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Neelakanta, Girish. "Genome variations in commensal and pathogenic E.coli." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974330329.
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Schlegel, Susan. "From protein production to genome evolution in Escherichia coli." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-94993.
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The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields. To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.
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Romero, Alvarez David. "Genome wide analyses of the Escherichia coli primary and secondary transcriptomes." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/6917/.
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Escherichia coli K12 serves as an important model for studying systems that are important to bacteria in their own right as well as those that are conserved in ‘higher' organisms, which are more difficult and costly to study. Like many model organisms, the genome of K12 has been sequenced, producing a catalogue of protein-coding and stable-RNA genes that enabled study using ‘omic’ approaches. This has led to a rapid expansion of our knowledge of patterns of gene expression and their dependency on growth conditions, cell physiology and individual genes. However, the underlying networks of gene regulation are less well understood, but are known to involve the control of steps in RNA processing and degradation as well as transcription and translation. With this in mind, this thesis describes the development of an approach based on RNA sequencing that produces nucleotide-resolution transcriptome maps that distinguish sites that correspond to RNA processing and steps in degradation from those of transcription initiation, while incorporating all classes of RNA. Comparison with results obtained previously validated the approach, which has been applied already to the study of other bacterial species. Within the E. coli map, many new features were identified, such as previously undetected small RNAs and processing at a site associated with the production of specialised ribosomes, which may ensure the translation of leaderless mRNAs, which were also mapped. The approach also showed the benefit of incorporating steps that can differentiate the 5’ status of transcripts in assigning sites of transcription initiation. RNA sequencing was also used to map sites of cleavage by RNase E, an essential endoribonuclease that is central to both the processing and degradation of RNA in bacteria and plant plastids. This aspect of the thesis has advanced from pilot studies to the point where the ‘code’ that determines one form of substrate recognition by RNase E is beginning to emerge. As a result of this success, equivalent data has been collected for other ribonucleases involved in RNA processing and degradation. Continuing analysis of the primary and secondary transcriptomes, consisting of native, unprocessed transcripts and of transcripts that have been modified from their native form via processing and/or degradation respectively, with the tools presented here promises to broaden and deepen our understanding of an important model organism.
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Coss, Dennis. "Insertion of genes and operons into the Escherichia coli genome through targeted recombination." Morgantown, W. Va. : [West Virginia University Libraries], 2005. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=3804.
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Thesis (Ph. D.)--West Virginia University, 2005.Title from document title page. Document formatted into pages; contains v, 125 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 71-87).
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Mosberg, Joshua Adam Weintrob. "Studying and Improving Lambda Red Recombination for Genome Engineering in Escherichia coli." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10777.
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The phage-derived Lambda Red recombination system utilizes exogenous DNA in order to generate precise insertion, deletion, and point mutations in Escherichia coli and other bacteria. Due to its convenience, it is a frequently-used tool in genetics and molecular biology, as well as in larger-scale genome engineering projects. However, limited recombination frequency constrains the usefulness of Lambda Red for several important applications. In this work, I utilize a mechanism-guided approach in order to improve the power and utility of Lambda Red recombination.
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Schmidt, Dorothea. "Molekulare Analyse des probiotischen Stamms Escherichia coli Nissle 1917." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1243973355362-88295.
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Der probiotische Stamm E. coli Nissle 1917 ist ein Fäkalisolat, das in der Medizin traditionell zur Behandlung verschiedener gastrointestinaler Erkrankungen eingesetzt wird. Durch erfolgversprechende klinische Studien zur Remissionserhaltung bei Colitis ulcerosa, bei denen EcN als therapeutische Alternative zur Standardmedikation eingesetzt wird, ist das Interesse an den Wirkmechanismen von Probiotika stark gestiegen. EcN gehört derzeit zu den am besten untersuchten Probiotika. Einige Wirkmechanismen konnten dadurch schon aufgeklärt werden. So sind vermutlich Strukturkomponenten und stammspezifische Syntheseleistungen an der Ausprägung des probiotischen Phänotyps von EcN beteiligt. Schlüssige Konzepte, die über Gene, Genprodukte und molekulare Mechanismen den probiotischen Effekt von EcN erklären, fehlen bislang. Im Rahmen dieser Arbeit wird das Genom von EcN analysiert und auf der Basis der Genomsequenz mit anderen E. coli-Stämmen verglichen. Mit Hilfe einer Promotor-Reporter-Fusionsbibliothek (Promotorbank) werden intestinal in vivo regulierte Gene identifiziert und dadurch neue Ansätze zur Untersuchung der probiotischen Eigenschaften von EcN geschaffen. Die Grundlage für die molekulare Analyse von EcN ist die manuelle Nachannotation seines sequenzierten Genoms. Die EcN-Sequenz wird mit 13 weiteren annotierten E. coli-Sequenzen verglichen. Nach dieser Analyse kodiert EcN derzeit 121 stammspezifische Gene. Die Genomstruktur ist mit den enthaltenen genomischen Inseln und Prophagen dem Genom des uropathogenen E. coli CFT073 sehr ähnlich. Mit wenigen Ausnahmen kodiert EcN alle in E. coli CFT073 vorhandenen Virulenz- und Fitnessfaktoren, so dass auf der Nukleotidebene die nahe Verwandschaft dieser beiden Stämme bestätigt werden kann. Zudem kann gezeigt werden, dass EcN in artifiziellen Systemen wie der Zellkultur oder gnotobiotischen Mäusen ein pathogenes Potenzial hat, obgleich die Kolonisierungsfähigkeit pathogener Bakterien durch Inkubation mit EcN herabgesetzt wird. Eine wichtige Rolle bei der Besiedlung des Intestinaltrakts und der Immunstimulation von Darmepithelzellen spielt auch die globale Regulation der Genaktivität bei EcN durch den alternativen Sigma-Faktor RpoS, der im Gegensatz zu rpoS-Deletionsmutanten zu einer gesteigerten mRNA-Expression des Tight-junction Proteins ZO-1 führt. Des Weiteren führte die Untersuchung von EcN-Deletionsmutanten zu der Schlussfolgerung, dass einige genomische Inseln für Eigenschaften, die das probiotische Verhalten erklären können, eine Rolle spielen. Durch den Einsatz einer Promotorbank von EcN in konventionellen und gnotobiotischen Mäusen werden erstmalig Sequenzen von intestinal in vivo aktiven Promotoren identifiziert. Der Aufbau eines Promotor-Reportergen-Assays mit dem Biolumineszenz erzeugenden luxCDABE-Operon ermöglichte die Untersuchung ausgewählter Promotoren in vitro. Mit einem In Vivo Imaging System (IVIS) kann in weiteren Experimenten die Aktivität dieser Promotoren in lebenden Mäusen untersucht werden. Im Rahmen dieser Arbeit wird gezeigt, dass EcN kein vollkommen harmloser probiotischer Stamm ist. Weitere Informationen über EcN sind dehalb wichtig für eine optimierte Anwendung als Therapeutikum. Die molekulare Analyse ist somit eine unbedingt notwendige Grundlage für weiterführende Untersuchungen der Eigenschaften von EcN, die für seinen probiotischen Charakter verantwortlich sindThe probiotic E. coli Nissle 1917 is a fecal isolate which is traditionally used for treatment of various gastrointestinal disorders. In clinical trials where EcN was used as therapeutic alternative for remission maintenance of ulcerative colitis compared to standard medication, promising results led to an increased interest in probiotics. Today, EcN is one of the best studied probiotics. Therefore, several mechanisms of action could be enlightened. Structural components and strain-specific products are responsible for its probiotic effects. But conclusive concepts about genes, gene products and molecular mechanisms that really contribute to the probiotic character of EcN have not been offered so far. In order to create new possibilities to elucidate the probiotic traits of EcN the genome is analysed by taking this as a basis for comparison to other E. coli genomes and identification of intestinal in vivo regulated genes using a promoter-trap-library. The sequenced EcN genome is annotated and compared to 13 other so far annotated E. coli genomes. Concerning these analyses EcN encodes 121 strain-specific genes. The genome structure including the genomic islands and prophages is highly homolog to the uropathogenic E. coli CFT073. EcN encodes most of the virulence and fitness factors that are present in E. coli CFT073. Therefore, the close relationship of these two strains is confirmed at nucleotide level. Furthermore, it is shown that in artificial systems like cell culture assays and gnotobiotic mice EcN reveals a pathogenic potential although EcN is able to decrease colonization efficiency of pathogenic bacteria. The alternative sigma factor RpoS that is responsible for global regulation and activity of several genes seems to play an important role during colonization of EcN in the intestine and its immunostimulatory effects on intestinal epithelial cells. Investigation of EcN-deletion mutants lacking genomic islands and prophages lead to the conclusion that some genomic islands may play a role for specific probiotic traits. This is the first time where a promoter-trap-library was used in conventional and gnotobiotic mice for collection of intestinal in vivo active promoters. Constructing and establishing a promoter-reporter gene assay with the bioluminescent luxCDABE operon made the investigation of selected promoters in vitro possible as well as establishing a bioluminescence assay using an In Vivo Imaging System (IVIS) for investigation of promoter activity in living mice. In this research project was shown that EcN is not a completely harmless probiotic. The genome structure and regulatory mechanisms of gene expression are the strain’s molecular traits that lead to probiotic activity and immunostimulatory effects. Therefore, the molecular analyses presented here, together with the complete genome sequence, are a basis for further investigations of mechanisms that are responsible for the probiotic effects of EcN
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Coulange, Frédérique. "Isolement et caracterisation de regions specifiques du genome des escherichia coli pathogenes aviaires (doctorat : microbiologie)." Paris 11, 1999. http://www.theses.fr/1999PA114802.
