Amino acids: inhibitors or promoters of anaerobic biocorrosion of steels?

Various authors have shown that some amino acids can act as corrosion inhibitors, which has generated an increasing interest in these compounds as substitutes to conventional corrosion inhibitors that are usually toxic [1,2]. Nevertheless, in the field of biocorrosion amino acids have been demonstrated to be able to enhance the corrosion process. Their role in biocorrosion is considered to be of two types: an acidifying role at the vicinity of the metal, or a chelating role that binds the metal ions issued from the corrosion process and can result in the formation of ion concentration cells, causing further corrosion on the metal surface [3,4]. The present study proposed another possible role of amino acids in anaerobic biocorrosion of steels. Voltammetric and potentiometric experiments were carried out with 316L stainless steel in solutions containing leucine or lysine at different concentrations and pH. The results demonstrated that the cathodic reaction was enhanced by the presence of amino acids, certainly because of the reduction of the hydrogen atoms linked to the amine or carboxylic acid groups. A so-called deprotonation mechanism, which produces molecular hydrogen, has already been demonstrated with phosphate ions and weak acids [5,6]. It introduces a new reversible cathodic reaction that may enhance the corrosion process, particularly when a downstream reaction of hydrogen consumption occurs

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10gl−1) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19h−1. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06h−1. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD+, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H2 removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H2 scavenging and acetate producing microorganism

A nonlinear mixed-effects approach for the mechanistic interpretation of time-series transcriptomics data

Mechanistic models are essential to unravel the molecular mechanisms driving cellular responses. However, the integration of high-throughput data with mechanistic knowledge is limited by the availability of scalable computational approaches able to disentangle biological and technical sources of variation. Results: We present an approach based on nonlinear mixed-effects modelling for the parameter estimation of large-scale mechanistic models from time-series transcriptomics data. It allows to factor out technical variability, to compensate for the limited number of conditions and time points by a population approach, and it incorporates mechanistic details to gain insight on the molecular causes of biological variability. We applied our approach for the biological interpretation of microarray and RNA-Seq gene expression profiles, with different levels of technical noise, but it is generalisable to numerous types of data. When integrated in a model describing the degradation kinetics of all cellular mRNAs, the data allowed to identify the targets of post-transcriptional regulatory mechanisms. Our approach paves the way for the interpretation of high-throughput biological data with more comprehensive mechanistic models. Availability: The Monolix script for estimation and output files are freely available at https://gitlab.inria. fr/tetienne/eccb_script, together with the microarray data. The RNA-Seq dataset is being prepared for publication (Roux et al., in preparation) and will be made available on demand upon acceptance of the article

Etude des relations structure-fonctions de l hydrogénase à fer de Clostridium acetobutylicum avec ses partenaires d oxydo-réduction

