Caractérisation fonctionnelle de gènes de Marinobacter hydrocarbonoclasticus lors du développement de biofilms sur composés organiques hydrophobes

Les composés organiques hydrophobes (HOCs), lipides et hydrocarbures, représentent une part significative de la matière organique dans l environnement marin. Leur faible solubilité dans l eau exige de la part des bactéries qui les dégradent des adaptations physiologiques permettant de stimuler leur transfert de masse de la phase organique vers la phase aqueuse où ils sont assimilés. La formation de biofilm à l interface HOC-eau est l une de ces adaptations. La bactérie marine Marinobacter hydrocarbonoclasticus (Mh), qui est capable d utiliser un catalogue assez large de HOCs comme les alcanes, les alcools gras et les triglycérides, a été utilisée comme modèle d étude de la formation de biofilms aux interfaces HOCs-eau. Le but de mes recherches était de : (i) mener la caractérisation fonctionnelle des gènes aupA et aupB, qui sont surexprimés en condition de biofilm sur hexadécane et (ii) dresser, par une étude de transcriptomique, une liste de gènes potentiellement impliqués dans l adhésion et la formation de biofilm aux interfaces HOCs-eau dans le but d appréhender les mécanismes moléculaires mis en jeu. L étude fonctionnelle de aupA et aupB a révélé que ces deux gènes forment un opéron dont l expression est activée par divers types de HOCs. Il a aussi été démontré qu ils sont impliqués dans le transport de l hexadécane et dans la formation de biofilm sur alcanes. La protéine AupA est localisée dans la membrane externe de Mh et AupB, une lipoprotéine présumée, est située dans la membrane interne. AupA appartient à une sous-famille de transporteurs FadL-like, spécifique des bactéries marines hydrocarbonoclastes (HCB). La distribution phylogénétique de l'opéron aupAB limitée aux bactéries marines ayant la capacité de dégrader les alcanes et sa présence en nombreuses copies chez certaines souches d Alcanivorax sp. suggèrent fortement que les protéines Aup joueraient un rôle primordial dans l adaptation des HCB à l utilisation d alcanes comme sources de carbone et d énergie. L analyse transcriptomique des cellules de Mh adhérées (après 15 min ou 3 h de contact) ou formant un biofilm aux interfaces HOCs-eau a révélé une modification importante et précoce de leur transcriptome. De nombreux gènes intervenant dans le métabolisme des HOCs, la production de polysaccharides, la synthèse d acides aminés et de protéines ribosomales présentent une expression modulée dès 15 min d adhésion. La surexpression des gènes de flagelle et du chimiotactisme conjointement avec celle de gènes de pili en condition d adhésion évoquent une possible mobilité des cellules de Mh à l interface dans les étapes précoces du développement du biofilm. De plus, il semblerait que le facteur de transcription RpoN soit impliqué dans la régulation de la formation de biofilm chez Mh et que les prophages puissent intervenir dans la structure et/ou la dispersion du biofilm. Enfin, le rôle potentiel d un îlot génomique dans la formation de biofilm sur trioléine a été suggéré.Hydrophobic organic compounds (HOCs), such as lipids and hydrocarbons, represent a significant part of the organic matter in the marine environment. Their low solubility in water requires from bacteria that degrade them physiological adaptations to stimulate their mass transfer from the organic to the aqueous phase where they are assimilated. Biofilm formation at the HOC-water interface is one of those adaptations. The marine bacterium Marinobacter hydrocarbonoclasticus (Mh) which is able to use a broad range of HOCs such as alkanes, fatty alcohols and triglycerides, was used as a model to study the biofilm formation at HOCs-water interfaces. The aim of my research was to (i) conduct the functional characterization of aupA and aupB genes which are overexpressed in biofilm on hexadecane, (ii) draw up a list of genes, through a transcriptomic study, that are potentially involved in adhesion and biofilm formation at HOCs-water interfaces in order to understand the molecular mechanisms involved.Functional study of aupA and aupB revealed that these two genes form an operon whose expression is activated by various types of HOCs. They have also been shown to be involved in the transport of hexadecane and in biofilm formation on alkanes. The AupA protein is localized in the outer membrane and the predicted lipoprotein AupB is located at the inner membrane. AupA belongs to a subfamily of the FadL-like transporters, specific to marine hydrocarbonoclastic bacteria (HCB). The phylogenetic distribution of the aupAB operon restricted to marine bacteria having the ability to degrade alkanes and its presence in multiple copies in somestrains of Alcanivorax sp. strongly suggest that Aup proteins play a key role in the adaptation of HCB to use alkanes as carbon and energy sources. The transcriptomic analysis of Mh cells adhering (after 15 min or 3 h of contact) or forming a biofilm at HOCs-water interfaces revealed significant and early changes in their transcriptome. The expression of many genes involved in the metabolism of HOCs, polysaccharides production, amino acids and ribosomal proteins synthesis is modulated as early as 15 min of adhesion. The overexpression of flagella and chemotaxis genes together with that of pili in adhesion condition suggest a possible motility at the interface during the early stages of biofilm development. In addition, it appears that the transcription factor RpoN is involved in the regulation of biofilm formation in Mh and that prophages could play a role in the structure and/or dispersal of the biofilm. Finally, a potential role of a genomic island in biofilm formation ontriolein was suggestedPAU-BU Sciences (644452103) / SudocSudocFranceF