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PRADEL, NATHALIE. "Escherichia coli producteurs de shiga-toxines : etude epidemiologique, recherche des caracteristiques des souches pathogenes par comparaison moleculaire et hybridation soustractive (doctorat : microbiologie)." Clermont-Ferrand 1, 2001. http://www.theses.fr/2001CLF1PP02.
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Brambilla, Elisa. "Investigation of E. coli genome complexity by means of fluorescent reporters of gene expression." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066607/document.
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Escherichia coli est capable de survivre dans de nombreux environnements différents. Les informations nécessaires à cette adaptation sont codées dans le chromosome. Cette molécule circulaire est condensé dans une structure compacte protéines-ADN, appelée nucléoïde. Le chromosome n¿est pas uniforme et montre notamment une distribution inégale de sites de fixation de protéines et de séquences riches en AT. Il a été montré que la position des gènes importants pour la cellule est hautement conservée dans les gamma-protéobactéries. Ces différences le long du chromosome et cette conservation de la position suggèrent que la position du gène peut influencer son expression. Pour tester cette hypothèse, on a étudié l'expression d'un gène fluorescent inséré dans différentes positions autour du chromosome. L'expression de ce gène est contrôlé par des promoteurs différemment régulés: un est réprimé par la protéine H-NS, un est non régulé et un est sensible au superenroulement de l'ADN. Nous avons étudié l'expression dynamique de ces promoteurs pendant les différentes phases de croissance dans différentes conditions. Nous avons montré que l'expression du promoteur dépendant de la protéine H-NS est liée à l'emplacement sur le chromosome. En effet, la répression par H-NS est accrue en présence de séquences riches en AT. Nous avons également étudié l'influence d'un gène divergent sur l'expression de gènes rapporteurs en fonction de la position chromosomique. Nous avons montré que cette influence dépend de la localisation du gène. Nous avons donc demontré l'impact de la position chromosomique sur l'expression des gènes tout en donnant une nouvelle perspective sur la complexité du génomeEscherichia coli is able to survive in many different environments. The information necessary for this adaptation is encoded in the chromosome. This circular molecule is condensed in a compact DNA-protein structure, called the nucleoid. The chromosome is not uniform, and shows uneven distributions of nucleoid-associated proteins (NAPs) binding sites, AT-rich sequences and general protein occupancy domains. It has been demonstrated that the position of important genes is highly conserved in ?-Proteobacteria. These differences along the chromosome and the conserved position of important genes suggest that the position of the gene can influence gene expression. To test this hypothesis, I studied the expression of a fluorescent reporter gene inserted in different positions around the chromosome. The expression of the reporter is driven by differently regulated promoters, one repressed by the important NAP H-NS, one non regulated and one subject to supercoiling and stringent control. We studied the dynamical expression of these promoters in different growth conditions, growth phases, upon nutritional upshift and under stress. We showed that the expression of the H-NS dependent promoter depends on the location on the chromosome, because H-NS repression is enhanced in presence of AT-rich sequences. We also studied the influence of a divergent gene on the reporter expression as a function of chromosomal position, and showed that this influence depends on the location of the gene. With our study we have been therefore able to show the impact of chromosomal position on gene expression and to give a new perspective on genome complexity
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Nguyen, Huong LE. "Etude des facteurs régulateurs de la traduction chez Escherichia coli." Thesis, Toulouse, INSA, 2019. http://www.theses.fr/2019ISAT0004.
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L’analyse des régulations de l’expression des gènes chez les bactéries permet de comprendre l’adaptation des bactéries à leur environnement et dans un contexte de biologie de synthèse d’optimiser la production microbienne de molécules d’intérêt. Notre objectif a été d’étudier la traduction au niveau du génome et ses relations avec les autres processus cellulaires par une approche de biologie des systèmes. Le traductome a été mesuré : pour chacun des ARN messagers, son pourcentage de copies en traduction et sa densité en ribosomes. Pour la première fois, une image complète de l’état traductionnel de E. coli en croissance rapide a été obtenue, caractérisée par une majorité de transcrits avec un très fort pourcentage de copies en traduction mais faiblement chargés en ribosomes. Notre modèle statistique a identifié des facteurs liés à la séquence comme déterminants de la traduction et le rôle important d’un paramètre physiologique : la concentration en ARNm. Pour la première fois, cet effet de la transcription sur la traduction a été validé à l’échelle moléculaire sur plusieurs gènes. Nous avons montré qu’une augmentation de la concentration d’un ARNm par induction transcriptionnelle entrainait une augmentation du pourcentage de copies en traduction et de la charge en ribosomesThe analysis of gene expression regulation is necessary to better understand bacterial adaptation to environment and to be able in a context of synthetic biology to optimize the production of molecules of interest. The goal of this thesis was to study translation at the genome-wide level and its relationship to other cellular processes using a systems biology approach. First, translation activity at the -omic scale (called the traductome) was measured : for each messenger RNAs, its percentage of copies in translation and ribosome density. For the first time, a complete picture of the translational state in fast growing E. coli cells was obtained, characterized by a majority of transcripts with a very high percentage of copies in translation but a low ribosome density. Our model identified sequence-related factors as determinants of translation but, more surprisingly, the model predicted the important role of a physiological parameter: the mRNA concentration. Thus, more concentrated mRNA would have higher percentage of copies in translation and higher ribosome density. For the first time, this effect of transcription on translation has been validated at the molecular level on several genes. We showed that an increase in mRNA concentration by transcriptional induction results in increases in percentage of copies in translation and in ribosome load
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Henze, Stefanie [Verfasser], and Claudia [Akademischer Betreuer] Acquisti. "The impact of nitrogen deprivation on genome evolution in Escherichia coli / Stefanie Henze ; Betreuer: Claudia Acquisti." Münster : Universitäts- und Landesbibliothek Münster, 2016. http://d-nb.info/1141682036/34.
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Ma, Chih-Yu. "Occurrence and characterization of antibiotic-resistant Escherichia coli in wastewater and surface water." Kyoto University, 2020. http://hdl.handle.net/2433/259030.
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Cowley, Lauren A. "Use of genome sequencing to investigate the molecular basis of bacteriaphage interaction of the Escherichia coli O157 typing phages and the elucidation of the biological and public health significance of phage type." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23583.
