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Bourbonneux, Valéry. "Identification des "Escherichia" par auxanogramme." Paris 5, 1993. http://www.theses.fr/1993PA05P139.
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Kerner, Michael Johannes. "The Escherichia coli GroEL interaction proteome identification and classification /." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=979094593.
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Kiel, Michael Christopher. "Identification and characterization of an Escherichia coli ribosomal ATPase." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0024/NQ50055.pdf.
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Jarboe, Laura Renee. "Identification and simulation of regulatory networks in Escherichia coli." Diss., Restricted to subscribing institutions, 2006. http://proquest.umi.com/pqdweb?did=1276395911&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Blanchard, Geneviève. "Versatilité nutritionnelle de l'espèce génomique "Escherichia Coli-Shigella"." Paris 5, 1990. http://www.theses.fr/1990PA05P055.
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DIEKERT, PASCALE. "Methodes d'identification et de denombrement des coliformes fecaux et d'escherichia coli." Strasbourg 1, 1990. http://www.theses.fr/1990STR15043.
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Christensen-Dalsgaard, Mikkel. "Identification and characterization of new messenger RNA interferases of Escherichia coli." Thesis, University of Newcastle Upon Tyne, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555996.
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Toxin-antitoxin (TA) loci, which are widely distributed in prokaryotes, are organized in small operons, consisting of genes encoding toxins and antitoxins. Transcription from the promoter is regulated by the TA protein complex. Activation of the toxin occurs when the toxin is in excess of the antitoxin, for example during nutritional stress when the labile antitoxins are rapidly degraded by cellular proteases. The biological function of TA systems is debated and several different models have been proposed. Several of the TA loci encode messenger RNA interferases (mls) that inhibit translation by cleaving mRNA. Two types are known: those that cleave mRNA codons at the ribosomal A-site and those that cleave any RNA site-specifically. In this work, it was shown that the ml, YoeB cleaved mRNA strictly dependent on translation in vivo. Furthermore, it was shown that site-specific mRNA cleavage by MazF occurs independently of translation but that translation can seriously influence MazF cleavage efficiency. Expression of the toxin Doc of phage P1 was shown to mediate mRNA cleavage, though activation of the endogenous E. coli RelE ml. In a different study, three new ml encoding TA loci of E. coli were identified and characterized. Ectopic expression of the three ml genes lead to a rapid growth arrest caused by inhibition the global rate-of-translation. Furthermore, ml induced degradation of several different model RNAs were observed in vivo. Two of the mls cleaved only translated RNAs, similar to RelE and YoeB, whereas one ml cleaved both in coding and non-coding regions of the RNAs, resembling the properties of MazF and ChpBK toxins. Transcription of the three TA mRNAs was induced by various stressful conditions and dependent on the proteases Lon and Clp. In a different section of this work, a new synthetic lethal screen was developed using antisense RNA regulated R1 plasmids. The screen was used to search for synthetic lethal of sick interactions with the molecule responsible for trans-translation, tmRNA. Two genes were found to be synthetic sick with tmRNA: the tatC gene, which encodes an essential component of the twin- ariginine translocation complex and the dksA gene, which encode a transcription factor responsible for regulation of ribosomal RNA promoters.
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Riley, Laura Maryse. "Identification of Bacteriophage 024B- Encoded Genes Expressed by Escherichia coli Lysogens." Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510970.
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Sarica, Nazim. "Identification de contraintes locales impactant l’expression des gènes chez Escherichia coli." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL021.
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L'expression des génomes dépend de toutes les « contraintes » qui s'appliquent à l'ADN, que ce soit au niveau nucléotidique et génique (1D), qu'au niveau de son organisation conformationnelle (3D). Les études menées chez les bactéries ont montré que l'organisation 3D de leur chromosome circulaire s'échelonne du niveau moléculaire au niveau cellulaire. Au niveau moléculaire, les NAPs (Nucleoid Associated Proteins) organisent l'ADN en microdomaines d'environ 10 kb qui présentent des surenroulements de l'hélice d'ADN indépendants (Postow et al., 2004). Au niveau cellulaire, le chromosome est organisé en 4 régions structurées et 2 autres non structurées, appelées macrodomaines, d'une taille proche de 1Mb (Valens et al., 2004). Le projet consiste à étudier l'impact de contraintes topologiques sur la transcription des gènes en fonction de la position chez E. coli. La taille élevée du chromosome et les éléments interagissant avec rendent variable et dynamique l'organisation chromosomique. Il est donc difficile d'étudier certains aspects de la structure du chromosome et de corréler ces résultats avec l'expression génique<br>A long list of constrains that affect gene expression have been discovered thanks to the thousands of sequenced genomes and comparative genomics. These contrains can be at the nucleotidic level, but also at the 3D scale. Studies showed that the spatial organization of bacterial genomes goes from the molecular level to the cellular level. At the molecular level, NAPs (Nuceloid Associated Proteins) are modeling the chromosome by inducing 10kb loops called microdomains, that present different supercoiling levels. At the cellular scale, it has been observed 4 different structured regions + 2 non-structured regions on E.coli's chromosome. These regions of approximatively 1Mbp are called macrodomains. The first goal of this project is to study the effects of positioning and constrains on gene expression in E.coli. With the chromosome being so large and precisely organized in time and space by several interacting elements simultaneously, it is often difficult for researchers to isolate one specific feature and to accurately correlate this to gene regulation. What becomes obvious when one begins to wade through the literature is that the field would greatly benefit from simplified model chromosomes
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Baertlein, Dawn Marie August. "Identification of phosphate starvation inducible mineral phosphate solubilization genes in Escherichia coli." Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184606.
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Under conditions of phosphate starvation Escherichia coli can solubilize mineral phosphates, such as dicalcium phosphate, to orthophosphate which is then available for uptake and cell growth processes. lac operon fusions were created using MudX phage, and mineral phosphate solubilization (Mps) mutants were identified by their inability to solubilize mineral phosphate on plate assays. Four of these mutants have been mapped on the E. coli chromosome via Hfr matings and are located at two distinct portions of the chromosome; between 23 and 50 minutes and between 60 and 90 minutes. One mutant in each region has phosphate starvation inducible (Psi) promoter activity. One of these mutants (DB1047) was mapped to between 69 and 75 minutes via F' matings, and fine structure mapped to 75 minutes by hybridization with λ clones from a genomic library of Escherichia coli. DB1047 was characterized more closely and found to exhibit pleiotropy with regard to several membrane related traits. Evidence that this is a single insertional event comes from the simultaneous loss of all traits tested in spontaneous revertants. Additionally, a Tn5 mutant was identified that was identical for these traits. Our data strongly support the hypothesis that the mutation carried by DB1047 is in the ompB locus. This locus consists of the two regulatory cotranscribed genes, ompR and envZ. This locus is involved in regulation of transcription of the ompC and ompF genes for outer membrane porin proteins, and is located at 75 minutes on the chromosome as is the DB1047 mutation. DB1047 lacks the outer membrane porin OmpF, a phenotype previously attributed to envZ mutants. However, the ompR321 mutant resembles DB1047 in reduced ability to solubilize phosphate. Additional supporting evidence for the DB1047 mutation belonging to the ompB locus comes from the most recent report that mutations in the himA gene, which I found to be deficient in the ability to solubilize phosphate, also affect the regulation of production of the outer membrane porin OmpF.
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Grenier, Frédéric. "Identification des gènes facultatifs chez Escherichia coli à l'aide de mutagénèses aléatoires." Mémoire, Université de Sherbrooke, 2017. http://hdl.handle.net/11143/11637.
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La biologie synthétique est une discipline émergente consistant principalement en la modification génomique d'organismes vivants. Ces modifications complexes visent la programmation de systèmes biologiques pour accomplir des fonctions spécifiques parfois absentes dans la nature. Ce domaine se divise en plusieurs branches dont l’élaboration d’un châssis cellulaire minimal. En effet, depuis déjà plus d’une dizaine d’années, un grand intérêt est porté vers la création d’une cellule simplifiée. Le génome d’une telle cellule contiendrait seulement ce qui est nécessaire à sa survie en condition de laboratoire. Cette cellule possèderait pour principal avantage de permettre une meilleure compréhension de son fonctionnement global en plus d’éventuellement devenir un modèle d’étude pour la modification et la restructuration des génomes. Elle sera également plus fiable dans un contexte d’ingénierie métabolique puisqu’elle contiendra moins de circuits géniques, diminuant le nombre d’interactions non désirées. Une cellule minimale serait donc un sujet d’étude fascinant en plus d’être un organisme facilement reprogrammable, constituant ainsi une pierre angulaire pour le développement de la biologie synthétique.
