Oxydation de l'arsenic chez Cenibactérium arsenoxidans, applications potentielles dans la biorémédiation des eaux contaminées par l'arsenic

L'arsenic est un métalloïde naturellement présent dans différents environnements. Les formes inorganiques, l'arsénite (As[III]) et l'arséniate (As[V]) sont les plus abondantes. Ce sont aussi les formes les plus toxiques. L'ingestion d'arsenic, en particulier via l'absorption d'eau contaminée, est à l'origine de graves problèmes de santé publique dans des nombreuses parties du monde. C est pourquoi, différentes méthodes de bio-réhabilitation ont été mises au point. La plupart de ces méthodes utilisent deux étapes : une oxydation chimique de As[III] en As[V], suivie de l'immobilisation de l'As[V]. L'utilisation d'oxydants puissants est à l'origine de pollutions secondaires. L'oxydation par voie microbiologique de l'As[III] permet de proposer une méthode alternative intéressante puisque non polluante. Notre travail s'est focalisé sur l'analyse d'une b-protéobactérie, Cenibacterium arsenoxidans, capable d'oxyder efficacement l'As[III] en As[V]. Nos études constituent des étapes préliminaires pour le développement de méthodologies destinées au traitement d'eaux contaminées par l'arsenic. Nous avons établi les conditions d'obtention de la biomasse d'intérêt en testant de nouveaux supports de culture, basé sur la valorisation de déchets d'industries agroalimentaires. L'oxydation d'As[III] par C. arsenoxidans a été testée avec des cellules en suspension ainsi qu avec des cellules immobilisées dans des billes d'alginate. En utilisant des cellules marquées avec la protéine GFP, nous avons étudié la survie et l'implantation de C. arsenoxidans en milieu non stérile. Enfin, dans le but d'isoler d'autres bactéries utilisables dans les processus de traitements de milieux contaminés par l'arsenic, nous avons développé une méthode simple et rapide pour le criblage de bactéries capables de réaliser l'oxydation d'As[III].Arsenic is a naturally occurring metalloid present in many organic and inorganic compounds. The most abundant arsenic species are the inorganic As[III] and As[V]. The prolonged exposure (occupational or natural) of humans to nonlethal arsenic doses causes chronic health effects, but in long time period usually causes death. Therefore, different chemical technologies were developed for arsenic decontamination of water. Most of them have two stages the oxidation of As[III] into As[V] and the subsequent immobilization of As[V]. The main disadvantage of these technologies is the use of strong chemical oxidants, which causes a secondary pollution of the environment. The replacement of the chemical oxidation step by a biological one has potential for development, mainly due to the lack of secondary pollution and the low impact on the environment. We focused our interest on the studies of an arsenic-oxidizing b-Proteobacterium, recently named Cenibacterium arsenoxidans, which possess high arsenic oxidation capacity. These studies are the preliminary step in order to develop a microbial oxidation step for an arsenic contaminated water cleanup technology. We investigated the optimal growth conditions of the strain, and new nutrient media were tested and developed. In addition to the studies of the As[III] oxidation from fre cells, the As[III] oxidation from immobilized C. arsenoxidans cells were studied. Thereafter, a tracking of the growth of C. arsenoxidans gfp-tagged cells in an open system was performed, which aimed to clarify the colonization and survival ability of the strain in such system, where randomly introduced microorganisms were presented. Also a method for rapid screening of arsenic-transforming bacteria was developed

Oxydation de l'arsenic chez Cenibactérium arsenoxidans, applications potentielles dans la biorémédiation des eaux contaminées par l'arsenic

L'arsenic est un métalloïde naturellement présent dans différents environnements. Les formes inorganiques, l'arsénite (As[III]) et l'arséniate (As[V]) sont les plus abondantes. Ce sont aussi les formes les plus toxiques. L'ingestion d'arsenic, en particulArsenic is a naturally occurring metalloid present in many organic and inorganic compounds. The most abundant arsenic species are the inorganic As[III] and As[V]. The prolonged exposure (occupational or natural) of humans to nonlethal arsenic doses cause

Oxydation de l'arsenic chez Cenibactérium arsenoxidans (Applications potentielles dans la biorémédiation des eaux contaminées par l'arsenic)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

"Unknown-genome" proteomics : a new NAD(P)-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR and high resolution mass spectrometry

We present here a new approach that enabled the identification of a newstrain with unknown genomic background, using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences, and high resolution peptide mass determination of proteolytic digests from two dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular weights of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P)-epimerase/dehydratase by specific assignment of peptide masses to a single open reading frame (ORF), excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity-isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations, identified in the domains (23 47) and (91 118) of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two dimensional gels by mass spectrometry provided a molecular mass of 35400 Da, consistent with an average degree of three hosphorylations

Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assay