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Background Shiga toxin producing Escherichia coli (STEC) O157 causes severe gastrointestinal disease and haemolytic uremic syndrome, and has a major impact on public health worldwide with regular outbreaks and sporadic infection. Phage typing, i.e. the susceptibility of STEC O157 strains to a bank of 16 bacteriophages, has been used in the UK to differentiate STEC O157 for the past 25 years and the phage type (PT) can be an epidemiological marker of strains associated with severe disease or associated with cases that occur from foreign travel. However, little is known about the molecular interactions between the typing phages (TP) and STEC O157. The aims of this thesis were to use whole genome sequencing to elucidate the genetic basis for phage typing of STEC O157 and through this understand genetic differences between strains relevant to disease severity and epidemiology. Results Sequencing the STEC O157 TPs revealed that they were clustered into 4 groups based on sequence similarity that corresponded with their infectivity. Long read sequencing revealed microevolutionary events occuring in STEC O157 genomes over a short time period (approximately 1 year), evidenced by the loss and gain of prophage regions and plasmids. An IncHI2 plasmid was found responsible for a change in Phage Type (PT) from PT8 to PT54 during two related outbreaks at the same restaurant. These changes resulted in a strain (PT54) that was fitter under certain growth conditions and associated with a much larger outbreak (140 as opposed to 4 cases). TraDIS (Transposon directed Insertion site sequencing) was used to identify 114 genes associated with phage sensitivity and 44 genes involved in phage resistance, emphasising the complex nature of identifying specific genetic markers of phage susceptibility or resistance. Further work is required to prove their phage-related functions but several are likely to encode novel phage receptors. Deletion of a Stx2a prophage from a PT21/28 strain led to a strain that typed as PT32, supporting the concept that the highly pathogenic PT21/28 lineage I strains emerged from Stx2c+ PT32 strains in the last two decades by acquisition of Stx2a-encoding prophages. Conclusions This body of work has highlighted the complexity of bacteriophage interaction and investigated the genetic basis for susceptibility and resistance in E. coli. The grouping of the TPs showed that resistance or susceptibility to all members of a typing group was likely to be caused by one mechanism. IncHI2 was identified as one of the markers for the PT54 phenotype. The Stx2a prophage region was associated with the switch from PT32 to PT21/28, although PT32 strains containing both Stx2a and Stx2c-encoding prophages have been isolated and can provide insights into phage variation underpinning the susceptibility to the relevant typing phages. The TraDIS results indicated that susceptibility or resistance was governed by multiple genetic factors and not controlled by a single gene. The significance of LPS for initial protection from phage adsorption was evident and a number of novel genes controlling phage susceptibility and resistance identified including the Sap operon and stringent starvation protein A respectively. While SNP-based typing provides an excellent indication of the evolution and relatedness of strains, phage typing can provide real insights into short term evolution of the bacteria as PTs can be altered by mobile elements such as prophages and plasmids. This study has shown that, although complex, genetic determinants for PT can be mined from the genome and allow us to understand the evolution of this zoonotic pathogen between host species and during outbreaks.
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Lember, Geivi. "Sepsis-associated Escherichia coli whole-genome sequencing analysis using in-house developed pipeline and 1928 diagnostics tool." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-19841.
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Sepsis is a life-threatening condition that is caused by a dysregulated host response to infection. Timely detection of sepsis and antibiotic treatment is important for the patient’s recovery from sepsis. Usually, when sepsis is detected, immediate antibiotic treatment is started with broad-spectrum antibiotics as it takes time to determine the correct antibiotic susceptibility. To overcome this problem, next-generation sequencing is seen as one possible development in clinical diagnostics in the future. Automated bioinformatics pipelines could be used initially for surveillance purposes but eventually for rapid clinical diagnosis. Therefore, the results of 1928 Diagnostics, an automated pipeline for whole-genome sequencing (WGS) data analysis, were compared with the results of an in-house developed pipeline for manual data processing by analyzing sepsis-associated Escherichia coli (SEPEC) WGS data. The pipelines were compared by assessing their predicted antimicrobial resistance (AMR) genes, virulence genes and epidemiological relatedness. In addition, the predicted resistance genes were compared to phenotypic antimicrobial susceptibility testing (AST) data from the clinical microbiology laboratory. All the results obtained from the 1928 Diagnostics and in-house pipeline were similar but differed in the number of virulence/predicted AMR genes, AMR gene variants, detection of species and epidemiologically related E. coli samples. Moreover, the predicted AMR genes from both pipelines did not show a good overall relation to the phenotypic AST result. More studies are needed to make predictions of genes from the WGS analysis more reliable so that WGS analysis can be used as a diagnostics tool in clinical laboratories in the future.
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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 géniqueA 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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Raeside, Colin. "Plasticité du génome au cours d'une expérience d'évolution au long terme chez Escherichia Coli." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV070/document.
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Les réarrangements d'ADN à grande échelle, tels que inversions, amplifications, duplications, délétions, insertions et transposition des éléments génétiques mobiles, sont des acteurs essentiels de l'évolution. Ils ont une forte incidence sur l'organisation et l'expression des chromosomes, ce qui affecte le phénotype des organismes. Toutefois, la dynamique de ces réarrangements au cours de l'évolution et leurs effets sur l'adaptation des organismes sont souvent inconnus. Nous avons abordé ces questions en utilisant la plus longue expérience d'évolution en cours. A partir d'un ancêtre commun d'Escherichia coli, douze populations indépendantes sont cultivées dans un milieu limité en glucose depuis plus de 60 000 générations, soit 26 ans. La plupart des études antérieures ont porté sur les mutations ponctuelles et les petites insertions et délétions (InDels). En utilisant des clones isolés au cours du temps dans ces 12 populations, nous avons caractérisé les réarrangements d'ADN à grande échelle à la fois par l'analyse des séquences de génomes et par cartographie optique. A 40 000 générations, nous avons identifié 110 réarrangements parmi lesquelles 82 délétions, 19 inversions et 9 duplications. Plusieurs régions du chromosome ont été touchées à plusieurs reprises par le même type de réarrangements dans des populations indépendantes. Dans une des populations au moins, les réarrangements se sont produits au début de l'expérience d'évolution, au moment où l'augmentation de la valeur sélective est la plus élevée. Par conséquent, certains de ces réarrangements pourraient être bénéfiques dans ces conditions. Même dans le cas contraire, nous avons montré que ces réarrangements affectaient fortement la structure du chromosome au cours de l'expérience d'évolution.Au niveau moléculaire, nous avons montré que ~ 70% des réarrangements se produisent par recombinaison entre séquences d'insertion (IS), ce qui illustre l'importance de ces dernières dans la plasticité du génome. Nous avons donc caractérisé la distribution et la dynamique de ces petits éléments génétiques mobiles dans l'ensemble des 12 populations. Nous avons montré que les éléments IS ont fortement contribué à l'ensemble des mutations après 40 000 générations. Dans une population, les IS représentent même la moitié des mutations, et un des types d'IS, IS150, présente une forte prolifération avec 6 fois plus de copies à 40 000 générations, intervenant dans la plupart des réarrangements détectés dans cette population. Nous avons montré une forte dynamique temporelle d'IS150, avec une forte expansion suivie d'une domestication par l'hôte. En testant trois scenarii évolutifs, nous avons démontré que l'expansion d'IS150 était liée à une forte augmentation de la valeur sélective conférée par les événements initiaux de transposition ayant eu lieu avant 2000 générations. Plus tard, entre 20 000 et 40 000 générations, nous avons mesuré une diminution de la fréquence de transposition, probablement en raison d'une régulation négative de la transposition imposée par l'hôte. Enfin, et pour la première fois, nous avons développé un modèle d'évolution de la dynamique des IS, qui confirme que leur expansion est liée à un nombre seuil d'insertions bénéfiques initiales. Ces résultats montrent que les réarrangements chromosomiques à grande échelle et les éléments IS ont joué un rôle actif dans la trajectoire évolutive au cours de 40 000 générations d'évolution bactérienneLarge-scale DNA rearrangements, including inversions, amplifications, duplications, deletions, insertions, and transposition of mobile genetic elements, are major drivers of evolution and strongly impact on chromosome organization and expression, thereby altering organismal phenotypes. However, their long-term evolutionary dynamics and effects on organismal fitness are often unknown. We addressed these questions by using the longest-running evolution experiment, during which twelve independent populations are propagated from a common E. coli ancestor in a glucose-limited environment for now over 60,000 generations (26 years). Most past studies have focused on point mutations and small InDels. Using evolved clones sampled over time in all 12 populations, we characterized all large-scale DNA rearrangements by using whole genome sequences and Whole Genome MappingTM (i.e optical mapping). After 40,000 generations, we identified a total of 110 rearrangements including 82 deletions, 19 inversions and 9 duplications. Many chromosomal regions were repeatedly affected by similar rearrangements and, at least in one population, they occurred early in evolution when fitness increase was strong. Therefore, many rearrangements may be under positive selection. At the very least, these rearrangements strongly affected the structure of the chromosome during evolution.At the molecular level, we showed that ~ 70% of all rearrangements occurred by recombination between Insertion Sequence (IS) elements, illustrating their importance in mediating genome plasticity. We therefore investigated the distribution and temporal dynamics of these small mobile genetic elements in all 12 populations. We showed that IS elements were strong contributors of the total mutations after 40,000 generations. In one population, they even represented about half of the total mutations and one IS type, IS150, revealed a strong 6-fold increase in copy number, accounting for the production of most of the rearrangements detected in this population. We showed that IS150 revealed a dynamic temporal behavior with a strong expansion followed by domestication by the host. By testing three evolutionary scenarios, we demonstrated that the IS150 expansion was related to a strong fitness increase conferred by the initial transposition events that occurred before 2000 generations. Later, between 20,000 and 40,000 generations, we measured a decreased transposition frequency, likely owing to a down regulation imposed by the host. Finally, and for the first time, we developed an evolution model of IS dynamics confirming that the IS expansion was related to a threshold number of initial IS beneficial insertions. All of our data showed that large-scale chromosomal rearrangements and IS elements have played an active role in the evolutionary outcomes after 40,000 generations of bacterial evolution
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Noll, Lance. "Detection and quantification of the top-seven Shiga toxin-producing Escherichia coli serogroups in feces and on hides of feedlot cattle and whole genome sequence-based analysis of O103 serogroup." Diss., Kansas State University, 2017. http://hdl.handle.net/2097/36204.