La présente étude concerne la réduction génomique de la bactérie Escherichia coli. Nous avons choisi cette dernière puisque de nombreux outils moléculaires permettant de modifier son génome sont disponibles et puisqu’une littérature très dense la concernant a été cumulée au fil des ans. Plusieurs projets de réduction ont été entamés avec cet organisme et les génomes produits ne semblent pas pouvoir atteindre moins de 3,0 Mpb sans présenter des défauts considérables de croissance. Nous avons donc sélectionné la souche simplifiée DGF298, dont le génome fait 3,0 Mpb et permet la croissance en milieu minimal, pour générer des mutants d’insertion ou de délétion et les avons suivis dans plusieurs conditions distinctes afin de déterminer si cette souche possédait encore des gènes facultatifs. Ce mémoire traite principalement des résultats obtenus dans un milieu riche et défini. Les données ainsi générées suggèrent que le génome de cette souche peut être réduit considérablement, puisque plus de 73 % de ses séquences codantes ne semblent pas être importantes en milieu riche. Une partie de ces éléments facultatifs peut cependant être impliquée pour la survie dans certaines conditions, telles qu’en présence d’une source de carbone autre que le glucose ou en absence d’oxygène. Lors de la réduction subséquente, il faudra donc déterminer le niveau d’adaptabilité désiré pour la souche.
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Koo, Jovanka Tepavcevic. "Identification of factors involved in processing of mRNA in a fimbrial operon of Escherichia coli /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/11506.
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Easton, James Allen. "Identification and Characterization of Zn(II)-responsive Genes and Proteins in E. coli." Oxford, Ohio : Miami University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1189685688.
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Bouvet, Jérôme. "Identification du danger lié aux Escherichia coli vérotoxiques (VTEC) et à Escherichia coli O157-H7 en abattoir et découpe de porc." Lyon 1, 2002. http://www.theses.fr/2002LYO10062.
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Les Escherichia coli verotoxiques (VTEC), et plus particulièrement le sérotype O157:H7, sont aujourd'hui des agents pathogènes d'origine alimentaire considérés comme importants en santé publique. La viande de porc n'est qu'exceptionnellement impliquée dans les accidents alimentaires dus aux VTEC. Cependant, la prévalence des VTEC dans la viande de porc n'est pas connue en France. Il est donc important de disposer de données sur l'épidémiologie de ces germes en abattoir et découpe de porc, afin d'évaluer l'importance du risque et de le maîtriser efficacement. Notre étude a été réalisée dans trois ateliers d'abattage et de découpe de porc. Sur les 4 469 échantillons analysés par PCR (produits carnés, fèces, environnement) aucun n'a été détecté positif en E. Coli O157:H7 verotoxique et 16 % ont été détectés positifs en VTEC (présence des gènes stx). Sur 2 800 échantillons de couenne et viande prélevés par excision (NF V04-501) sur carcasses réfrigérées et pièces de découpe, 12 % sont positifs en VTEC par PCR. Les porcs vivants sont des sources d'introduction de VTEC à l'abattoir. Un tiers (56/182) des porcs est détecté porteur fécal et près de la moitié des carcasses (83/182) est contaminée en surface. Au cours des opérations d'abattage la contamination de surface des carcasses diminue, elle reste stable lors du ressuage. La moitié des carcasses réfrigérées est contaminée en VTEC (75/150), mais la contamination n'est pas massive. Les pièces de découpe sont peu contaminées en VTEC, 12 % en moyenne, les pièces brutes l'étant davantage (19 %) que les pièces découennées désossées (5 %). La contamination des locaux d'abattage et de découpe augmente significativement au cours de l'activité d'où le rôle vraisemblable de l'environnement dans les contaminations croisées et la nécessité d'un nettoyage-désinfection efficace en fin d'activité. A partir de 598 échantillons positifs en VTEC par PCR, 116 isolats ont été obtenus par hybridation sur boîtes. Un seul semble potentiellement pathogène au vu de l'analyse de ses facteurs de virulence. En conséquence, les VTEC dont E. Coli O157:H7 ne représentent pas un risque sanitaire majeur en abattage-découpe de porc. Outre les bonnes pratiques hygiéniques habituellement préconisées en abattage-découpe de porc, aucune mesure préventive spécifique à ce danger microbiologique ne semble nécessaire.
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Sancak, Aziz Arda. "Identification and characterisation of pathogenic Escherichia coli associated with intestinal disease in dogs." Thesis, Royal Veterinary College (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284817.
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Bernier-Febreau, Christine. "Identification et caractérisation de facteurs de pathogénicité produits par les Escherichia coli entéroagrégatifs." Paris 7, 2003. http://www.theses.fr/2003PA077012.
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Kathayat, Dipak. "Identification of Novel Small Molecule Growth Inhibitors Specific to Avian Pathogenic Escherichia coli." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu150032603130236.
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Scott, David Lee Jr. "Identification of a region in the central regulatory segment of plasmid R6K responsible for complexing to membranes of escherichia coli." Thesis, Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/31050.
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Mercier, Romain. "Organisation du chromosome d'Escherichia coli en macrodomaines : identification et rôle du système spécifique de site matS-MatP." Paris 11, 2009. http://www.theses.fr/2009PA112361.
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Le génome d’E. Coli est composé d’un chromosome unique et circulaire d’une taille de 4,6Mb. Le chromosome est organisé selon différentes échelles : (i) nucléotidique par les séquences codantes et les motifs d’ADN fonctionnels ; (ii) locale par les domains topologiques ou plectonèmes ; (iii) globale par les réplichores et les macrodomaines. Mon travail de thèse s’est attaché à l’étude de l’organisation globale du chromosome en macrodomaines. Le chromosome d’E. Coli est composé de quatre macrodomaines et de deux régions Non‐Struturées. Les macrodomaines divisent le chromosome en quatre unités structurales séparées les unes des autres. Ce projet de recherche s’articule selon deux axes : la compréhension de la dynamique des macrodomaines au cours du cycle cellulaire et la caractérisation de déterminants moléculaires responsables de la structuration du chromosome en macrodomaines. Le premier axe de recherche a permis de mettre en évidence des proprieties dynamiques du chromosome qui suivent la topographie des macrodomaines : cohésion des chromatides sœurs, confinement fort et isolement spatial. A chaque instant du cycle cellulaire toutes les régions composant un macrodomaine présentent une localisation subcellulaire spécifique dépendante du macrodomaine concerné. Les macrodomaines sont spatialement confinés dans des territoires cellulaires distincts. Ce mode d’organisation est comparable à celui des chromosomes interphasiques eucaryotes en territoires chromosomiques. Le deuxième axe de recherche a permis de caractériser un mécanisme moléculaire responsable de la structuration d’un macrodomaine. Ce mécanisme est composé d’un motif d’ADN répété (matS) dans le macrodomaine Ter reconnu spécifiquement par un facteur protéique (MatP). Le complexe matS/MatP confère toutes ses propriétés de dynamique au macrodomaine Ter : forte cohésion des chromatids soeurs, fort confinement et isolement spatial. De plus, ces propriétés de dynamique sont requises pour le bon déroulement de la fin du cycle cellulaire et la formation de deux cellules filles. Finalement, la co‐conservation de MatP avec un groupe de protéines contenant les protéines Dam, SeqA, MukBEF et plusieurs protéines de fonctions inconnues suggère que le complexe matS/MatP fait partie d’un système général de métabolisme de l’ADN lié à la méthylation<br>The organization of the E. Coli chromosome has been defined genetically as consisting of four insulated macrodomains and two less constrained regions. During my Ph. D. Thesis, we have analyzed the positioning, the segregation pattern and the motility of fluorescent markers in the macrodomains or the Non Structured regions. We have demonstrated that the organization into macrodomains influences the segregation of sister chromatids and the mobility of chromosomal DNA in a radically different way than the NS regions. Moreover we have demonstrated that the organization of the Terminus region into a macrodomain relies on the presence of a 13 bp motif called matS repeated 23 times in the 800 kb-long domain. MatS sites are the main targets in the E. Coli chromosome of a newly identified protein designated MatP. MatP accumulates in the cell as a discrete focus that colocalizes with the Ter macrodomain. The effects of MatP inactivation reveal its role as main organizer of the Ter macrodomain : in the absence of MatP, DNA is less compacted, the mobility of markers is increased, and segregation of Ter macrodomain occurs early in the cell cycle. Our results indicate that a specific organizational system is required in the Terminus region for bacterial chromosome management during the cell cycle
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Haines, Sara. "Identification of environmental and genetic factors influencing virulence gene expression in Enterotoxigenic Escherichia coli." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10017.
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Yang, Yang. "Investigation of enterotoxigenic Escherichia coli (ETEC) vaccine candidates and identification of inhibitor of enterohemorrhagic Escherichia coli (EHEC) Type III secretion system effector NleB." Thesis, Kansas State University, 2017. http://hdl.handle.net/2097/38174.
