, the 2Fe subsite exists in the FeIIFeI and FeIFeI states (Hox and Hred, respectively). The cubane is reduced in the so-called super-red state (Hsred). In theoretical and experimental studies of the H-cluster, CO is often considered as an innocent ligand which competes for hydrogen, although early experiments with the H2ase from Clostridium pasteurianum (Cp) showed that CO may also alter the enzyme in an irreversible manner. Recent experiments clarified this by demonstrating that CO inhibition is fully reversible when the enzyme is oxidizing H2, not when it is producing H2. Here we elucidate the nature of the irreversible damage induced by exogenous CO.

Lautier T., Ezanno P., Baffert C., Fourmond V., Cournac L., Fontecilla-Camps J. C. , Soucaille P., Bertrand P., Meynial-Salles I. and Leger C. (2011)
 The quest for a functional substrate access tunnel in FeFe hydrogenase.
 Faraday Discussions 148: 385.
L'article complet en PDF: Lautier et al 2011.pdf
Summary
We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative ‘‘static’’ H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzyme’s kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.

Liebgott, P.P., Leroux, F., Burlat, B., Dementin, S., Baffert, C., Lautier, T., Fourmond, V., Ceccaldi, P., Cavazza, C., Meynial-Salles, I., Soucaille, P., Fontecilla-Camps, J., Guigliarelli, B., Bertrand, P., Rousset, M., Léger, C. (2010)
Hydrogenases: the relation between diffusion along the substrate tunnel and oxygen sensitivity.
Nature Chemical Biology 6: 63-70.
L'article complet en PDF: Liebgott et al 2010.pdf
Summary
In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O(2) in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O(2) toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H(2) and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O(2) because access to the active site is slow.

Fourmond, V., Lautier, T., Baffert, C., Leroux, F., Liebgott, P.P., Dementin, S., Rousset, M., Arnoux, P., Pignol, D., Meynial-Salles, I., Soucaille, P., Bertrand, P., Léger, C. (2009)
Correcting for electrocatalyst desorption and inactivation in chronoamperometry experiments.
Analytical Chemystery 15;81: 2962-8.
L'article complet en PDF: Fourmond et al 2009.pdf
Summary
Chronoamperometric experiments with adsorbed electrocatalysts are commonly performed either for analytical purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concentration is being increased to determine a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such experiments, the fact that the catalyst's coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. We propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric experiments. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control experiment. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. We describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case we discuss the trustfulness and the benefit of the correction. This method also applies to experiments where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concentration of catalyst.

Lautier, T. and Nasser, W. (2007)
The DNA nucleoid-associated protein Fis coordinates the expression of the main virulence genes in the phytopathogenic bacterium Erwinia chrysanthemi.
Molecular Microbiology 66: 1474-90.
L'article complet en PDF: Lautier and Nasser 2007.pdf
Summary
Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases (Pels) play a key role in soft rot symptoms but the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include the harpin HrpN, the cellulase Cel5, proteases (Prts), flagellar proteins and the Sap system, involved in the detoxification of plant antimicrobial peptides. HrpN and flagellum are mostly involved in the early steps of infection whereas the degradative enzymes (Pels, Cel5, Prts) are mainly required in the advanced stages. Production of these virulence factors is tightly regulated by environmental conditions. This report shows that the nucleoid-associated protein Fis plays a pivotal role in the expression of the main virulence genes. Its production is regulated in a growth phase-dependent manner and is under negative autoregulation. An E. chrysanthemi fis mutant displays a reduced motility and expression of hrpN, prtC and the sap operon. In contrast, the expression of the cel5 gene is increased in this mutant. Furthermore, the induction of the Pel activity is delayed and increased during the stationary growth phase in the fis mutant. Most of these controls occur through a direct effect since purified Fis binds to the promoter regions of fis, hrpN, sapA, cel5 and fliC. Moreover, potassium permanganate footprinting and in vitro transcription assays have revealed that Fis prevents transcription initiation at the fis promoter and also transcript elongation from the cel5 promoter. Finally, the fis mutant has a decreased virulence. These results suggest a coordinated regulation by Fis of virulence factors involved in certain key steps of infection, early (asymptomatic) and advanced (symptomatic) phases.