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Doctor of PhilosophyDepartment of Diagnostic Medicine/Pathobiology
Tiruvoor G. Nagaraja
Cattle are a reservoir for major Shiga toxin-producing Escherichia coli (STEC), which includes STEC O157 and the top six non-O157 serogroups (STEC-6; O26, O45, O103, O111, O121, O145). Collectively known as the STEC-7, these organisms are harbored in the hindgut and shed in the feces of cattle, which can contaminate hides. The de-hiding step during beef cattle processing can introduce fecal contaminants from the hide onto the carcass surface, creating the potential for contaminated beef products. The STEC-7 have been declared by the USDA-Food Safety and Inspection Service as adulterants in ground beef and non-intact beef products, and are monitored during beef cattle processing. However, many of the culture- and PCR-based tests for detection and/or quantification of the STEC, particularly of the STEC-6, are not established or require improvement and also virulence characteristics of STEC strains from cattle have not been fully analyzed. Therefore, the following studies were conducted: 1. Immunomagnetic separation (IMS)-based culture-method for detection of STEC-6 in cattle feces was developed and compared to a PCR-based method; 2. Detection sensitivity of pooled vs. individual IMS beads for isolation STEC-6 from cattle feces was evaluated; 3. Real-time PCR assay, based on the clustered regularly interspaced short palindromic repeat sequence polymorphisms (CRISPR), was developed and validated for serotype-specific detection and quantification of STEC O157:H7 in cattle feces; 4. Virulence gene profiles of bovine enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype E. coli O103 strains were examined with whole genome sequence (WGS)-based comparative analysis; 5. Prevalence and concentration of STEC-7 of fed-beef, cull beef and cull dairy cattle were determined. The culture and PCR methods detected all six serogroups in samples negative by the other method. Based on noninferiority tests, detection with pooled IMS beads was not inferior to detection with individual beads. Detection limits of the CRISPR-based qPCR assay for cattle feces spiked with pure cultures were 2.1 x 10³ and 2.3 x 10⁰ colony-forming units/g before and after enrichment, respectively. WGS-based analysis of E. coli O103 strains revealed key differences in the virulomes and mobilomes of EHEC, EPEC, and putative non-pathotype strains. The prevalence study revealed that a significantly higher (P < 0.01) proportion of hide samples from fed beef cattle (4.8%) were positive for STEC O157:H7, compared to samples from cull beef (1.6%) or cull dairy (0.2%); the majority of quantifiable STEC O157:H7 from each cattle type was at concentrations between 3 to 4 log CFU/100 cm². These data contribute to a knowledge gap on prevalence and concentration of STEC-7 and surrogate bacteria on cattle hides and carcasses, respectively. Furthermore, the development and refinement of culture- and PCR-based screening assays may lead to increased surveillance of major STEC serogroups, especially if the potential of WGS-based comparative genomics in identifying novel gene targets can be harnessed.
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CHEVALLIER, BRUNO. "Structure et expression du genome de lactobacillus plantarum : clonage et etude de la stabilite de l'adn chez escherichia coli." Université Louis Pasteur (Strasbourg) (1971-2008), 1994. http://www.theses.fr/1994STR13184.
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Afin de cloner et d'etudier les genes intervenant dans la voie de biosynthese des pyrimidines d'une bacterie lactique, lactobacillus plantarum, nous avons entrepris la construction d'une banque d'adn genomique de l. Plantarum dans un vecteur navette escherichia coli-bacterie gram positive chez e. Coli. Nous avons commence par estimer, par electrophorese en champ pulse, en utilisant les endonucleases asci, sfii et i-ceui, la taille du genome de 8 souches de l. Plantarum et obtenu des valeurs comprises entre 3 a 4,2 mb. Au moins 5 loci codant pour les arn ribosomiques ont ete mis en evidence dans le genome de ces souches. Les profils de restriction et de rapd (random amplified polymorphic dna) du genome obtenus se sont reveles etre des outils taxinomiques interessants. Nous avons mis en evidence une instabilite de l'adn genomique de l. Plantarum clone dans plusieurs plasmides (pam239, pdl278, pnz12, ptg262, puc19) chez e. Coli. Une faible proportion de clones recombinants et une majorite de plasmides deletes sont les deux caracteristiques de cette instabilite. L'utilisation de differents plasmides caracterises par des terminateurs de transcription presente de chaque cote du site de clonage ou un faible nombre de copies par cellule, ne reduit pas cette instabilite ; par contre la methylation dam de l'adn a une influence. L'instabilite de l'adn genomique de l. Plantarum, independante des systemes de recombinaison homologue, est probablement due, chez e. Coli, a un mecanisme de recombinaison illegitime encore inconnu. L'efficacite de transformation par electroporation de l. Plantarum ccm 1904 a ete amelioree de 100 a 1000 fois de maniere a obtenir 100 000 transformants par microgramme d'adn plasmidique. L. Plantarum ccm 1904 se transforme 5 a 30 fois mieux par un plasmide non methyle dam. L'ensemble des resultats obtenus nous permet d'esperer construire une banque d'adn genomique chez l. Plantarum ou cloner directement les genes par complementation des mutants de cette espece obtenus au laboratoire. Nous eviterons ainsi l'instabilite de l'adn genomique de l. Plantarum observee chez e. Coli
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Gunasekera, Samantha Thilini. "Evaluating whole-genome sequencing strategies in AMR research through characterisation of mobile genetic elements in wildlife-origin Escherichia coli." Thesis, Gunasekera, Samantha Thilini (2019) Evaluating whole-genome sequencing strategies in AMR research through characterisation of mobile genetic elements in wildlife-origin Escherichia coli. Honours thesis, Murdoch University, 2019. https://researchrepository.murdoch.edu.au/id/eprint/50510/.
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Background
Resistance against antimicrobials critically important to human medicine has been frequently observed in livestock and clinical settings where the selection pressure imposed by antimicrobial use is high. Today, it is detected in wildlife even in the absence of a direct selection pressure. On Penguin Island, 660 m from the coast of Western Australia, Escherichia coli isolated from little penguin and feral pigeon faecal samples (n = 20) was found to be phenotypically resistant to extended-spectrum cephalosporins and fluoroquinolones. It was hypothesised that the frequent observations of resistance against CIAs in this group of birds were a result of horizontal gene transfer. However, mobile genetic elements have historically been challenging to characterise using second-generation sequencing technology alone. These difficulties have been linked to coverage bias caused by library preparation, and to insufficient read length to assemble long, repetitive genomic regions. To address the limitations of read length, second-generation sequencing technologies were used in conjunction with third-generation sequencing technologies to produce more contiguous assemblies while maintaining accuracy. To address coverage bias, the performance of the Nextera XT and Nextera Flex library preparation kits were evaluated to assess whether either library preparation kit was associated with reduced sequencing bias.
Results
The results of the library preparation kit comparison found that the Nextera Flex library preparation kit produced more even coverage than the Nextera XT kit, however tagmentation bias, GC content bias, de novo assembly quality and antimicrobial resistance (AMR) gene detection was either unchanged or poorer than Nextera XT.
Resistance to extended-spectrum cephalosporins observed in the E. coli isolates was mediated mainly by blaCTX-M-15 and was shown to have circulated through the different isolates via mobile genetic elements such as ISEcp1, IS26 and Tn2. Resistance against fluoroquinolones occurred primarily through mutations in quinolone resistance-determining regions, with only a minority of isolates harbouring plasmid-mediated quinolone resistance genes. Using a combination of second and third generation sequencing technology, a composite transposon conferring multidrug-resistance to macrolides, folate pathway inhibitors and aminoglycosides was found in varying conformations in over half of the E. coli isolates.
Conclusions
This study was the first to assess the Nextera Flex library preparation kit in the context of AMR research and found that coverage bias was markedly improved. However, this did not impact the practical applications of whole-genome sequencing data such as de novo assembly or AMR gene detection.
Using a combination of second and third-generation sequencing technologies, this study established the presence of multiple mobile genetic elements conferring resistance to critically important antimicrobials in E. coli isolated from avian wildlife on Penguin Island, Western Australia.
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Launay, Adrien. "Etude de l'émergence de la diversité d'Escherichia coli in vivo par séquençage de génomes complets." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066692/document.
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Escherichia coli est une espèce commensale du tube digestif, mais elle peut aussi se révéler être un dangereux pathogène intra ou extra intestinal. Un même clone pouvant passer d'un état commensal à pathogène, la compréhension des mécanismes impliqués dans la diversification d'E. coli dans ces deux habitats représente un enjeu majeur de santé publique. Des expériences d'évolution expérimentale utilisant E. coli ont permis de révéler différentes facettes de l'adaptation bactérienne. Cependant, ces expériences de laboratoire utilisant des conditions artificielles, on peut s'interroger sur la pertinence des observations qui en découlent en milieu naturel et plus globalement s’interroger sur la part de la sélection naturelle dans la diversification de E. coli dans la nature. Pour répondre à ces questions, j'ai analysé les profils génomiques de diversification de E. coli au cours (1) d’une adaptation au tube digestif de souris ou (2) dans des infections extra-intestinales. Dans les deux cas, j’ai pu montrer une importante convergence au niveau du gène : un même gène étant muté plusieurs fois indépendamment, un signe que l’adaptation est active. Dans les infections aigues, des mutations touchant des régulateurs globaux ont été retrouvées, alors que dans le tube digestif les cibles de l’adaptation semblaient plus spécifiques. Enfin, les échantillons issus des infections incluant des souches a fort taux de mutation dites mutatrices, j'ai pu documenter pour la première fois la génomique de l'émergence de bactéries mutatrices en milieu naturel.En conclusion, mes travaux montrent que l’adaptation joue un rôle important dans la diversification de E. coli en milieu naturel et que ce processus s’apparente à celui observé dans des milieux artificiels de laboratoire. L’adaptation semble néanmoins plus active en conditions d’infections aigues que dans le tube digestif de sourisEscherichia coli is a commensal species living in the digestive tract of vertebrates, but can also be a harmful pathogen involved in both intra and extraintestinal diseases. As clones can behave both as commensals and pathogens, the comprehension of the mechanisms involved in the diversification of E. coli in those two habitats represents a major public health concern. In vitro experimental evolution studies using E. coli have unraveled the different faces of bacterial adaptation. However, as those experiments used artificial conditions, the relevance of these observations and more generally the contribution of adaptation to the diversification of E. coli in the wild remain questionable. To answer these questions, I analyzed the genomic profiles of diversification of E. coli during (1) adaptation to the mice digestive tract or (2) during acute extraintestinal infections. In both cases, I found a strong convergence at the gene level, i.e. observation of several impendent mutations in the same gene, suggesting a dynamic adaptation. In acute infections, mutations in global regulators were recovered, while more specific genes were recruited in the mice gut. Finally, the existence of clones with high mutation rate in the infections, allowed me to document for the first time the genomics of mutator emergence in the wild. In conclusion, my work shows that adaptation is playing an important role in the diversification of E. coli, and that this process is fairly similar to the one observed in the laboratory. Nevertheless, adaptation seems more active during infections than in the mice gut
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Yesil, Mustafa. "Enhancing the inactivation of Escherichia coli O157:H7 by bacteriophage and gaseous ozone to improve postharvest fresh produce safety." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512103801957122.
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Stoesser, Nicole Elinor. "Applications of whole genome sequencing to understanding the mechanisms, evolution and transmission of antibiotic resistance in Escherichia coli and Klebsiella pneumonia." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:10ed1097-b2a1-4e3e-a4b3-58318d325f89.
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Whole genome sequencing (WGS) has transformed molecular infectious diseases epidemiology in the last five years, and represents a high resolution means by which to catalogue the genetic content and variation in bacterial pathogens. This thesis utilises WGS to enhance our understanding of antimicrobial resistance in two clinically important members of the Enterobacteriaceae family of bacteria, namely Escherichia coli and Klebsiella pneumoniae. These organisms cause a range of clinical infections globally, and are increasing in incidence. The rapid emergence of multi-drug resistance in association with infections caused by them represents a major threat to the effective management of a range of clinical conditions. The reliability of sequencing and bioinformatic methods in the analysis of E. coli and K. pneumoniae sequence data is assessed in chapter 4, and provides a context for the subsequent study chapters, investigating resistance genotype prediction, outbreak epidemiology in two different contexts, and population structure of an important global drug-resistant E. coli lineage, ST131 (5-8). In these, the advantages (and limitations) of short-read, high-throughput, WGS in defining resistance gene content, associated mobile genetic elements and host bacterial strains, and the relationships between them, are discussed. The overarching conclusion is that the dynamic between all the components of the genetic hierarchy involved in the transmission of important antimicrobial resistance elements is extremely complicated, and encompasses almost every imaginable scenario. Complete/near-complete assessment of the genetic content of both chromosomal and episomal components will be a prerequisite to understanding the evolution and spread of antimicrobial resistance in these organisms.
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Maistrenko, Oleksandr. "Variation in Core and Accessory Parts of Genome of Escherichia Coli Isolated from Soil from Riparian Areas in New York State." Thesis, North Dakota State University, 2016. https://hdl.handle.net/10365/28022.
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Escherichia coli is commensal bacteria and is a symbiont of the digestive system of vertebrates. Due to frequent deposition of E. coli into extrahost habitats (soil, water), approximately half of its population exists as free living organisms. It is unclear what genome-wide variation stands behind adaptation for extrahost habitat. This thesis applies a genome-wide association study approach to find genetic variation in core and accessory parts of genome of E. coli that is associated with 1) forest or agricultural field soil habitats and 2) with survival phenotype in soil microcosm. Gene composition analysis suggests that pan-genome of environmental E. coli is unlimited. Core and accessory genome contained variation associated with survival phenotype and with forest or field habitat.Federal Formula Funding (Hatch-Act)
ND-EPSCoR
Fulbright-STEP scholarship
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Royer, Guilhem. "Génomique comparée à grande échelle de souches d’Escherichia coli responsables de bactériémies chez l’Homme : implications cliniques et analyse des réseaux métaboliques PlaScope: a targeted approach to assess the plasmidome from genome assemblies at the species level Mortality in Escherichia coli bloodstream infections: antibiotic resistance still does not make it." Thesis, université Paris-Saclay, 2021. https://www.biblio.univ-evry.fr/theses/2021/interne/2021UPASL012.pdf.
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Escherichia coli est la bactérie aéro-anaérobie facultative majoritaire du tube digestif de l'Homme et, également, l'espèce la plus fréquemment isolée au cours de bactériémies dans les pays industrialisés. La population de E. coli présente une diversité importante mais néanmoins structurée, avec certains groupes phylogénétiques préférentiellement associés à un mode de vie (e.g. A/B1 et commensal, B2/D et pathogène extraintestinal). De nombreux facteurs de virulence ont été décrits chez les souches pathogènes extraintestinales (ExPEC), mais le pronostic des bactériémies à E. coli semble dépendre des facteurs associés à l'hôte. Cependant, certains clones virulents et multirésistants aux antibiotiques ont récemment émergé, modifiant drastiquement l'épidémiologie de ces infections. En parallèle, la démocratisation des méthodes de séquençage offre aujourd'hui une granularité encore jamais atteinte dans l'analyse des génomes bactériens. L'objectif de ce travail de thèse est de tirer profit des méthodes de génomique comparée pour améliorer notre compréhension de la physiopathologie des bactériémies à E. coli, tout en prenant en compte les modifications épidémiologiques majeures qui sont observées. La réalisation de telles comparaisons génomiques a nécessité, tout d'abord, la mise en place d'une stratégie d'analyse, incluant notamment le développement d'une approche ciblée pour l'identification des séquences plasmidiques au sein d'assemblages de génomes.Cette stratégie, appliquée à 545 souches recueillies au cours de l'étude Septicoli en 2016-2017, a permis de confirmer le rôle mineur des déterminants bactériens dans l'issue des bactériémies à E. coli. De plus, par la comparaison de ces souches à celles de l'étude Colibafi, nous avons étudié la dynamique de la population sur 12 ans. Si celle-ci apparaît globalement stable, l'exploration plus fine des principaux clones montre d'importants remaniements, parfois associés à des facteurs de virulence typiques des ExPEC. D'autre part, la diversité antigénique de ces clones est variable et suggère des pressions de sélection différentes en fonction de leur niche écologique respective. Enfin, dans une dernière partie nous avons réalisé la reconstruction des réseaux métaboliques de plus de 1400 souches de E. coli afin d'étudier les liens entre métabolisme et mode de vie. Les résultats soulignent la conservation du métabolisme chez E. coli et son association forte avec la phylogénie. Par ailleurs, une analyse détaillée des principaux clones retrouvés dans les bactériémies, dont notamment le ST131, met en évidence une voie métabolique impliquée dans la dégradation de composés aromatiques dérivés de la lignine. Cette voie, habituellement absente des souches de phylogroupe B2, pourrait procurer un avantage sélectif à ce clone pandémique mondial d'émergence récenteEscherichia coli is the predominant aero-anaerobic bacterium of the human gut and also the leading cause of bacteremia in industrialized countries. The E. coli population presents a high but structured diversity, with certain phylogenetic groups preferentially associated with a given lifestyle (e.g. A/B1 and commensal, B2/D and extraintestinal pathogen). Many virulence factors have been described in extraintestinal pathogenic strains (ExPEC), but the prognosis of E. coli bacteremia appears to be linked mainly to host-associated factors. However, some virulent and multi-resistant clones have recently emerged and dramatically modified the epidemiology of these infections. At the same time, the democratization of sequencing methods now offers a granularity yet never reached in the analysis of bacterial genomes. The objective of this thesis is to take advantage of comparative genomic methods to improve our understanding of the physiopathology of E. coli bacteremia, while taking into account the major epidemiological changes that are observed. Carrying out such genomic comparisons required, first of all, the implementation of an analysis strategy, including in particular the development of a targeted approach for the identification of plasmid sequences within genome assemblies.This strategy, applied on 545 strains collected during the Septicoli study in 2016-2017, confirmed the minor role of bacterial determinants in the outcome of E. coli bacteremia. In addition, by comparing these strains to those of the Colibafi study, we studied the population dynamics over 12 years. While the population appears to be stable overall, further exploration of the main clones shows significant changes, sometimes associated with virulence factors typical of ExPEC. On the other hand, the antigenic diversity of these clones is variable and suggests different selective pressures according to their respective ecological niches. Finally, in a last part we reconstructed the metabolic networks of more than 1400 E. coli strains in order to study the links between metabolism and lifestyle. The results highlight the conservation of metabolism in E. coli and its strong association with phylogeny. In addition, a detailed analysis of the main clones found in bacteremia, including the ST131, highlights a metabolic pathway involved in the degradation of aromatic compounds derived from lignin. This pathway, usually absent in phylogroup B2 strains, could provide a selective advantage to this recently emerging global pandemic clone
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Walz, Paul S. "Influence of pathogenic bacterial determinants on genome stability of exposed intestinal cells and of distal liver and spleen cells." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Biology, c2011, 2011. http://hdl.handle.net/10133/3405.
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Most bacterial infections can be correlated to contamination of consumables such as food and water. Upon contamination, boil water advisories have been ordered to ensure water is safe to consume, despite the evidence that heat-killed bacteria can induce genomic instability of exposed (intestine) and distal cells (liver and spleen). We hypothesize that exposure to components of heat-killed Escherichia coli O157:H7 will induce genomic instability within animal cells directly and indirectly exposed to these determinants. Mice were exposed to various components of dead bacteria such as DNA, RNA, protein or LPS as well as to whole heat-killed bacteria via drinking water. Here, we report that exposure to whole heat-killed bacteria and LPS resulted in significant alterations in the steady state RNA levels and in the levels of proteins involved in proliferation, DNA repair and DNA methylation. Exposure to whole heat-killed bacteria and their LPS components also leads to increased levels of DNA damage.xiv, 132 leaves : ill. (chiefly col.) ; 29 cm
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Cavalcanti, Leonardo Sousa. "Papel da celulase XF-0818 na interação Xylella fastidiosa X Citros." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-16052005-162932/.
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A partir do sequenciamento do genoma da bactéria Xylella fastidiosa, agente causal da clorose variegada dos citros (CVC), estudos sobre os mecanismos de patogenicidade expressos durante a doença foram iniciados em projetos de genômica funcional, com o objetivo de confirmar a função das sequências gênicas identificadas no projeto Genoma de X. fastidiosa. Dentre esses mecanismos destacam-se as enzimas envolvidas no processo de ataque da bactéria. O presente trabalho teve como alvo de estudo a seqüência gênica denominada Xf-0818, clonada em Escherichia coli através da inserção em plasmídeo pET 28b(+), que presumivelmente codifica uma celulase de cerca de 60 kDa, possivelmente envolvida na degradação de fibras de celulose presentes nas membranas dos vasos pontuados do xilema de plantas de citros. A degradação desta membrana permitiria a movimentação de células bacterianas entre os vasos do xilema, o que é determinante para o desenvolvimento dos sintomas da CVC. A proteína foi superexpressada em E. coli por meio de indução com lactose e purificada através de ultrafiltração associada à cromatografia de afinidade a metal. A enzima apresentou alta atividade sobre a celulose carboximetilada 1%, avaliada através da quantificação de açúcares redutores. A obtenção de anticorpos foi realizada mediante injeção da enzima purificada em camundongos, os quais foram avaliados através de ELISA. A atividade da enzima sobre celulose carboximetilada foi reduzida após prévia incubação com os anticorpos. Esses anticorpos representam uma poderosa ferramenta em pesquisas visando à identificação das condições que favoreçam a expressão desta enzima in vivo. A presença de partículas de ouro coloidal em seções de tecido provenientes de plantas infectadas com X. fastidiosa indica a atuação da celulase durante a infecção, porém fazse necessária a confirmação do papel da proteína Xf-0818, através de novas análises com imunolocalização, além de estudos utilizando linhagens mutantes de X. fastidiosa para o gene que codifica esta celulase.From the genome sequencing of the bacteria Xylella fastidiosa, the agent responsible for the citrus variegated chlorosis (CVC), studies on the pathogenicity mechanisms expressed during the occurrence of the disease were started in genomic function projects, aiming to confirm the function of the genic sequences identified in the project X. fastidiosa Genome. Among these mechanisms, the enzymes involved in the bacterium attaching process are highlighted. The present study had, as a goal, the orf denominated Xf-0818, cloned in Escherichia coli by means of the insertion into pET 28b(+) plasmid, which codifies a cellulase of a size nearly 60 kDa, possibly involved in the degradation of cellulose fibers present in the membranes of the xylem vessels of citrus plants. The degradation of this membrane would allow for the movement of bacterial cells among the xylem vessels, which would be important for the development of CVC symptoms. The enzyme was over expressed into E. coli by means of their induction using lactose and purified using ultra-filtration associated to metal affinity chromatography. The enzyme presented high activity over 1%-carboximethyl cellulose (CMC), which was evaluated through quantification of released reduced sugars. The antibodies were obtained by injection of the purified enzyme into mice, which were evaluated by using the ELISA. The enzyme activity over CMC was reduced after previous incubation with the antibodies. These antibodies represent a tool for the researches aiming the identification of conditions that favor the expression of this enzyme in vivo. The presence of gold particles on the citrus diseased tissue samples indicate the participation of this cellulase on the disease, but other studies using immunocytochemistry and mutants of X. fastidiosa are necessary to confirm this hypothesis.
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Mendes, Renata de Siqueira. "Caracterização de proteínas de membrana de Leptospira interrogans expressas em Escherichia coli." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-27092011-151543/.
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O sequenciamento genômico da L. interrogans sorovar Copenhageni e os avanços das análises bioinformáticas permitiram a identificação de seis novos candidatos vacinais. Esses genes de Leptospira foram submetidos a ensaios de conservação do DNA genômico, RNA mensageiro e proteína nativa correspondente em doze sorovares de Leptospira, nos quais o gene LIC11469 foi o mais conservado. As proteínas recombinantes rLIC11469 e rLIC11030 foram purificadas por cromatografia de afinidade a metal, e submetidas a ensaio de dicroísmo circular. Em ensaios de localização celular pudemos observar a presença das proteínas nativas correspondentes na membrana externa de Leptospira. Ensaios de adesão mostraram a ligação da proteína rLIC11469 à laminina e ao plasminogênio. Ensaios de imunização e desafio demonstraram que a proteína rLIC11030 conferiu proteção contra infecção letal de L. interrogans em hamsters. Ambas as proteínas apresentaram reatividade com anticorpos presentes em soros de pacientes com leptospirose, sugerindo sua expressão durante a infecção.The genomic sequencing of the L. interrogans serovar Copenhageni and the advances of bioinformatics analysis allowed the identification of six new vaccine candidates. These genes were subjected to genomic DNA, mRNA and native protein conservation assays in twelve serovars from Leptospira, and the gene LIC11469 was the most conserved among these serovars. The recombinant proteins rLIC11469 and rLIC11030 were purified by metal affinity chromatography, and subjected to circular dichroism. Through cellular localization assays we could observe the presence of the corresponding native proteins on the outer membrane of Leptospira. In adhesion assays, the protein rLIC11469 showed binding to laminin and plasminogen. Immunization and challenge assays showed that the protein rLIC11030 afforded protection against lethal leptospiral inoculation in hamsters. Both proteins showed reactivity against sera of patients diagnosed with leptospirosis, suggesting that these proteins are probably expressed during infection.
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Souza, Natalie Michele de. "Avaliação da atividade imunogênica de três proteínas de Leptospira interrogans expressas em Escherichia coli." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-07062013-110750/.
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A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. No mundo, aproximadamente 500.000 casos são reportados a cada ano, com 10% de taxa de mortalidade. Atualmente, vacinas contra leptospirose são compostas por células inativadas e são ineficazes em diferentes aspectos. Após analise do genoma, os genes LIC11121, LIC11087, LIC11228 e LIC11084 foram escolhidos para caracterização da imunogenicidade de suas respectivas proteínas. Esses genes foram clonados no vetor de expressão pAE e as proteínas recombinantes foram purificadas. Os resultados sugerem que essas proteínas podem estar localizadas na membrana externa, são imunogênicas, possivelmente expressas durante a infecção e que podem ter envolvimento em mecanismos de evasão do sistema imune e de patogenicidade da bactéria. Além disso, em um de dois experimentos, a proteína rLIC11084 induziu imunidade protetora parcial em hamsters imunizados frente desafio letal.Leptospirosis is a zoonotic disease caused by pathogenic bacteria of genus Leptospira. In the world, nearly 500,000 cases are reported each year, with 10% of mortality rate. Currently, vaccines against leptospirosis are composed by inactivated cells that are ineffective in many aspects. After genome analysis, the genes LIC11121, LIC11087, LIC11228 e LIC11084 were chosen for immunogenicity characterization of their respective proteins. These genes were cloned in the pAE expression vector and the proteins encoded by LIC11087, LIC11228 and LIC11084 were purified. The results suggest the localization of these proteins in the bacterial outer membrane, are immunogenic, are possibly expressed during infection and may have involvement in mechanisms of immune system evasion and pathogenicity. Moreover, in one of two experiments, the rLIC11084 protein induced partial protective immunity of immunized hamsters against lethal challenge.
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Walters, D. E. "A further nar gene in Escherichia coli." Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383761.
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Santos, Ana Carolina da Silva. "Caracterização fenotípica, sequenciamento e anotação do genoma total de uma cepa de Escherichia coli isolada de uma paciente com Doença de Crohn." Botucatu, 2015. http://hdl.handle.net/11449/144081.
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Orientador: Josias RodriguesBanca: Silvio Luís de Oliveira
Banca: Rogéria Keller
Resumo: A Doença de Crohn (DC) é uma variante das doenças inflamatórias intestinais que não tem uma causa definida, porém há o envolvimento de fatores ambientais e genéticos, entre eles a microbiota. Portadores da DC apresentam desequilíbrio na composição de espécies da microbiota intestinal, incluindo o aumento da Escherichia coli. A E. coli é uma bactéria versátil em sua relação com o hospedeiro, isto é, inclui cepas comensais e várias categorias patogênicas. Estas compreendem dois grupos: linhagens causadoras de infecções intestinais e linhagens causadoras de infecção extraintestinais. As E. coli isoladas de portadores da DC em geral pertencem ao segundo grupo, e uma das principais características apresentadas por estas bactérias é a interação com células epiteliais (aderência e invasão). Estudos feitos na década de 90 levaram a identificação de um novo patótipo de E. coli, capaz de aderir e invadir células epiteliais e ainda invadir e se replicar dentro de macrófagos, produzindo granulomas, característica histopatológica da DC. Este novo patótipo, chamado de Adherent and Invasive E. coli (AIEC), têm sido usada como referência na caracterização de E. coli isoladas a partir de portadores da DC. Neste trabalho, foi feito o estudo de caracterização fenotípica e genética de E. coli isoladas a partir de 9 portadores da DC e 5 pacientes controles, as quais foram submetidas a testes de adesão e invasão celular em células epiteliais, teste de formação de biofilme e identificação de filogrupos da coleção de referência de E. coli (EcoR). Dentre o conjunto de amostras bacterianas estudado, foi encontrada uma amostra isolada de uma paciente com DC que apresentou invasibilidade superior às demais, e foi intensivamente estudada, tendo-se verificado que ela apresenta um perfil de virulência distinto de AIEC. O genoma da amostra em questão foi sequenciado e anotado. Os resultados da caracterização...
Abstract: Not available
Mestre
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Mahmoud, Nada. "Next-generation diagnostics of escherichia coli from community-onset sepsis patients in sweden : Studying the biodiversity of escherichia coli genomes." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-19893.
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Escherichia coli (E. coli) is among the gram-negative bacteria that can cause several infections including sepsis. Confirmed sepsis patients must show a sequential organ failure assessment (SOFA) score of ≥2 with verified infection. Understanding the genotypic characteristics of E. coliclinical isolates from sepsis patients can help directing treatment strategies, tracking antibiotic resistance, and monitoring acquired virulence factors that can contribute to the severity of sepsis. The isolates included in this thesis were collected from confirmed sepsis patients (SOFA 2-3)during a prospective observational study that was conducted in Sweden. The aim was to study the biodiversity in E. coli clinical isolates using whole genome sequencing (WGS) paired-end reads that were produced by the next-generation sequencer Illumina. To perform the WGS-based analysis, two bioinformatics pipelines were used. The first is the in-house developed pipeline and the second is the 1928Diagnostics E. coli pipeline. The obtained in silico results were compared with the phenotypic findings for species identification and the in vitro predictions of antibiotic resistance. Species identification by the bioinformatics pipelines matched the phenotypic method, except for three isolates that were highly contaminated with other species. Both pipelines predicted the exact multi locus sequence types, which revealed that the most common sequence types (STs) were ST73(17%), ST95(9%), and ST131(6%). The phenotype of the isolates resulted in 5% resistant to at least one of the assessed antibiotics. The 1928Diagnostics predicted 28% of the isolates were resistant to at least one class of the tested antibiotic classes, while the in-house pipeline predicted 33% of the isolates to be resistant. Out of the predicted resistant isolates, 52% coded for multi-drug resistance. The in-house pipeline reported virulence genes. The common reported genes were coding for iron reuptake, adhesins, cell outer membrane and increased serum survival. It was observed that the isolates that belonged to ST73 and ST95 showed a more susceptible antibiotic profile than isolates that belonged to ST131, those harbored the highest mean of virulence genes. In conclusion, the present study provided an evidence of the usefulness of the WGS-based analysis to study the biodiversity in E. coli. The obtained results are valuable for surveillances, tracking antibiotic resistance and identifying virulence factors, but with a limited use in clinical settings.
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Mahfouz, Norhan, Serena Caucci, Eric Achatz, Torsten Semmler, Sebastian Guenther, Thomas U. Berendonk, and Michael Schroeder. "High genomic diversity of multi-drug resistant wastewater Escherichia coli." Nature Publishing Group, 2018. https://tud.qucosa.de/id/qucosa%3A32482.
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Wastewater treatment plants play an important role in the emergence of antibiotic resistance. They provide a hot spot for exchange of resistance within and between species. Here, we analyse and quantify the genomic diversity of the indicator Escherichia coli in a German wastewater treatment plant and we relate it to isolates’ antibiotic resistance. Our results show a surprisingly large pan-genome, which mirrors how rich an environment a treatment plant is. We link the genomic analysis to a phenotypic resistance screen and pinpoint genomic hot spots, which correlate with a resistance phenotype. Besides well-known resistance genes, this forward genomics approach generates many novel genes, which correlated with resistance and which are partly completely unknown. A surprising overall finding of our analyses is that we do not see any difference in resistance and pan genome size between isolates taken from the inflow of the treatment plant and from the outflow. This means that while treatment plants reduce the amount of bacteria released into the environment, they do not reduce the potential for antibiotic resistance of these bacteria.
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Bin, Thani Ali Salman. "Genomic diversity of ten Escherichia coli strains associated with bloodstream infections." Thesis, University of Leicester, 2009. http://hdl.handle.net/2381/4246.