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Master of Science in Biomedical Sciences<br>Department of Diagnostic Medicine/Pathobiology<br>Philip R. Hardwidge<br>Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in travellers and young children in developing countries. We previously characterized three vaccine candidates (MipA, Skp, and ETEC_2479) which effectively protected mice in an intranasal ETEC challenge model after immunization. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. In this study, we examined the potential of these antigens to affect the host intestinal microbiota and subsequently found no significant impact on healthy of host after vaccination. In addition, we also optimized the types of adjuvants and forms of antigens and evaluated the efficacy in a mouse intranasal challenge model.
Enterohemorrhagic Escherichia coli (EHEC) is an emerging zoonotic pathogen that cause global public health threads. EHEC possesses the potential to cause gastroenteritis, hemorrhagic colitis and hemolytic uremic syndrome (HUS), which may lead to renal failure. Type III secretion system (T3SS) is a hallmark of EHEC, characterized by the needle-like structure and a variety of effectors injected into host cells. NleB, one of T3SS effectors, is a glycosyltransferase with the ability to catalyze the transfer of N-acetyl-D-glucosamine (N-GlcNAc) to host proteins to suppress the activation of NF-kB signaling pathway. In this study, we employed luminescence-based glycosyltransferase assay and high-throughput screening using a chemical library of various compounds. A total of 128 chemicals was selected with significant inhibition on NleB glycosyltransferase activity for further pharmaceutical study as novel therapy against EHEC infection.
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Reynaud, Alain. "Pathologie digestive du lapin sevré : caractéristiques des E. coli 0103, 015 et 02 : isolement et identification de Campylobacter." Clermont-Ferrand 1, 1993. http://www.theses.fr/1993CLF1PP03.
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Mukhopadhyay, Suman. "Identification and characterization of genes that protect Escherichia coli from hydrogen peroxide mediated oxidative stress." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0018/NQ30161.pdf.
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Chouikha, Iman. "Identification et caractérisation d'un îlot génomique d'une souche d'Escherichia coli pathogène aviaire." Tours, 2006. http://www.theses.fr/2006TOUR4010.
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Certaines souches d'E. Coli sont pathogènes et provoquent des infections intestinales ou extra-intestinales chez l'homme et les animaux. Chez les volailles, les APEC (Avian Pathogenic E. Coli), provoquent une infection systémique à point de départ respiratoire pouvant conduire à la mort de l'animal. Plusieurs facteurs de virulence ont été identifiés pour ces souches, mais ne suffisent pas à expliquer tout le processus infectieux. L'identification de nouveaux facteurs de virulence devrait permettre de mieux comprendre le schéma infectieux de ces souches. Les facteurs de virulence bactériens sont fréquemment situés au sein d'éléments mobiles (plasmides, transposons ou îlots de pathogénicité (PAIs)). Les PAIs sont souvent associés à leurs extrémités au loci d'ARNt. Nous avons mis en évidence chez la souche APEC BEN2908, un nouvel îlot génomique nommé AGI-3 pour "APEC Genomic Island 3" inséré au locus selC et possédant la plupart des caractéristiques des PAIs. AGI-3 est encadré par des séquences directes répétées, possède des gènes codant pour des éléments de mobilité potentiels et est de taille importante (49 600 pb). Cet îlot est composé de 49 ORFs, dont 3, aec35 à aec37, semblent impliqués dans le métabolisme des sucres. Par comparaison du phénotype de la souche sauvage et de son mutant isogénique délété des ORFs aec35-37, nous avons mis en évidence l'implication de ce groupe de gènes dans l'assimilation des hydrates de carbone. Des reproductions expérimentales de la colibacillose chez le poulet ont permis de déterminer leur implication dans les étapes précoces de l'infection. La présence d'une intégrase de phage ainsi que des sites att indiquent qu'AGI-3 a probablement été acquis par transfert horizontal. En faveur de cette hypothèse, nous avons montré qu'AGI-3 pouvait exister sous forme épisomale dans le cytoplasme bactérien et être transféré à une souche d'E. Coli K-12 par conjugaison. Ces données montrent qu'AGI-3 est un vecteur potentiel de dissémination de gènes de virulence entre souches bactériennes<br>Escherichia coli is also of causing a variety of intestinal or extraintestinal infections in humans and animals. In poultry flocks APEC (Avian pathogenic E. Coli) are involved in a systemic infection initiated in the respiratory tract that can be lethal for the animals. Several bacterial factors have been associated with their virulence, however the mechanisms underlying pathogenicity are not fully understood yet. Identification of new factors involved in the pathogenesis of APEC should lead to a better understanding of the infectious process. In bacteria, virulence factors are often located in mobile genetic elements (transposons or pathogenicity islands (PAIs)). PAIs are often associated with tRNA loci. We identified a new PAI in the APEC strain BEN2908, that we named AGI-3 for "APEC genomic island 3". AGI-3 is located at the 3'-end of the selC tRNA and possesses several characteristics of PAIs (flanked by direct repeats, presence of genes coding for putative mobile elements and large size 49 600 pb). AGI-3 shows a mosaic structure. It is composed of 49 ORFs three of them, aec35 to aec37, seem to be implicated in sugar metabolism. Comparing the abilities of the wild type strain BEN2908 and of the isogenic mutant, deleted for the three genes, to assimilate carbohydrates and to induce colibacillosis in a chicken experimental model, we demonstrated the implication of the cluster in the uptake of seven carbohydrates and in pathogenesis at the early steps of the infection. The presence of a putative active integrase gene as well as att sites suggested that AGI-3 could have been acquired by horizontal gene transfer. We demonstrated that AGI-3 is able to excise from the chromosome and to persist in the bacterial cytoplasm as a circular form and be transferred to an E. Coli K-12 strain by conjugation. These data show that AGI-3 could be a potential vector for dissemination of virulence genes between bacterial strains
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Lacroix, Olivier. "Etude préliminaire pour l'application en routine d'un test de chimiotaxie aux acides aminés utilisé pour la détection de l'altération de la perméabilité chez les entérobactéries." Paris 5, 1991. http://www.theses.fr/1991PA05P153.
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Xue, Mingyu. "Identification and Characterization of Intermediates during Folding on the β-Barrel Assembly Machine in Escherichia coli". Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11594.
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β-barrel membrane proteins play important structural and functional roles in Gram negative bacteria and in mitochondria and chloroplasts of eukaryotes. A conserved machine is responsible for the folding and insertion of β-barrel membrane proteins but its mechanism remains largely
unknown. In E. coli, a five protein β-barrel assembly machine (Bam) assembles β-barrel proteins into the outer membrane (OM). Among all β-barrel membrane proteins in E. coli
, the β-barrel component of the OM LPS translocon is one of
only two essential β-barrels, the other being the
central component of the Bam machinery itself. The OM LPS translocon, which consists of OM β-barrel protein LptD (lipopolysaccharide transport) and OM lipoprotein LptE, is responsible for the final export of LPS molecules into the outer leaflet of the OM, resulting in an asymmetric bilayer that blocks the entry of toxic molecules such as antibiotics. This thesis describes the characterization of the biogenesis pathway of the OM LPS translocon and its folding and insertion
into the OM by the Bam machinery.
An in vivo S35-Methionine pulse-labeling assay was developed to identify intermediates along the biogenesis of the OM LPS translocon. Seven intermediates were identified along the
pathway. We show that proper assembly of the OM LPS translocon involves an oxidative disulfide bond rearrangement from a nonfunctional intermediate containing non-native disulfides. We also found that the rate determining step of OM LPS translocon biogenesis is β-barrels folding process by the Bam machinery.
Using in vivo chemical crosslinking, we accumulated and trapped a mutant form of LptD on BamA, the central component of the Bam machinery. We extended the S35-Methionine pulse-labeling method to allow chemical crosslinking of substrates on the Bam complex and trapped LptD while it was being folded on the Bam machine. We demonstrated that the interaction between LptD and BamA is independent of LptE, while that between LptD and BamD, the other
essential component of the Bam complex beside BamA, is LptE dependent. Based on these findings, we proposed a model of Bam-assisted folding of the OM LPS translocon in which LptE
templates the folding of LptD.<br>Chemistry and Chemical Biology
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Huerta, Uribe Alejandro. "Identification and characterisation of small-molecule inhibitors of Shiga toxin expression in Escherichia coli O157:H7." Thesis, University of Glasgow, 2019. http://theses.gla.ac.uk/40993/.
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Shiga toxin (Stx) producing E. coli (STEC) infections represent an important public health problem given the severity of the disease and sequelae associated to it. Since the use of antibiotics enhances the virulence of STEC, new therapeutic strategies are urgently required. Thus, the main aim of this project is the study of small molecules that are able to block expression of Shiga toxin in Escherichia coli O157:H7. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimized compound interferes with the SOS response by directly affecting the activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants. As a result of the successful identification of a small molecule capable of inhibiting Stx2 expression in E. coli O157:H7, an additional high-throughput screening (HTS) of small molecules was performed. Two new compounds with activity against Stx2 production were successfully identified and characterised in biological assays. Finally, we describe the use of a small molecule with previously reported anti-quorum sensing activity. Our findings suggest that the compound furanone C-30 blocks stx expression in vitro.