Bacteriophage-Mu Repressor - a Target Protein for the Escherichia-Coli Atp-Dependent Clp Protease

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Assembly of both the head and tail of bacteriophage Mu is blocked in Escherichia coli groEL and groES mutants

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Behavior of Marinobacter hydrocarbonoclasticus SP17 cells during initiation of biofilm formation at the alkane-water interface

International audienceHexadecane assimilation by Marinobacter hydrocarbonoclasticus SP17 occurs through the formation of a biofilm at the alkane-water interface. In this study we focused on the interactions of cells with the alkane-water interface occurring during initiation of biofilm development. The behavior of cells at the interface was apprehended by investigating alterations of the mechanical properties of the interface during cell adsorption, using dynamic drop tensiometry measurements. It was found that after having reached the hexadecane-water interface, by a purely thermal diffusion process, cells released surface-active compounds (SACs) resulting in the formation of an interfacial viscoelastic film. Release of SACs was an active process requiring protein synthesis. This initial interaction occurred on metabolizable as well as non-metabolizable alkanes, indicating that at this stage cells are not affected by the nature of the alkane forming the interface. In contrast, at a later stage, the nature of the interface turned out to exert control over the behavior of the cells. The availability of a metabolizable alkane at the interface influenced cell activity, as revealed by cell cluster formation and differences in the interfacial elasticity

Physiological adaptation of Desulfitobacterium hafniense strain TCE1 to tetrachloroethene respiration

Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of D. hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways such as PspA, GroEL and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its metabolic pathways to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration

Imaging bacterial cells and biofilms adhering to hydrophobic organic compounds-water interfaces

International audienceAssimilation of Hydrophobic Organic Compound (HOC) entails frequently the formation of biofilm at the HOC-water interface. Knowledge on the behavior of cells at the oil-water interface and within the structured biofilm is therefore important to understand the degradation of the HOC in ecosystems. The adhesion and biofilm formation on oil-water interface is best documented by microscopic observations. In this chapter we thus describe two methods for observation of bacterial cells and biofilms growing at the HOC-water interface. The first method uses CLSM (confocal laser scanning microscopy) to obtain in situ images of biofilm developing on thin paraffin strip which offers a flat transparent surface allowing imaging directly through the bottom of the culture dish without sampling. Alternatively, the biofilm can be grown on a paraffin strip deposited on a glass microscope slide and then imaged from the top when high resolution is needed. The second method addresses the problematic of the ultrastructure of biofilm developing on HOC. It enables to obtain by TEM (transmission electron microscopy) images of cross sections of biofilms with identification of the side in contact with the HOC