Source d énergie renouvelable et non polluante, le biohydrogène connaît un intérêt croissant. En culture continue sur glucose, la productivité d hydrogène la plus élevée est réalisée par la bactérie anaérobie Clostridium acetobutylicum avec 2,4 litres d hydrogène produit l-1 h-1. Cette production d hydrogène est catalysée par la [FeFe]-hydrogénase HydA. Pour comprendre et améliorer les capacités de ce micro-organisme pour la production d hydrogène, nous avons voulu caractériser les relations structure-fonctions de HydA avec ses partenaires d oxydo-réduction. Par homologie avec l hydrogénase à fer I de Clostridium pasteurianum, les centres [4Fe-4S] FS4C et [2Fe-2S] FS2, situés en surface de la protéine, pourraient être impliqués dans le transfert inter-moléculaire d électrons entre HydA et ses partenaires d oxydo-réduction. Notre objectif a alors été de déterminer l implication de FS4C et FS2 dans ce transfert. Pour cela, des mutations par substitutions d acides aminés et par délétions de domaines ont été effectuées au niveau de FS4C et FS2. Pour palier des problèmes d instabilité des hydrogénases à fer native et modifiées, le protocole de purification a été amélioré. L hydrogénase native a nettement été stabilisée, mais l instabilité persistante des hydrogénases modifiées est restée une limitation importante pour leur caractérisation catalytique. La perte d activité des hydrogénases modifiées a pu être corrélée à une perte importante de fonctionnalité de leur site actif et à un nombre d atomes de fer incorporés inférieur à la valeur théorique. Les partenaires physiologiques d oxydo-réduction de HydA chez C. acetobutylicum, la ferrédoxine CAC0303 et la flavodoxine, ont été purifiés. Le profil catalytique complet et les paramètres cinétiques des activités de consommation et de production d hydrogène de HydA native avec différents partenaires d oxydo-réduction ont été déterminés. L amélioration du protocole de purification a permis d augmenter significativement les activités de consommation et production d hydrogène. Nous avons confirmé la préférence in vitro de HydA à catalyser la consommation de l hydrogène par rapport à la production. Une valeur très élevée de kcat a été obtenue avec le substrat artificiel, méthyl viologène, en consommation d hydrogène. Cela semble indiquer que le méthyl viologène pourrait interagir plus ou moins directement avec le site actif de l enzyme, en évitant le transfert intra-moléculaire d électrons. Des efficacités catalytiques élevées de consommation et de production de l hydrogène ont été obtenues avec le méthyl viologène (sauf sous sa forme réduite), la ferrédoxine et la flavodoxine. Ce résultat reflète le haut potentiel de HydA pour les réactions liées à l hydrogène, potentiel conservé aussi bien pour ses partenaires redox physiologiques qu artificiel. Ainsi, en condition de croissance en carence en fer, la substitution de la ferrédoxine par la flavodoxine ne serait pas une limitation pour l'activité hydrogénase in vivoThe biological production of hydrogen, a "clean" energy carrier, has recently aroused a great interest. The most efficient reported micro-organism for hydrogen production from hexose is the anaerobic bacterium Clostridium acetobutylicum with a chemostat production of 2.4 l H2 l-1 h-1, catalyzed by the HydA [FeFe]-hydrogenase. In order to understand and improve C. acetobutylicum hydrogen potentialities, we have attempted to characterize the structure-function relationships of HydA with its redox partners. By homology with the [FeFe]-hydrogenase of Clostridium pasteurianum, the [4Fe-4S] FS4C and [2Fe-2S] FS2 clusters located on the protein surface were predicted to be involved in the inter-molecular electron transfer between HydA and its redox partners. Our goal was to determine the implication of the FS4C and FS2 clusters in this transfer. To do so, mutagenesis by amino acid substitutions and domain deletions was performed on FS4C and FS2. In order to stabilize native and modified hydrogenases, the purification protocol was improved. Native hydrogenase was successfully stabilized, while the persisting instability of modified hydrogenase was an obstacle of their catalytic characterization. Decreased activity of modified hydrogenases was due to a loss of active site functionality and to a lower iron content than the theoretical value. The physiological redox partners of HydA, ferredoxine CAC0303 and flavodoxin, were purified. The complete catalytic profile of HydA and its kinetic parameter with different redox partners for hydrogen uptake and hydrogen production were determined. The optimized purification protocol led to a significant increase of both hydrogen uptake and hydrogen production activities. We confirmed that the in vitro hydrogen uptake was more favourable than the hydrogen production. A very high kcat was determined with the artificial substrate methyl viologen for the hydrogen uptake activity. This result might indicate that methyl viologen could be prone to interact more or less directly with the active site, shunting the intra-molecular electron transfer chain. High catalytic efficiencies for both H2 uptake and H2 production activities were observed with either artificial (except for reduced methyl viologen) and physiological redox partners. This result reflects the high potential of HydA for hydrogen-related activities which is conserved with three different electron carriers. Hence, in iron-limited growth conditions, the in vivo substitution of ferredoxin by flavodoxin might not be a limitation for in vivo hydrogenase activityTOULOUSE-INSA (315552106) / SudocSudocFranceF