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Tydings, Heather Anne. "Identification and Optimization of the Antagonistic Potential of Native Spinach Microbiota towards Escherichia coli O157:H7." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/43364.
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Leafy greens such as spinach have been the object of several recent food-borne pathogen outbreaks. The purpose of this study was to isolate bacteria spinach epiphytic bacteria that inhibit growth of E. coli O157:H7, which we describe as antagonism. The mechanism of antagonism was investigated and we attempted to improve the antagonistic potential in vitro and on spinach leaves when cellobiose, a carbon source utilized by the antagonists but not E. coli O157:H7, was added.
There were larger culturable populations of bacteria on the leaves of savoyed cultivars compared to flat. From the isolated colonies, 47 displayed antagonism towards E.coli O157:H7, and were identified as members of 11 different genera and sixteen species. A representative isolate from each species was evaluated for three possible mechanisms of antagonism: acid production, secretion of an inhibitory compounds or secreted protease. The majority (14/16) produced at least a moderate level of acid. Two of these strains, Paenibacillus polymyxa and Pseudomonas espejiana, were found to secrete a non- protease antagonistic compound.
These antagonists varied in their reduction of E.coli O157:H7 numbers in vitro, but all significantly reduced numbers in 48 hours of co-culturing in nutrient rich media. Five antagonists resulted in a significant reduction in E.coli O157:H7 populations when co-cultured on spinach leaves. Application of cellobiose did not improve the amount of antagonism in vitro or on the leaf surface after 24 hours.<br>Master of Science
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Mainil, Jacques. "Escherichia coli entérotoxinogènes bovins: identification des facteurs et des plasmides de virulence par hybridation ADN-ADN." Doctoral thesis, Universite Libre de Bruxelles, 1988. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/212877.
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Rycke, Jean de. "Mise en évidence et caractérisation biologique de deux toxines colibacillaires à activité cytotoxique et nécrosante (CNF1 et CNF2) et identification immunochimique de CNF1." Lyon 1, 1990. http://www.theses.fr/1990LYO10181.
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L'etude des proprietes cytotoxiques de souches d'e. Coli isolees de diarrhees du veau a permis de mettre en evidence deux types originaux de toxines dont la caracteristique commune est de produire un effet de multinucleation sur les cellules de lignee en culture. Les modalites de l'effet cytopathique et les epreuves de seroneutralisation ont montre que l'un des types de cytotoxicite est identique a celui exerce par la toxine cnf (pour cytotoxic necrotizing factor) decrite anterieurement dans les souches d'e. Coli provenant de diarrhees de l'enfant. Nous avons denomme ce type cnf1 pour le distinguer de l'autre, appele cnf2. La toxine cnf1 a ensuite ete identifiee apres electrophorese en gel de polyacrylamide en presence de sodium dedecyl sulfate et par trois approches complementaires: purification, immunoempreinte a l'aide d'un antiserum neutralisant specifique, comparaison des profils de souches produisant ou non la toxine. Il s'agit d'une proteine de 115 kda, associee a la fois a la cytotoxicite et a la toxicite in vivo. Un test elisa de detection de cnf1 dans les extraits bacteriens bruts a ete mis au point (methode double-sandwich). Enfin l'effet toxique en letal de cnf1 a ete confirme chez l'agneau nouveau-ne inocule par voie intraveineuse. L'ensemble de ces travaux montrent que, outre l'interet fondamental qu'elles peuvent presenter dans l'etude du fonctionnement de la cellule eucaryote, les toxines cnf constituent des facteurs de pathogenicite potentiels dont la contribution exacte dans la virulence des souches productrices d'e. Coli devra etre evaluee
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Bordi, Christophe [Etienne Charles]. "Identification par approche génomique des gènes contrôlés par le système de régulation TorS/TorR en réponse au triméthylamine N-oxyde (TMAO) chez Escherichia coli et Shewanella oneidensis." Aix-Marseille 2, 2004. http://www.theses.fr/2004AIX22011.
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Gardette, Marion. "Virulence des Escherichia coli entérohémmoragiques : rôle central du monoxyde d'azote dans le devenir de l'infection et identification de nouveaux déterminants impliqués dans l'adaptation du pathogène à l'envirronement digestif." Thesis, Université Clermont Auvergne (2017-2020), 2019. http://www.theses.fr/2019CLFAC075.
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Les Escherichia coli entérohémorragiques (EHEC) représentent un enjeu majeur en santé publique. En effet, ces pathogènes sont responsables chaque année de milliers de cas de toxi-infections alimentaires à travers le monde et peuvent engendrer des complications graves, notamment des atteintes rénales chez les jeunes enfants et cérébrales chez les personnes âgées. Actuellement, le principal problème réside dans le fait que les traitements thérapeutiques disponibles sont limités puisque l’antibiothérapie peut favoriser le développement des complications liées à l’infection. Il est donc primordial et d’actualité de mettre en évidence les facteurs bactériens associés à la virulence des EHEC et de comprendre les interactions entre le pathogène et l’hôte afin de développer des stratégies thérapeutiques visant à éliminer le pathogène et limiter l’apparition des symptômes graves. Ainsi, le premier objectif de cette thèse était d’identifier de nouveaux facteurs bactériens potentiellement impliqués dans le processus infectieux. L’utilisation de la technologie RIVET sur la souche de référence O157:H7 EDL933 en modèle murin, a permis de mettre en évidence 31 gènes dont l’expression est spécifiquement induite lors de l’infection. La caractérisation de ces gènes a démontré que certains codent des facteurs de niche qui pourraient accroître le potentiel des souches d’EHEC à s’adapter à l’environnement intestinal et ainsi participer à la virulence du pathogène. Le second volet de cette thèse avait pour but de caractériser in vivo la réponse des EHEC au monoxyde d’azote (NO), un médiateur de la réponse immunitaire de l’hôte, et ainsi d’évaluer le potentiel rôle protecteur du NO lors d’une infection en modèle murin. En utilisant une souche d’EHEC rapportant la présence de NO, nous avons démontré que le NO est produit dès les premiers stades de l’infection et que celui-ci limite l’adhésion du pathogène à la muqueuse colique. En revanche, nous avons également mis en évidence un effet néfaste du NO pour l’hôte puisqu’il favorise la production des Shigatoxines (Stx), le facteur de virulence majeur des EHEC, conduisant au développement d’un dysfonctionnent rénal. Enfin, nous avons montré l’importance de la NO réductase NorVW dans la virulence de certaines souches d’EHEC. En effet, l’inactivation de l’opéron norVW chez la souche O157:H7 620 réduit la capacité du pathogène à coloniser efficacement le tractus digestif et à produire Stx. Cette observation est toutefois souche dépendante et suggère que la réponse des EHEC au stress nitrosant lors d’une infection est complexe et probablement multifactoriel. L’ensemble de ces travaux contribue à une meilleure compréhension du processus infectieux des EHEC, une étape indispensable au développement de futures stratégies anti-infectieuses<br>Enterohemorrhagic Escherichia coli (EHEC) are a major public health concern. Indeed, these pathogens are responsible for thousands of food-borne illness cases worldwide every year and can lead to serious complications, including kidney damages in young children and brain damages in the elderly. Currently, the main issue is the limited number of available therapeutic treatments since antibiotic therapy can promote the development of infection-related complications. Therefore, it appears essential and topical to identify bacterial factors associated with EHEC virulence and to understand the interactions occurring between the pathogen and the host, in order to develop new anti-infective strategies. The first objective of this thesis was to identify new bacterial factors potentially involved in the infectious process. Application of the RIVET technology to the reference strain O157:H7 EDL933 revealed 31 genes specifically induced during mouse infection. Characterization of these genes showed that some of them encode niche factors potentially involved in the adaptation of EHEC to the intestinal environment, therefore contributing to virulence. The second aim of this thesis was to characterize in vivo the response of EHEC to nitric oxide (NO), a mediator of the host’s immune response, and thus assess the protective role of NO against EHEC infection in a mouse model. By using a NO-sensing reporter EHEC strain, we demonstrated that NO is produced by the host at the early stages of infection and this NO limits adhesion of the pathogen to the colonic mucosa. On the other hand, we also showed that NO is detrimental to the host since it promotes the production of Shigatoxins (Stx), which is the major EHEC virulence factor, and leads to the development of renal dysfunction. Finally, we showed that the NO reductase NorVW is important for the virulence of some, but not all, EHEC strains. Inactivation of the norVW operon in strain O157:H7 620 reduces the ability of the pathogen to efficiently colonize the digestive tract and to produce Stx. However, this observation is strain-specific and this suggests that EHEC response to nitrosative stress during infection is complex and probably multifactorial. This work contributes to a better understanding of the EHEC infectious process, an essential step for the development of future anti-infective strategies
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Matheson, Stephanie L. "Preparatory studies towards the identification of genes regulated by the Ner-like protein (Nlp) of Escherichia coli." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21607.