Cells dispersed from Marinobacter hydrocarbonoclasticus SP17 biofilm exhibit a specific protein profile associated with a higher ability to reinitiate biofilm development at the hexadecane-water interface

cited By 14International audienceBiofilm formation by marine hydrocarbonoclastic bacteria is commonly observed and has been recognized as an important mechanism for the biodegradation of hydrocarbons. In order to colonize new oil-water interfaces, surface-attached communities of hydrocarbonoclastic bacteria must release cells into the environment. Here we explored the physiology of cells freshly dispersed from a biofilm of Marinobacter hydrocarbonoclasticus developing at the hexadecane-water interface, by combining proteomic and physiological approaches. The comparison of the dispersed cells' proteome with those of biofilm, logarithmic- and stationary-phase planktonic cells indicated that dispersed cells had lost most of the biofilm phenotype and expressed a specific proteome. Two proteins involved in cell envelope maturation, DsbA and CtpA, were exclusively detected in dispersed cells, suggesting a reshaping of the cell envelopes during biofilm dispersal. Furthermore, dispersed cells exhibited a higher affinity for hexadecane and initiated more rapidly biofilm formation on hexadecane than the reference planktonic cells. Interestingly, storage wax esters were rapidly degraded in dispersed cells, suggesting that their observed physiological properties may rely on reserve mobilization. Thus, by promoting oil surface colonization, cells emigrating from the biofilm could contribute to the success of marine hydrocarbonoclastic bacteria in polluted environments. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd

Electrochemical Benzylic C-H Functionalization with Isocyanides

We report the challenging direct carbamoylation or cyanation of benzylic C(sp3)-H bonds with an isocyanide via an electrochemical process giving rise to structures which are encountered in several biologically relevant compounds and drugs. This transformation proceeds in mild conditions without the need of external oxidant and avoids the necessity to start from a prefunctionalized benzylic substrate or the deployment of the cation pool method. Anodic oxidation of the benzylic position and subsequent addition of the isocyanide leads to the formation of a C-C bond and to a nitrilium cation which hydrolysis leads to alpha-aryl acetamide derivatives, while elimination of a t-butyl cation delivers alpha-aryl acetonitrile derivatives

Cytoplasmic wax ester accumulation during biofilm-driven substrate assimilation at the alkane–water interface by Marinobacter hydrocarbonoclasticus SP17

International audienceDuring growth on n-alkanes, the marine bacterium Marinobacter hydrocarbonoclasticus SP17 formed a biofilm at the alkane–water interface. We showed that hexadecane degradation was correlated with biofilm development and that alkane uptake is localized in the biofilm but not in the bulk medium. Biofilms were observed in cultures on metabolizable n-alkanes (C8–C28) and n-alcohols (C12 and C16), but were formed neither on non-metabolizable alkanes (pristane, heptamethylnonane and n-C32) nor on inert substrata (glass, polystyrene and Permanox®). This substratum specificity indicates that biofilm formation is determined by the presence of an interface between an insoluble substrate and the aqueous phase. Simultaneously with biofilm growth, planktonic cells were released from the biofilm. Detached cells were in a non-growing state, implying that the growing population was exclusively located within the biofilm. Planktonic and sessile cells exhibited differences in their ultrastructure and lipid content. Biofilm cells contained a large amount of wax esters (0.47 mg/mg protein) in rounded or irregularly shaped cytoplasmic inclusions, whereas detached cells displayed rod-shaped inclusions and contained 5 times fewer wax esters (0.10 mg/mg protein) than their sessile counterparts. This study points out the inter-relationship between biofilm formation, insoluble substrate uptake and lipid storage

Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp protease

Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe