Academic literature on the topic 'Spirosomes'

The bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Å resolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering.

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcoholviaan aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD+pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophileGeobacillus thermoglucosidasiushas been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain.In silicomodelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

A bacterial strain, designated RHs22T, was isolated from a soil sample cultivated with rice in the Suwon region of South Korea. The cells were aerobic, Gram-stain-negative, non-spore-forming, non-flagellated rods or occasionally filaments. The strain grew at 10–37 °C (optimum, 28–30 °C), at pH 5.0–10.0 (optimum, 7.0) and in the presence of 0–1 % (w/v) NaCl (optimum, 0 %). Phylogenetically, the strain was closely related to members of the genus Spirosoma , as its 16S rRNA gene sequence had similarity of 90.3–92.1 % with respect to those of members of the genus Spirosoma , showing the highest sequence similarity with Spirosoma panaciterrae DSM 21099T. Strain RHs22T revealed relatively low sequence similarities of less than 90 % with all the other species with validly published names. It contained MK-7 as the predominant menaquinone and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C16 : 1ω5c, iso-C15 : 0 and iso-C17 : 0 3-OH as the main fatty acids. The polar lipids of strain RHs22T were phosphatidylethanolamine, one unknown aminolipid, two unknown aminophospholipids, one unknown phospholipid and five unknown lipids. The DNA G+C content was 57.0 mol%. Phylogenetic, phenotypic and chemotaxonomic data obtained in this study indicate that strain RHs22T represents a novel species of the genus Spirosoma , for which the name Spirosoma oryzae sp. nov. is proposed. The type strain is RHs22T ( = KACC 17324T = DSM 28354T). An emended description of the genus Spirosoma is also proposed.

A Gram-reaction-negative, yellow-pigmented strain, designated EX36T, was characterized using a polyphasic approach comprising phylogenetic, morphological and genotypic analyses. The endophytic strain was isolated from Zn/Cd-accumulating Salix caprea in Arnoldstein, Austria. Analysis of the 16S rRNA gene demonstrated that the novel strain is most closely related to members of the genus Spirosoma (95 % sequence similarity with Spirosoma linguale ). The genomic DNA G+C content was 47.2 mol%. The predominant quinone was and the major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), C16 : 1ω5c, iso-C17 : 0 3-OH and iso-C15 : 0. On the basis of its phenotypic and genotypic properties, strain EX36T should be classified as a novel species of the genus Spirosoma , for which the name Spirosoma endophyticum sp. nov. is proposed. The type strain is EX36T ( = DSM 26130T = LMG 27272T).

A bacterial strain, designated MSd3T, was isolated from a freshwater sample collected from the Hosoda River in Japan. The cells of strain MSd3T were Gram-stain-negative, non-spore-forming, aerobic, non-motile, curved rods forming rings, coils and undulating filaments. The 16S rRNA gene sequence of strain MSd3T showed closest similarity to that of Spirosoma linguale DSM 74T (97.6 % similarity) and similarity to other members of the genus Spirosoma ranged from 90.3 to 95.9 %. Strain MSd3T contained menaquinone 7 as the sole respiratory quinone. The major cellular fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and C16 : 1ω5c. The polar lipids were phosphatidylethanolamine, three unidentified aminophospholipids and three unidentified polar lipids. The DNA G+C content was 53.3 mol%. The DNA–DNA relatedness between strain MSd3T and S. linguale DSM 74T was 19 % or 25 % (reciprocal value). From the chemotaxonomic and physiological data and the levels of DNA–DNA relatedness, strain MSd3T should be classified as the representative of a novel species of the genus Spirosoma, for which the name Spirosoma fluviale sp. nov. (type strain MSd3T = JCM 30659T = DSM 29961T) is proposed.

Cette thèse est divisée en deux thématiques.La première porte sur le système de Sécrétion de TypeIV cag (cag-SST4) d’Helicobacter pylori. Il s’agit d’une machinerie de sécrétion complexe enchâssée dans l’enveloppe cellulaire de la bactérie, lui permettant d’injecter l’oncoprotéine CagA dans les cellules épithéliales gastriques humaines. Cette toxine est considérée comme un facteur de virulence majeur d’H. pylori. Elle interagit avec des protéines de l’hôte, perturbant la signalisation cellulaire et entraînant des modifications pouvant favoriser le développement de maladies gastrointestinales, y compris des ulcères et des cancers gastriques. Le cag-SST4 est subdivisé en trois parties : (i) un complexe de membrane interne, composé essentiellement d’ATPases fournissant l’énergie nécessaire à son assemblage et/ ou son fonctionnement ; (ii) un complexe de membrane externe, ou complexe core, formant un canal qui relie les membranes interne et externe et (iii) un pilus extracellulaire, dont l’existence est toujours controversée, et qui permettrait d’établir un contact entre la bactérie et sa cible, et éventuellement de transférer les substrats à travers la membrane de l’hôte.Un premier projet porte sur le pilus extracellulaire. L’objectif est d’obtenir des données concernant une interaction supposée entre les protéines CagI et CagL, essentielles à la sécrétion et pressenties pour entrer dans la composition du pilus. Nous avons surexprimé des versions recombinantes de ces protéines chez Escherichia coli et nous les avons co-purifiées par chromatographie d’affinité, démontrant ainsi une interaction directe entre elles. La capacité de DARPins et Nanobodies de lier ce complexe a été testée. L’analyse de ces complexes a également été entreprise par cryo-microscopie électronique (cryoME).Le second projet porte sur le complexe core avec pour objectif d’obtenir sa structure à haute résolution afin d’éclaircir les zones d’ombre qui persistent concernant cet imposant assemblage. Différentes techniques ont été mises en œuvre afin de pouvoir solubiliser ce complexe. Sa purification reste à optimiser afin de pouvoir envisager une analyse en cryoME. L’obtention de telles structures pourrait permettre de mieux comprendre le fonctionnement du cag-SST4 et d’envisager des stratégies permettant d’inhiber son assemblage et/ ou son fonctionnement, privant ainsi H. pylori d’un facteur de virulence majeur.La seconde thématique porte sur les spirosomes bactériens. L’enzyme AdhE est très conservée dans le règne bactérien et chez certains organismes eucaryotes. Il s’agit d’une enzyme bifonctionnelle alcool/ aldéhyde déshydrogénase, responsable de la conversion de l’acétyl-CoA en acétaldéhyde puis en éthanol au cours de la fermentation alcoolique en anaérobie. Cette enzyme est communément retrouvée sous sa forme oligomérique, appelée spirosome. En fonction des ligands présents dans le milieu, les spirosomes d’E. coli peuvent se présenter dans une conformation compacte ou étendue, cette dernière constituant la forme active de l’enzyme. Contrairement aux spirosomes d’E. coli, ceux de Streptococcus pneumoniae sont naturellement stabilisés dans leur conformation étendue.L’objectif de ce projet est de comprendre quels sont les mécanismes à l’origine de cette différence de conformation. La cryoME nous a permis d’obtenir une structure à haute résolution du spirosome de S. pneumoniae et ainsi de pouvoir la comparer à celle du spirosome étendu d’E. coli. Des expériences de mutagenèse fonctionnelle avec complémentation nous ont permis de déterminer quels sont les résidus impliqués dans l’extension de ces spirosomes. Etant impliqués dans la pathogénicité et révélés indispensables à la physiologie bactérienne en l’absence d’oxygène, l’étude approfondie de leur conformation pourrait donc mener à la découverte de molécules capables de réguler leur activité, ce qui pourrait présenter un intérêt majeur dans les domaines des biotechnologies et de la santé
This thesis is divided into two themes.The first theme focuses on the cag Type IV secretion system (cag-T4SS) of the bacterium Helicobacter pylori. This is a complex secretion machinery embedded in the bacterium's cellular envelope, enabling it to inject the CagA oncoprotein into human gastric epithelial cells. This toxin is considered a major virulence factor of H. pylori. It interacts with host proteins, disrupting cell signaling and leading to changes that can promote the development of gastrointestinal diseases, including gastric ulcers and cancers. The cag-T4SS is subdivided into three parts: (i) an inner membrane complex, composed essentially of ATPases providing the energy required for its assembly and/or its function; (ii) an outer membrane complex, or core complex, forming a channel that connects the inner and outer membranes; and (iii) an extracellular pilus, the existence of which is still controversial, and which would establish contact between the bacterium and its target, and possibly transfer substrates across the host membrane.The first project focuses on the extracellular pilus. The aim is to obtain data concerning a putative interaction between the CagI and CagL proteins, which are essential for secretion and are thought to be involved in the composition of the cag-T4SS pilus. We overexpressed recombinant versions of these proteins in Escherichia coli and co-purified them by affinity chromatography, demonstrating a direct interaction between them. The ability of DARPins and Nanobodies to bind this complex was tested. Analysis of these complexes was also undertaken by cryo-electron microscopy (cryoEM).The second project focuses on the core complex, with the aim of obtaining its structure at high resolution in order to shed light on the remaining grey areas concerning this imposing assembly. Various techniques have been used to solubilize this complex. Its purification remains to be optimized before it can be analyzed by cryoEM. Obtaining such structures could lead to a better understanding of how cag-T4SS functions, and to consider strategies to inhibit its assembly and/or function, thus depriving H. pylori of a major virulence factor.The second theme concerns bacterial spirosomes. The AdhE enzyme is highly conserved in the bacterial kingdom and in certain eukaryotic organisms. It is a bifunctional alcohol/aldehyde dehydrogenase enzyme, responsible for the conversion of acetyl-CoA to acetaldehyde and then to ethanol during anaerobic alcoholic fermentation. This enzyme is commonly found in its oligomeric form, known as spirosome. Depending on the ligands present in the medium, E. coli spirosomes can have a compact or extended conformation, the latter constituting the active form of the enzyme. Unlike E. coli spirosomes, Streptococcus pneumoniae ones are naturally stabilized in their extended conformation.The aim of this project is to understand the mechanisms behind this conformational difference. CryoEM enabled us to obtain a high-resolution structure of the S. pneumoniae spirosome and thus comparing it with the extended E. coli spirosome. Functional mutagenesis experiments with complementation enabled us to determine which residues are involved in the extension of these spirosomes. As they are involved in pathogenicity and have been shown to be essential to bacterial physiology in the absence of oxygen, in-depth study of their conformation could lead to the discovery of molecules capable of regulating their activity, which could be of major interest in the fields of biotechnology and healthcare