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A lacZ transcriptional fusion library was constructed without the cloned gene of interest (i.e. nlp). To do this, the nlp gene was interrupted using luxAB and a tetracycline resistance gene in the Deltalac strain E. coli 40. This strain (LF20300) was subsequently transformed with an expression vector possessing the ara PBAD promoter and a chloramphenicol resistance marker (pBAD18Cm). The plasmid-containing strain was then lysogenized with a Mu d1 phage containing the lacZYA reporter gene and an ampicillin resistance marker. The resulting lysogens were selected on TCMG plates containing 0.2% glucose, tetracycline, chloramphenicol, and ampicillin. These lysogens were then mastered onto TCMG plates containing either 0.2% glucose or 0.2% arabinose plus X-gal and the appropriate antibiotics. Differences in blue colour were noted for eight different clones on glucose in comparison to their growth on arabinose. Total genomic DNA was isolated from each arabinose-responsive clone, digested with several different restriction endonucleases, and subjected to Southern hybridization (using labeled lacZ as a probe). Putative restriction maps were determined for those clones with single Mu d1 inserts, and used to identify candidate genes from the complete restriction map of E. coli. (Abstract shortened by UMI.)
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Matheson, Stephanie L. "Preparatory studies towards the identification of genes regulated by the Ner-like protein, Nlp, of Escherichia coli." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0027/MQ50834.pdf.
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Ceccaldi, Pierre. "Identification des déterminants moléculaires de la réactivité d'une molybdoenzyme modèle : La nitrate réductase A d' Escherichia coli." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4016/document.
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Les molybdoenzymes (MoEs) à bisPGD sont des métalloprotéines dont le site actif est constitué d'un cofacteur à molybdène (Mo) mononucléaire. Elles sont impliquées dans les cycles biogéochimiques de l'azote, du carbone et du soufre et catalysent essentiellement des réactions d'oxydoréduction à 2 e-/2 H+ envers une grande variété de substrats. En dépit de la connaissance de la structure cristallographique de nombreuses MoEs, leur fonctionnement reste encore largement incompris. Ces enzymes présentent un intérêt biotechnologique car certains de leurs substrats sont des composés toxiques notoires, tels que les oxydes de sélénium ou d'arsenic. L'objectif de ma thèse a été d'identifier quels facteurs structuraux gouvernent la réactivité d'une MoE à bisPGD, la nitrate réductase A d'E. coli. Le premier axe de mes travaux de thèse a consisté à étudier l'activité de l'enzyme envers différents substrats et examiner le rôle du ligand protéique du Mo dans sa réactivité, en combinant des approches de mutagenèse dirigée, de biochimie et de spectroscopie RPE. J'ai montré que le ligand protéique du Mo est impliqué dans une étape clé du cycle catalytique. Le second axe a consisté à identifier les relations existantes entre la structure atomique du site actif et ses signatures spectrales. Pour augmenter la résolution et permettre d'identifier les transitions structurales mises en jeu lors de l'interconversion entre les différentes formes spectrales, j'ai utilisé la spectroscopie RPE impulsionnelle, qui permet de détecter les noyaux magnétiques (1H et 14N, …). Mes résultats constituent un pré-requis nécessaire pour l'étude structurale à haute résolution du site actif de la nitrate réductase<br>Molybdenum (Mo) is a rare transition metal that is indispensable to most living organisms. In particular, it makes part of the active site of metalloenzymes involved in the biogeochemical cycles of carbon, nitrogen and sulphur. In this context, prokaryotic molybdoenzymes (MoEs) with the bisPGD cofactor at their active site essentially catalyze oxidoreduction reactions with 2 e-/2 H+ towards a wide range of substrates. Given that some MoEs can activate substrates that are well-known pollutants, understanding the mechanism of these enzymes accounts for a major prerequisite for future enzymatic engineering strategies aimed at optimizing enzyme reactivity towards bioremediation processes. To identify the molecular determinants of the reactivity of MoEs, we have explored the importance of the Mo proteic ligand aspartate in the respiratory Nitrate Reductase from E. coli. We have combined biochemistry and EPR spectroscopy to analyze the impact of the Mo-ligand substitution on both the enzymatic and the structural properties of the molybdenum cofactor. Our results show that the nature of the proteic ligand plays a critical role in the reactivity of the active site. A second part of my thesis work consisted in establishing the link between spectroscopic data on the MoV centre and its atomic structure. To get a high level of resolution and to identify which kind of structural modification is responsible for the spectroscopic differences between every Mo(V) signature, pulsed EPR spectroscop is most promising. Our results constitute a pre-requisite for structural studies of every species of the MoV center of the NRA
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Damdimopoulos, Anastasios E. "Identification and functional characterization of novel thioredoxin systems /." Stockholm,, 2003. http://diss.kib.ki.se/2003/91-7349-661-8/.
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Roux, Agnès. "Identification et étude du rôle de nouvelles adhésines impliquées dans la formation des biofilms chez Escherichia et Salmonella." Paris 7, 2006. http://www.theses.fr/2006PA077152.
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Les biofilms sont des populations microbiennes associées aux surfaces et englobées dans une matrice extracellulaire. Ce mode de vie fixé est présent dans tous les environnements et notamment dans les domaines médicaux et industriels où il est fréquemment nuisible. La présence des biofilms est en effet problématique en raison de leurs capacités importantes de dissémination et de résistance aux agents anti-bactériens. Au cours de la formation du biofilm, les bactéries synthétisent différentes structures d'adhésion de surface soumises à un réseau complexe de régulations. En effet, la formation des biofilms est associée à une modification du profil d'expression des gènes et à la mise en place de réponses physiologiques spécifiques. L'objectif de mon travail de thèse est l'exploration de la diversité des facteurs d'adhésion impliqués dans la formation des biofilms chez E. Coli et Salmonella. J'ai développé une technique génétique permettant la modulation fine de l'expression de gènes cibles. Cet outil a été utilisé pour mettre en évidence de nouvelles adhésines chez E. Coli et S. Enteritica. J'ai également identifié deux gènes, ychA et ycbB, présents sur le plasmide conjugatif F qui codent pour des adhésines autotransporteurs et j'ai étudié leur rôle dans l'adhésion et la formation de biofilms chez E. Coli. Enfin, j'ai étudié le rôle des gènes rhs dans la formation des biofilms. Ces gènes n'ont pas de fonction connue et sont considérés comme cryptiques dans les conditions de laboratoire. J'ai montré qu'ils étaient exprimés en biofilm ce qui suggère qu'ils jouent un rôle dans sa formation. Mon travail met en évidence la diversité des structures d'adhésion impliquées dans la formation des biofilms<br>Biofilms are heterogeneous microbial populations associated to surfaces and encased in an extracellular matrix. They are very common in all type of ecological niches and they are often found in medical and industrial environments where they are frequently harmful. The presence of biofilms is indeed problematic because of their high scattering capacities and resistances to anti-bacterial agents. During biofilm formation, bacteria synthesize various adhesive surface structures subjected to a complex network of regulations. Indeed, biofilm formation leads to a modification of the gene expression profile and to specific physiological answers. The objectives of my phD work is the exploration of factors involved in biofilm formation in E. Coli and Salmonella. I first developed a new genetical tool which allows thé modulation of target genes expression, to identify news adhesins in E. Coli and S. Enteritica. I also identified two genes, ychA and ycbB, located on the F conjugative plasmid, which encode autotransporters adhesins and I studied their role in E. Coli biofilm formation. Finally, I investigated the role of the rhs genes in biofilm formation. These genes of unknown function are considered as cryptic in laboratory conditions. I showed that they are specifically expressed in biofilm suggesting that they play a role in its formation. All these results show the wide variety of adhesion factors that are involved in biofilm formation
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Srimanote, Potjanee. "Analysis of putative virulence factors of a locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21 strain." Title page, contents and abstract only, 2003. http://web4.library.adelaide.edu.au/theses/09PH/09php863.pdf.
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"February 2003." Addendum and corrigenda inserted at back Includes bibliographical references (leaves 249-272) Aims to identify and characterise potential virulence-associated factors from the locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21 strain 98NK2 which was responsible for an outbreak of haemolytic uremic syndrome. Particular attention was focused on putative virulence genes encoded on the megaplasmid of this strain.
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Wood, Jayde Lian. "Examination of microbiological quality of in-field leafy vegetables and identification of on-farm generic Escherichia coli transmission dynamics." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44613.