Lautier, T., Blot, N., Muskhelishvili, G. and Nasser, W. (2007)
Integration of two essential virulence modulating signals at the Erwinia chrysanthemi pel gene promoters: a role for Fis in the growth phase regulation.
Molecular Microbiology 66: 1491-505.
L'article complet en PDF: Lautier et al 2007.pdf
Summary
Production of the essential virulence factors, called pectate lyases (Pels), in the phytopathogenic bacterium Erwinia chrysanthemi is controlled by a complex regulation system and responds to various stimuli, such as the presence of pectin or plant extracts, growth phase, temperature and iron concentration. The presence of pectin and growth phase are the most important signals identified. Eight regulators modulating the expression of the pel genes (encoding Pels) have been characterized. These regulators are organized in a network allowing a sequential functioning of the regulators during infection. Although many studies have been carried out, the mechanisms of control of Pel production by growth phase have not yet been elucidated. Here we report that a fis mutant of E. chrysanthemi showed a strong increase in transcription of the pel genes during exponential growth whereas induction of expression in the parental strain occurred at the end of exponential growth. This reveals that Fis acts to prevent an efficient transcription of pel genes at the beginning of exponential growth and also provides evidence of the involvement of Fis in the growth-phase regulation of the pel genes. By using in vitro DNA-protein interactions and transcription experiments, we find that Fis directly represses the pel gene expression at the transcription initiation step. In addition, we show that Fis acts in concert with KdgR, the main repressor responding to the presence of pectin compounds, to shut down the pel gene transcription. Finally, we find that active Fis is required for the efficient translocation of the Pels in growth medium. Together, these data indicate that Fis tightly controls the availability of Pels during pathogenesis by acting both on their production and their translocation in the external medium.
 

Le Bourgeois, P., Bugarel, M., Campo, N., Daveran-Mingot, M.L., Labonté, J., Lanfranchi, D., Lautier, T., Pagès, C. and Ritzenthaler, P. (2007)
The Unconventional Xer Recombination Machinery of Streptococci/ Lactococci.
PLoS Genetics 3: e117.
L'article complet en PDF: Le Bourgeois et al 2007.pdf
Summary
Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (difSL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine difSL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when difSL sites are located on chromosome dimers. Moreover, the XerS/difSL recombination requires the streptococcal protein FtsKSL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs.

Lautier, T. et Nasser, W. (2005)
Régulation des gènes de virulence par la phase de croissance chez les bactéries à Gram négatif.
Regard sur la Biochimie 1: 19-28.
L'article complet en PDF: Lautier et Nasser 2005.pdf
Résumé:
L’un des moyens couramment utilisé par les pathogènes pour échapper aux réactions de défense de l’hôte est de n’induire une synthèse massive de leurs facteurs de virulence susceptibles d’éveiller les réactions de défense de l’hôte que lorsqu’ils sont en quantité suffisamment importante pour conduire une attaque brusque et forte de l’hôte. Ce type de contrôle communément appelé régulation par la phase de croissance s’exerce sur les gènes de virulence chez les bactéries Gram négatif essentiellement via trois mécanismes :
– Lors de conditions environnementales défavorables, le transcriptome est modifié par un facteur global de transcription : le facteur Sigma S. Il met en place la réponse générale au stress et contrôle également des gènes de virulence. Sigma S permet de coupler le déclenchement de la virulence avec la réponse générale au stress.
– La plupart des bactéries à Gram négatif synthétisent un type de molécule diffusible, appelé autoinducteur dont la concentration est le reflet de la densité cellulaire ; la perception de la densité cellulaire via les autoinducteurs est appelée « quorum sensing ». Ce système initie une réponse concertée de la population bactérienne. Le « quorum sensing » permet d’assujettir la synthèse des facteurs de virulence à la densité cellulaire.
– Enfin, les bactéries possèdent des protéines « histone-like » impliquées dans la structuration de l’ADN génomique. La plupart de ces protéines ont une fonction de régulateur en plus de leur rôle architectural dans le nucléoïde. L’expression de certains gènes de virulence est également modulée par les protéines « histone-like ».
Par ailleurs, il existe des connexions entre les trois systèmes de régulation de la virulence par la phase de croissance. Ce mode d’organisation intégrée permet au pathogène d’assurer une synthèse des facteurs de virulence appropriée aux différentes phases du processus infectieux.

Liste des posters:

Lautier T., Ezanno P., Baffert C., Fourmond V., Cournac L., Fontecilla-Camps J.C., Soucaille P., Bertrand P., Léger, C and Meynial-Salles, I (2010)
             The quest for a functional gas substrate access tunnel in FeFe hydrogenase from Clostridium acetobutylicum.
             Congrès Clostridium (San Diego, California).

Le poster en PDF: poster-congres-clostridium-2010.pdf

Ezanno P., Lautier T., Baffert C., Fourmond V., Cournac L., Fontecilla-Camps J.C., Soucaille P., Bertrand P., Meynial-Salles I., Léger, C. (2010)
            A site-directed mutagenesis study of the [FeFe] hydrogenase HydA of Clostridium acetobutylicum.
            Congrès Hydrogenase (Uppsala, Suède).

Zghidi-Abouzid, O., Reverchon, S., Lautier, T., Muskhelishvili, G. and Nasser, W. (2010)
Expression of the main virulence genes in Dickeya dadantii (formely Erwinia chrysanthemi) is sensitive to the DNA supercoiling state.
Congrès 35TH FEBES, (Gothenburg, Suède).

Lautier, T. and Nasser, W. (2007)
Central regulatory role of the nucleoid associated protein Fis in the virulence of Erwinia chrysanthemi.
Congrès Molecular Plant Microbe Interactions, Sorrento, Italy.

Le poster en PDF: poster Erwinia.pdf