Etude des relations structure-fonction de la Glycéraldéhyde 3-phosphate oxydoréductase et Ingénierie métabolique de Clostridium acetobutylicum pour la production de Biohydrogène

La bactérie Clostridium acetobutylicum est l un des meilleurs microorganismes producteurs d hydrogène. Elle nécessite toutefois une amélioration de son rendement de conversion du glucose en hydrogène avant de pouvoir être utilisée dans un bioprocédé industriel économiquement viable. Il est possible de lever ce verrou métabolique en déviant une partie supplémentaire du flux électronique de la glycolyse vers la formation d hydrogène par l introduction dans le métabolisme central de la bactérie d une Glycéraldéhyde 3-phosphate oxydoréductase (GAPOR), une tungsto-enzyme atypique issue d archaea hyperthermophiles. La création de cette nouvelle chaîne de transfert électronique implique l interaction de trois protéines clés: la GAPOR; un transporteur d électrons: la Ferrédoxine, et l accepteur final responsable de la production d hydrogène: l Hydrogénase. Des améliorations significatives ont ainsi été apportées au système d expression hétérologue de la GAPOR afin d obtenir une enzyme stable et fonctionnelle. Le rôle essentiel de l import du tungstène dans la cellule hôte a été souligné, ainsi que la synthèse et l incorporation des différents cofacteurs dans la protéine. La ferrédoxine étant l intermédiaire de cette nouvelle chaîne, il est indispensable qu elle soit capable d interagir efficacement avec la GAPOR mais également avec l hydrogénase à fer native de C. acetobutylicum. Une analyse comparative des activités de réduction de plusieurs ferrédoxines clostridiales et d archaea par l hydrogénase a permis de confirmer la flexibilité naturelle de cette enzyme vis-à-vis de ce type de partenaire redox et de mettre en évidence la ferrédoxine CAC0303 comme étant potentiellement le meilleur candidat dans le cadre de l approche d ingénierie métabolique envisagée. L influence des différents facteurs biochimiques et biophysiques qui gouvernent le transfert électronique lors de l interaction entre ces deux protéines a été étudié et a notamment fait l objet d une discussion approfondieClostridium acetobutylicum is one of the most efficient reported micro-organism for hydrogen production. However, it requires the improvement of its hydrogen production yield from glucose before being used in an industrial bioprocess economically attractive. It is possible to remove this metabolic restriction by diverting an additional part electronic flow of glycolysis to the formation of hydrogen by the introduction in the central metabolism a 3-phosphate Glyceraldehyde oxidoreductase (GAPOR), an atypical tungsto-enzyme present in some hyperthermophilic archea. The creation of this new electronic chain involves the interaction between three key proteins: a GAPOR, a specific electron carrier: Ferredoxin, and a Hydrogenase enzyme, the final electron acceptor and the catalyst of the hydrogen synthesis. Significant improvements have been achieved to the heterologous system expression of GAPOR in order to obtain a stable and a functional enzyme. The essential contribution of tungsten import into the host cell, cofactors synthesis and incorporation into the protein was demonstrated. The shuttle protein ferredoxin should be able to efficiently interact with both GAPOR and C. acetobutylicum hydrogenase. We have selected several ferredoxins naturally present in the in vivo surrounding of either GAPOR or hydrogenase, and compare their reduction rates by the C. acetobutylicum iron hydrogenase. Although the observed rates confirmed the natural substrate flexibility of iron hydrogenase, the ferredoxin CAC0303 was identified as a potential best partner than the others for the selected metabolic engineering strategy. The influence of different biochemical and biophysical factors that govern the electronic transfer between these two proteins has been particularly studied and discussedTOULOUSE-INSA (315552106) / SudocSudocFranceF

RNA dynamics to control gene expression

International audienc