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Fresh produce has increasingly been linked to foodborne outbreaks in North America. Although contamination of produce may occur at any point along the food continuum, significant risks are thought to occur at the farm level. Data illustrating bacterial transmission dynamics on farm, however, are lacking. The aim of this project was to produce baseline data describing the occurrence of indicator bacteria on in-field leafy vegetables; examine antimicrobial resistance (AMR) of recovered Escherichia coli; and identify on-farm microbiological reservoirs impacting leafy vegetables. In-field plants (n=1093) and environmental samples (irrigation water, compost, soil, and hand swabs; n=316) were collected from two production systems (conventional and organic) weekly between July-October during 2011 and 2012. Aerobic colony, coliform, and Escherichia coli counts were determined using 3M Petri-films. Escherichia coli prevalence was determined by enrichment with recovered isolates subjected to BOX-PCR and multiplex PCR phylogenetic typing. Mean coliform count for in-field plants was 1.2 ± 0.1 log₁₀ CFU/g. The prevalence of E. coli was 0.8 and 7% for conventional and organic leafy vegetable samples, respectively. No AMR to therapeutically critical antibiotics was observed in E. coli recovered from in-field plants, though nine (13%, n=67) multi-drug resistant strains were identified. Escherichia coli was recovered from an irrigation water reservoir (27%, n = 15), sprinkler (40%, n = 35), soil (58%, n = 19), hand swabs (4%, n = 27), and compost (6%, n = 16) from the organic production system. Escherichia coli was recovered from ditch water (100%, n = 10), and soil (12%, n = 25) from the conventional production system. Four phylogenetic groups were recovered, with B1 E. coli being the predominant phylogroup (78%). Though 92% of recovered E. coli were unrelated, BOX-PCR revealed identical fingerprints for E. coli recovered from irrigation water or compost and in-field plants. In summary, based on E. coli levels (n=5, c=2, m=100 CFU/g, M=1,000 CFU/g), the microbiological quality of leafy vegetables from both farms was acceptable. BOX-PCR data demonstrated transmission of E. coli from on-farm reservoirs to in-field plants, suggesting possible transmission routes for foodborne pathogens. The limitation of B1 E. coli as fecal indicators was highlighted.
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Strutinsky, Jeanna Brandy Lee. "Identification and genetic mapping of perR, a novel stationary phase gene that mediates oxidative stress protection in Escherichia coli." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/MQ53229.pdf.
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Stokes, Matthew Oliver. "Comparative genomics of blaCTX-M plasmids from veterinary and human 'Escherichia coli' and methods for their identification and differentiation." Thesis, Kingston University, 2014. http://eprints.kingston.ac.uk/29889/.
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The blaCTX-M gene confers resistance to penicillins and cephalosporins and is now the most widely disseminated plasmid mediated Extended Spectrum beta-lactamase (ESBL). Plasmids harbouring blaCTX-M have been recovered from both human and animals isolates, with increasing evidence for the transmission between hosts which is a major public health concern. The aim of this study was to investigate the relationship of blaCTX-M plasmids from UK human and veterinary E. coli isolates with plasmids previously sequenced around the world, and develop molecular markers to identify and differentiate plasmids. Molecular markers were first established as a suitable method for identifying plasmids by studying the prevalence of IncK pCT-like plasmids, which were found to be associated with 30% of CTX-M-14 producers in the UK, with plasmids mobilising the gene between unrelated isolates from cattle, turkeys and humans. Seven blaCTX-M plasmids, belonging to four incompatibility groups, from E. coli were isolated, fully sequenced and annotated. The only human sequenced plasmid was pH19 the first IncZ blaCTX-M-14 to be sequenced. The plasmids sequenced from animals included pSAM7 from cattle, the first IncX4 plasmid to be sequenced with blaCTX-M-14b in a novel transposition unit. Plasmid pCH01 was isolated from a chicken isolate and harboured the blaCTX-M-3 and is the first IncA/C group CTX-M to be sequenced. The four IncI1γ blaCTX-M-1 plasmids from chicken (pCH02 and pCH03), cattle (pCT01) and turkey (pT01), are the first ST3 and IncI1γ blaCTX-M-1 plasmids to be sequenced. Comparative analysis found UK plasmids shared approximately 70-99% sequence coverage with previously published sequences from different hosts and bacterial species around the world. This demonstrated that plasmids in the UK were closely related to plasmids found elsewhere, and no genetic characteristics were identified why these plasmid could not exist in either human or animals isolates, with the main differences observed in the inserted resistance regions. In all plasmids the blaCTX-M was associated with ISEcp1, and included a novel blaCTX-M-14b and blaCTX-M-3 transposition unit in pSAM7 and pCH01 respectively. In 6/7 plasmids the ISEcp1-blaCTX-M was not associated with any other resistance regions, inserting as separate events. Molecular markers were designed from the comparative analysis between plasmids that were capable of both identifying and differentiating plasmids belonging to the same incompatibility group. Five groups were identified for IncX4, eight for IncZ, B and K, 12 for IncI1γ and 14 for IncA/C. Markers were used in screening of field isolates to identify similar plasmids, with novel combinations being observed, not previously identified in silico. These markers represent a new non-sequencing based tool to identify and characterise plasmids further, benefitting the study of plasmids and their epidemiology.
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Camprubí, Font Carla. "Genetics and transcriptomics of adherent-invasive Escherichia coli (AIEC): new approaches to uncover molecular markers for its rapid identification." Doctoral thesis, Universitat de Girona, 2019. http://hdl.handle.net/10803/672302.
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The adherent-invasive Escherichia coli (AIEC) pathotype could play a role in the course of Crohn’s disease. This is characterized by its capacity to adhere to and to invade intestinal epithelial cells as well as to replicate and survive within macrophages. At present, identification of the AIEC pathotype relies on time-consuming techniques based on the phenotypic screening of cultured bacteria, which are not standardized. In this thesis, we focused on the study of AIEC genetics in order to look for key characteristics that could assist the development of a molecular tool for the identification of the pathotype.
To sum up, the results of this work provide meaningful information that contributes to our understanding of AIEC genomics. In this case, two putative molecular markers resulting from a combination of genetic and/or phenotypic features have been presented and these could assist in AIEC screening. Finally, gene expression results provide new insights to better describe genes putatively involved in AIEC virulence<br>El patotip adherent-invasiu d’Escherichia coli (AIEC) podria jugar un paper en el transcurs de la malaltia de Crohn. Aquest es caracteritza per tenir capacitat d’adhesió i invasió a cèl·lules de l’epiteli intestinal a més de replicar-se i sobreviure en macròfags. Actualment la única manera d’identificar aquests bacteris és analitzant aquestes característiques fenotípiques, un mètode poc estandarditzat i que requereix molt temps i dedicació. En la present tesi ens hem centrat en estudiar genèticament el patotip AIEC per tal de buscar característiques clau que puguin ajudar en el desenvolupament d’una eina molecular per a la seva identificació.
En resum, els resultats d'aquest treball proporcionen informació significativa que contribueix a la comprensió de la genètica del patotip AIEC. En aquest cas, s'han presentat dos possibles marcadors moleculars resultants d'una combinació de característiques genètiques i/o fenotípiques que podrien ajudar en la detecció d’AIEC. Finalment, els resultats d'expressió gènica proporcionen noves idees per descriure millor els gens implicats en la virulència del patotip AIEC<br>Programa de Doctorat en Biologia Molecular, Biomedicina i Salut
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Hafidi, Ghizlane. "Application de la commande prédictive non-linéaire à la commande de culture de bactéries escherichia coli." Phd thesis, Université Paris Sud - Paris XI, 2008. http://tel.archives-ouvertes.fr/tel-00345850.
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Cette thèse propose une méthodologie de commande d'un bioréacteur fed-batch de culture E. coli. La stratégie consiste à maximiser la croissance de la bactérie, c'est-à-dire à maintenir le bioréacteur à un point de fonctionnement « optimal », caractérisé par la frontière entre les régimes oxydatif et oxydo-fermentatif. La démarche proposée comprend une première étape de modélisation mathématique et de détermination d'un modèle paramétrique simplifié. Une deuxième phase consiste en une identification paramétrique du modèle en se basant sur l'analyse de sensibilité du modèle vis-à-vis de ses paramètres. Un profil optimal d'alimentation est ensuite élaboré pour la maximisation de la croissance de la biomasse. A partir de toutes ces données, la synthèse et l'application de la commande prédictive non-linéaire au bioprocédé E. coli sont mises en œuvre. L'objectif est de réguler la concentration en acétate à une valeur faible donnée, tout en forçant le débit d'alimentation à suivre un profil de référence. La stratégie de commande proposée se base sur la transformation du problème de commande prédictive non-linéaire classique en un problème de programmation non-linéaire non contraint, résolu par des techniques de CVP. Pour une meilleure robustesse de la structure proposée, la différence entre le système et le modèle est explicitement incluse dans l'algorithme. Enfin, une étude de robustesse par une approche statistique de type Monte Carlo permet de juger de l'applicabilité de la loi de commande proposée sur le système réel. Ce travail constitue une étude préliminaire en vue d'une implantation de cette loi de commande à un bioréacteur à l'échelle du laboratoire ou industrielle
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Rongy, Manuelle. "Identification of bacterial genes regulated by the Escherichia coli transposable phage ner protein homologue, NlpSfs 7, a histone-like protein." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37642.
Full textAbstract:
DNA-binding proteins such as IHF play a crucial role in bacterial survival. IHF is involved in many biological processes in E. coli and possibly in other bacteria. It was hypothesized that another protein, Nlp, belongs to a family of conserved DNA-binding proteins, and this protein was shown to be non-essential for cell viability, but highly conserved among the Enterobacteriaceae. As Nlp appears to play a role in gene regulation, we wished to identify all Nlp-regulated genes. nlp was cloned into pBAD18-Kan generating pMM5, where Nlp expression was under the control of arabinose. Strain LF20300 was transformed with pMM5 and then lysogenized with the lacZ transcriptional fusion reporter vector Mu dI to create a library of approximately 10,000 clones. This library was screened on plates containing either glucose/X-gal or arabinose/X-gal in order to identify genes whose expression was altered upon production of Nlp. Two clones, 90-6 and 205-15, were identified which displayed increased beta-galactosidase expression in the presence of Nlp. Further studies with these two clones have shown the insertion of a single Mu dI phage within clone 90-6 and a double insertion within clone 205-15. Cloning and sequencing of one of the Mu dI insertions of strain 205-15 has identified the yqhG gene as being one of the genes whose expression may be altered in the presence of Nlp.
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Williams, Kyle Brandon. "The DamX cell division protein of Escherichia coli: identification of amino acid residues critical for septal localization and peptidoglycan binding." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/625.
Full textAbstract:
In the bacterium Escherichia coli, cell division involves the concerted inward growth of all three layers of the cell envelope: the cytoplasmic membrane, the peptidoglycan (PG) cell wall, and the outer membrane. This is a complex, highly regulated process that involves over 20 proteins. Four of these proteins contain a domain of ~70 amino acids known as a SPOR domain (Pfam no. 05036). One of these SPOR domains (from a protein named FtsN) has been shown previously to bind PG. In this thesis we show that six additional SPOR domains, three from E. coli and three from other bacterial species, also bind PG. Thus, PG binding is a general activity of SPOR domains. We then examine the SPOR domain from DamX of E. coli in detail. In collaboration with Dr. Andrew Fowler of the NMR Core Facility, we determined the solution structure of the domain. The domain adopts an "RNP fold," characterized by a four-stranded anti-parallel β-sheet that is buttressed on one side by α-helixes. Several mutant forms of the DamX SPOR domain were constructed and studied both in vivo and in vitro. These studies support the following inferences: 1) The β-sheet is the PG-binding site; 2) The β-sheet contains critical information for targeting the SPOR domain to the midcell; 3) The SPOR domain probably localizes to the midcell by binding preferentially to septal PG; and 4) It follows, then, that septal PG must differ from PG elsewhere around the cell. We suggest that further studies of the SPOR:PG interaction will yield novel insights into PG biogenesis during septation.
This thesis also presents an in vivo characterization of several mutant forms of a cytoplasmic membrane protein named FtsW, homologs of which are found in all bacteria that contain a PG cell wall. FtsW recruits a PG synthase named FtsI to the division site and might also transport PG precursors across the cytoplasmic membrane. We systematically mutagenized each of FtsW's ten transmembrane (TM) helixes and investigated the ability of the mutant proteins to support division, localize to the division site, and recruit FtsI. This characterization leads us to propose that TM1 is involved in targeting FtsW to the division site, TM4 is involved in the putative transport activity, and TM10 is involved in recruitment of FtsI.
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Ndlovu, Thando. "Comparison of diagnostic tools and molecular based techniques for the rapid identification of Escherichia coli and coliforms in contaminated river water." Thesis, Cape Peninsula University of Technology, 2013. http://hdl.handle.net/20.500.11838/794.
Full textAbstract:
Thesis submitted in fulfilment of the requirements for the degree
Master of Technology: Environmental Health
in the Faculty of Applied Sciences
at the Cape Peninsula University of Technology, 2013<br>Water is an important daily requirement and in a clean, pure form, it promotes health and well-being. In addition to South Africa being one of the driest countries in the world, water availability is also being compromised by massive pollution of remaining water sources. The Berg- and Plankenburg Rivers are two of the surface water sources in the Western Cape, South Africa, which are highly polluted by sewage, industrial and agricultural run-off. The current investigation was aimed at comparing diagnostic tools, which are employed by municipalities and food industries, and molecular based techniques to routinely monitor water for indicator organisms in time- and cost-effective manner. These rivers were sampled twice a month (July 2010 to January 2011) at the sites closest to the informal settlements of Kayamandi in Stellenbosch (Plankenburg River) and Mbekweni in Paarl (Berg River).
The contamination levels of the two river systems were evaluated by the enumeration of Escherichia coli and coliforms using the Colilert 18® system, Membrane Filtration (MF) and Multiple Tube Fermentation (MTF) techniques. The highest faecal coliform count of 9.2 × 106 microorganisms/100 ml was obtained in weeks 21 and 28 from the Plankenburg River system by the MTF technique, while the lowest count of 1.1 × 103 microorganisms/100 ml was obtained in week one for both river systems by the MTF technique. The highest E. coli count of 1.7 × 106 microorganisms/100 ml was obtained from the Berg River system (week 9) using the MTF technique, while the lowest count of 3.6 × 102 microorganisms/100 ml was obtained by the MF technique from the Plankenburg River system. The coliform and E. coli counts obtained by the enumeration techniques thus significantly (p > 0.05) exceeded the guidelines of 2000 microorganisms/100 ml stipulated by the Department of Water Affairs and Forestry (DWAF, 1996) for water used in recreational purposes.
Overall the results obtained in this study showed that the water in the Berg- and Plankenburg River systems is highly polluted, especially where these water sources are used for irrigational and recreational purposes. For the coliform and E. coli counts obtained using the three enumeration techniques, it was noted that the MTF technique was more sensitive and obtained higher counts for most of the sampling weeks. However, the media (Membrane lactose glucuronide agar) used in the MF technique also effectively recovered environmentally stressed microbial cells and it was also better for the routine selection and growth of coliforms and E. coli. While E. coli and total coliforms were detected utilising the Colilert 18® system, accurate enumeration values for these two indicator groups was not obtained for the entire sampling period for both river systems. It has previously been shown that dilutions (up to 10-3) of highly polluted waters increase the accuracy of the Colilert 18® system to enumerate colifoms and E. coli in marine waters. As the results obtained utilising
the Colilert 18® system were also not comparable to the MF and MTF techniques it is recommended that highly polluted water samples be diluted to increase the accuracy of this system as a routine enumeration technique.
Water samples were directly inoculated onto MacConkey, Vile Red Bile (VRB) agar and the Chromocult Coliform agar (CCA) and single colonies were inoculated onto nutrient agar. Chromocult coliform agar proved to be more sensitive than MacConkey and VRB agar for the culturing of E. coli and coliforms. Preliminary identification of these colonies was done using the RapID ONE and API 20 E systems. The most isolated Enterobacteriaceae species by both systems, included Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli and Enterobacter cloacae in both river systems. The API 20 E system was more sensitive in the preliminary identification of the various isolates, as greater species diversity was obtained in comparison to the RapID ONE system.
The Polymerase Chain Reaction (PCR) was firstly optimised using positive Enterobacteriaceae species. The optimised method was then applied to the analysis of river water samples, which were centrifuged to harvest the bacterial cells, with DNA extracted using the boiling method. The extracted DNA was amplified using conventional PCR with the aid of species specific primers. The Enterobacteriaceae species that were detected throughout the study period in both river systems include Serratia marcescens, Escherichia coli, Klebsiella pneumoniae and Bacillus cereus. Conventional PCR was the most reliable and sensitive technique to detect Enterobacteriaceae to species level in a short period of time when compared to RapID ONE and the API 20 E systems. Multiplex PCR was optimised using the positive pathogenic E. coli strains namely, Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAEC). It was then employed in river water sample analysis and enabled the detection of EAEC, EHEC, and EIEC strains in Berg River system, with only the EAEC detected in the Plankenburg River system. Real-time PCR was used to optimise the multiplex PCR in the amplification of E. coli strains and successfully reduced the time to obtain final results when using control organisms. Real-time PCR was found to be more sensitive and time-effective in the identification of E. coli strains, and also more pronounced DNA bands were observed in real-time PCR products compared to conventional-multiplex PCR amplicons.
To sustain the services provided by the Berg- and Plankenburg Rivers in the Western Cape (South Africa), these water sources should frequently be monitored, results assessed and reported according to the practices acknowledged by responsible bodies. It is therefore recommended that the enumeration techniques be used in conjunction with the very sensitive PCR technique for the accurate detection of coliforms and E. coli in river water samples.
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Branchu, Priscilla. "Pathogénicité des Escherichia coli entérohémorragiques : identification de voies de régulation contrôlant la mobilité, la formation de biofilm et le locus d'effacement des entérocytes." Phd thesis, Université Blaise Pascal - Clermont-Ferrand II, 2012. http://tel.archives-ouvertes.fr/tel-00866934.
Full textAbstract:
Les Escherichia coli entérohémorragiques (EHEC) sont responsables de toxi-infections alimentaires conduisant à des colites hémorragiques pouvant se compliquer d'un syndrome hémolytique et urémique. Une fois arrivés dans l'intestin, les EHEC adhèrent aux cellules épithéliales en causant des lésions d'attachement-effacement. Le système de sécrétion de type III et les protéines effectrices requis pour ce phénotype sont codés majoritairement par le locus d'effacement des entérocytes (LEE), constitué de plusieurs opérons (LEE1-5). Notre étude a permis de clarifier une des cascades de régulation contrôlant l'expression du LEE. Par des analyses en qRT-PCR et des immuno précipitations de la chromatine, nous avons déterminé que les régulateurs GadE et GadX sont des répresseurs indirects de l'expression du LEE. GadE active l'expression de gadX, et GadX réprime l'expression de ler, codant pour le principal activateur des opérons LEE2-5. De plus, GadE réprime aussi l'expression des opérons LEE4 et LEE5 indépendamment de Ler. En retour, Ler réprime l'expression de gadE et de gadX. Le monoxyde d'azote (NO) est un effecteur majeur de la réponse immune innée, produit en particulier par les cellules épithéliales intestinales. Il avait été montré que le NO réprime l'expression du LEE et active celle de gadE et de gadX. Notre étude a permis d'identifier le régulateur clé responsable de ces régulations, NsrR. NsrR réprime indirectement l'expression de gadE et gadX et active l'expression des opérons LEE1, LEE4 et LEE5 en se fixant sur leurs promoteurs respectifs. En présence de NO, NsrR devient inactif. Ainsi, le NO réprime directement l'expression du LEE en supprimant la fixation de NsrR aux promoteurs du LEE1, LEE4 et LEE5, et indirectement en activant l'expression de gadE et donc de gadX. Un modèle de régulation intégrant l'ensemble de ces résultats est proposé. D'autre part, nous avons identifié et caractérisé une nouvelle phosphodiestérase spécifique des EHEC les plus pathogènes, VmpA. Par son activité d'hydrolyse du di-GMPc, VmpA contrôle la mobilité bactérienne, la formation de biofilm, et probablement l'expression du LEE, mais aurait aussi un rôle plus général dans la physiologie des EHEC.
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E, Guangqi. "La cyclopropane fatty acid synthase d'Escherichia coli : études mécanistiques et identification de nouveaux inhibiteurs." Paris 6, 2008. http://www.theses.fr/2008PA066302.
Full textAbstract:
La tuberculose est l’une des maladies les plus mortelles dans le monde. L'apparition de souches de Mycobacterium tuberculosis résistantes aux antibiotiques actuellement utilisés, impose de trouver de nouvelles cibles et de nouveaux antituberculeux. Les enzymes responsables de la biosynthèse des acides mycoliques, composants essentiels de la paroi des mycobactéries, constituent de très bonnes cibles thérapeutiques, et en particulier, les Cyclopropane Synthase (CS) de Mycobacterium tuberculosis. La Cyclopropane Fatty Acid Synthase (CFAS) d’Escherichia coli est une enzyme homologue de ces CS et constitue un très bon modèle d’étude pour élucider le mécanisme enzymatique des CS et pour trouver de nouveaux agents antituberculeux. En effet, les CS de M. Tuberculosis ne sont pas fonctionnelles in vitro. La CFAS transfère le méthyle de la S-adénosyl-L-méthionine vers une double liaison non activée de phospholipides insaturés pour former le motif cyclopropane. Les travaux réalisés, au cours de cette thèse, ont permis de mieux comprendre le mécanisme réactionnel de la CFAS : En utilisant diverses approches (mutagenèse dirigée, échanges isotopiques, mesures d'effets isotopiques cinétiques…) nous avons pu préciser quelques points concernant le mécanisme réactionnel de cette réaction. En ce qui concerne la recherche d’inhibiteurs, grâce à un test colorimétrique mis au point au laboratoire nous avons pu cribler la chimiothèque de l’ICSN, et nous avons identifié quelques inhibiteurs de faibles IC50. Le mode d'action de certain de ces inhibiteurs a été précisé. D'autre part, nous avons montré qu'un des inhibiteur inhibe la cyclopropanation in vivo chez E. Coli. C'est le premier inhibiteur actif contre la CFAS in vivo. Une méthode de synthèse de cet inhibiteur et d'analogues a été mise au point et permettra de faire des études de structure-activité afin d'identifier des inhibiteurs plus puissants de la CFAS.
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Kihara, Akio. "Identification,characterization,and regulation of the proteolytic system that degrades uncomplexed SecY subunit of protein translocase in the Escherichia coli plasma membrane." 京都大学 (Kyoto University), 1998. http://hdl.handle.net/2433/157158.
Full textAbstract:
本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである<br>Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(理学)<br>甲第7164号<br>理博第1938号<br>新制||理||1043(附属図書館)<br>UT51-98-G93<br>京都大学大学院理学研究科化学専攻<br>(主査)教授 伊藤 維昭, 教授 井上 丹, 教授 三木 邦夫<br>学位規則第4条第1項該当
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Sauget, Marlène. "Identification de marqueurs épidémiologiques par spectrométrie de masse de type MALDI-TOF : application aux principales espèces bactériennes responsables d'infections nosocomiales." Thesis, Besançon, 2016. http://www.theses.fr/2016BESA3006/document.
Full textAbstract:
Le typage bactérien est une mesure de contrôle essentielle pour lutter contre la diffusion des bactéries multirésistantes, mais les techniques actuelles sont longues et coûteuses. L'objectif de cette thèse était d'évaluer la capacité de la technique MALDI-TOF MS à typer les 3 principales espèces bactériennes responsables d'infections nosocomiales - Escherichia coli, Staphylococcus aureus et Pseudomonas aeruginosa. L'analyse par MALDI-TOF MS permet d'identifier les souches de E. coli appartenant au phylogroupe B2, souches les plus virulentes parmi les souches extra-intestinales, et au STI 31, clone très impliqué dans la dissémination des P-lactamases à spectre étendu. Chez S. aureus, cette technique reconnait les souches appartenant au CC398, impliquées dans une proportion importante d'infections graves chez des personnes fragiles. La technique MALDI-TOF MS identifie également cinq clones de P. aeruginosa à haut risque épidémique - STI 11, STI 75, ST235, ST253, ST395. Si nous avons confirmé des pics caractéristiques du phylogroupe B2 décrit également par d'autres auteurs, les pics biomarqueurs identifiant E. coli ST131 ou des clones épidémiques de S. aureus varient suivant les études. Différents paramètres peuvent influencer les résultats de typage par MALDI-TOF MS et doivent donc être standardisés. La technique MALDI-TOF MS permet d'identifier certains clones épidémiques. En gardant à l'esprit que le choix d'une méthode de typage doit être fait en fonction des objectifs mais aussi des performances des différents systèmes disponibles, la technique MALDI-TOF MS pourrait se positionner comme un outil de typage de première ligne<br>Bacterial typing is crucial to tackle the spread of bacterial pathogens but current methods are time-consuming and costly. The objective of this study was to evaluate the ability of MALDI-TOF MS to type the 3 main bacterial species re.sponsible for nosocomial infections - Escherichia coti, Staphylococcus aureus and Pseudomonas aeruginosa. Analysis of MALDI-TOF mass spectra allow the identification (i) of E. coti isolates belonging to phylogroup B2, that fosters the most virulent extra-intestinal strains, and (ii) of E. coli STl 31, a clone involved in the dissemination of extended-spectrum β-lactamases. MALDI-TOF MS also recognizes S. aureus isolates belonging to CC398, a pathogen responsible for invasive infections in fragile patients and associated with a higher 30-day mortality. Furthermore the MALDI-TOF MS based typing method identifies the major high risk epidemic clones of P. aeruginosa STl 11, STl 75, ST235, ST253, and ST395. We confirmed phylogroup B2 specific peaks also described by other authors. However the identification of E. coli STl 31 or epidemic clones of S. aureus is based on biomarkers that differs between studies. Different parameters can influence the MALDI-TOF MS typing results and must therefore be standardized. MALDI-TOF-based typing methods allow the detection of epidemic pathogens. Keeping in mind that the choice of a method for routine bacterial typing should be made according to the objectives but also the performance of the different systems available, the MALDI-TOF MS technique could become a first-line typing method