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Escherichia coli are usually regarded as a harmless human colonic flora. However, pathogenic strains of E. coli have been associated with infections that could range from infected mucosal surfaces by intestinal pathogenic E. coli to the more severe cases of disseminated infections throughout the body by the extraintestinal pathogroups. The main focus of this project was to investigate the genomic contents of pathogenic bloodstream infection (BSI)-associated E. coli strains. This is because the genome contents of the E. coli BSI-associated isolates have not been well studied, with only few reports indicating that the pathogenincity of these strains could be attributed to horizontally acquired DNAs known as genomic islands (GEIs). The genomic contents of 10 clinical BSI-associated E. coli strains, isolated at the Leicester Royal Infirmary were investigated in this study. The first approach used to investigate the genomic contents of these strains was by interrogating the downstream ends of tRNA genes for their GEI contents by the sequential PCR strategy tRIP-PCR (tRNA interrogation for pathogenicity islands) followed by the SGSP-PCR (single genome specific primer-PCR). In this approach the flanking regions of the tRNA sites were used to first screen the tRNA genes for their GEIs followed by amplifying the boundaries of the identified GEIs. In the second approach termed Microarray-Assisted mobilome Prospecting (MAmP), the physical genome size of the tested strains obtained by the pulsed-field gel electrophoresis (PFGE) is compared to the sum total of the bits of the genome detected or visualized by the array. The difference between the two measurements is used to estimate the size of the novel, non-microarray-represented mobile genome (mobilome) present in the tested strains. Remarkably, despite only studying 10 E. coli strains, associated with a single disease type the tRIP-PCR method has identified at least 3 GEIs that contain novel sequences, and 46 GEIs, resembling uropathogenic E. coli CFT073-like entities. One particular strain E105 had 13 tRNA sites occupied with GEIs. On the other hand, an average novel, non-microarray-borne mobilome of (219 kb /strain) was obtained by the MAmP which, corresponds with previous studies. The strategies used in this study had proved successful in addressing and identifying mobilome-rich strains. Therefore, using such approaches in combination with whole genome sequencing progects could prioritize the strains and the genomic regions that need to be sequenced. Such prioritization would avoid sequencing of hundreds of isolates to identify their novel gene pool and would reduce the cost of genomic sequencing. Moreover, applying such approaches for the identification of new virulence genes and/or pathogenic mechanisms could lead to significant improvements in the treatment of E. coli infections.
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Eger, Nicole. "Advanced Detection Methods of Genomic Barcodes for Genotyping Escherichia coli Libraries." Thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-439038.
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Pooled cell strain libraries are a powerful tool allowing to investigate the influence of genetic modifications on phenotypes in high throughput single-cell assays. To link the genotype to phenotype in each cell of the library, unique 20 base pairs (bp) long barcodes are used to allow in situ genotyping after phenotyping via fluorescence microscopy. In previous studies, these barcode sequences were expressed from high copy number plasmids resulting in a high number of targets for detection via fluorescence in situ hybridization (FISH) and thus, a strong readout signal. However, constant selection pressure must be applied on the cells to maintain the foreign plasmid DNA which may influence the phenotype. Inserting unique barcodes on the chromosome ensures stability of the construct which is required for some genomic library applications. However, the low copy number of the barcode sequence often requires an additional step of DNA amplification for efficient detection. In this study, two methods for barcode amplification were investigated. First, amplification from the double stranded DNA upon binding of peptide nucleic acids and subsequent amplification via rolling circle amplification (AmPPR). Second, amplification from genomic DNA or cDNA via loop-mediated isothermal amplification (LAMP). Whereas the AmPPR approach remained unsuccessful, chromosomal barcode sequences were successfully amplified in situ via LAMP and subsequently detected using FISH. I show that LAMP can potentially be a quick, specific, and elegant amplification technique for in situ genotyping in microfluidic devices. However, nonspecific amplification and partly nonspecific readout signals when using LAMP remain a problem and need to be further investigated before implementing this method on pooled libraries.
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Albrecht, Huguette. "Les produits des genes i et iv du virus de la mosaique du chou-fleur (camv)." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13178.
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Nelson, Adam Michael. "Genomic analysis of pathogen evolution virulence gene acquisition and genetic erosion in Escherichia coli /." Diss., Connect to online resource - MSU authorized users, 2008.
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Dufour, Delphine. "Recherche de déterminants génétiques permettant l'adaptation d'une souche Escherichia coli à la mamelle bovine." Thesis, Vandoeuvre-les-Nancy, INPL, 2008. http://www.theses.fr/2008INPL050N/document.
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L’objectif de ce travail a été de caractériser la souche MPEC Escherichia coli P4. Une étude phylogénétique a montré qu’elle appartenait au groupe phylogénétique A de l’espèce E. coli et que son génome “core” se rapprochait de celui de la souche commensale non pathogène E. coli K12 MG1655 appartenant également au groupe A. Une recherche au sein de son génome de gènes codant différents facteurs de virulence connus chez les autres pathotypes de l’espèce E. coli a permis de détecter uniquement la présence du gène traT, codant un facteur de résistance au sérum. Un criblage de quinze loci d’ARNt connus pour accueillir fréquemment des îlots génomiques, effectué au sein de son génome, a révélé, pour sept d’entre eux la présence de telles structures. Le séquençage partiel ou complet des régions aval à ces sept loci a montré la présence systématique de séquences nucléotidiques différentes de celles présentes chez E. coli K12 MG1655. Si l’analyse du contenu de ces îlots n’a pas encore permis d’expliquer directement la virulence d’E. coli P4, leur mise en évidence est une première de ce type au sein du pathotype MPEC et laisse envisager la découverte d’autres régions génomiques spécifiques à ce pathotype, pouvant expliquer son tropisme et sa nature. Par ailleurs, afin d’évaluer le rôle d’E. coli P4 dans la caséinolyse élevée du lait observée lors d’une mammite bovine, une sécrétion apparemment constitutive de quatre protéases extracellulaires a été mise en évidence par zymographie caséines. L’activité caséinolytique de ces enzymes ne semble toutefois pas significative, laissant envisager plutôt un rôle dans la virulence de la soucheThe objective of this work was to characterize the MPEC Escherichia coli P4 strain. A phylogenetic study showed that it belongs to the phylogenetic group A of the E. coli species and that its core genome is similar to the one of the commensal non-pathogenic E. coli K12 MG1655 strain which also belongs to the group A. A search in its genome of different genes encoding virulence factors known among other pathotypes of the E. coli species was done and only the traT gene, encoding a serum resistance factor, was detected. A screening of fifteen tRNA loci known for frequently hosting genomic islands, made in its genome, revealed for seven of them the presence of such structures. The partial or complete sequencing of the regions downstream from these seven loci showed the systematic presence of nucleotide sequences different from those present in E. coli K12 MG1655. If the content analysis of these islands does not yet explain the virulence of E. coli P4, their highlighting is the first of this kind in the pathotype MPEC and suggests the discovery of other genomic regions specific to this pathotype, which may explain its tropism and its nature. In addition, to assessing the role of E. coli P4 in milk caseinolysis observed during bovine mastitis, a constitutive secretion of four extracellular proteases was highlighted by casein zymography. However, the caseinolytic activity of these enzymes does not seem significant. This fact may suggest a role in virulence of the strain
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Santos, Ana Carolina da Silva [UNESP]. "Caracterização fenotípica, sequenciamento e anotação do genoma total de uma cepa de Escherichia coli isolada de uma paciente com Doença de Crohn." Universidade Estadual Paulista (UNESP), 2015. http://hdl.handle.net/11449/144081.
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000868600.pdf: 2036190 bytes, checksum: 3872e6084fbe526a55b084da83826c3a (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A Doença de Crohn (DC) é uma variante das doenças inflamatórias intestinais que não tem uma causa definida, porém há o envolvimento de fatores ambientais e genéticos, entre eles a microbiota. Portadores da DC apresentam desequilíbrio na composição de espécies da microbiota intestinal, incluindo o aumento da Escherichia coli. A E. coli é uma bactéria versátil em sua relação com o hospedeiro, isto é, inclui cepas comensais e várias categorias patogênicas. Estas compreendem dois grupos: linhagens causadoras de infecções intestinais e linhagens causadoras de infecção extraintestinais. As E. coli isoladas de portadores da DC em geral pertencem ao segundo grupo, e uma das principais características apresentadas por estas bactérias é a interação com células epiteliais (aderência e invasão). Estudos feitos na década de 90 levaram a identificação de um novo patótipo de E. coli, capaz de aderir e invadir células epiteliais e ainda invadir e se replicar dentro de macrófagos, produzindo granulomas, característica histopatológica da DC. Este novo patótipo, chamado de Adherent and Invasive E. coli (AIEC), têm sido usada como referência na caracterização de E. coli isoladas a partir de portadores da DC. Neste trabalho, foi feito o estudo de caracterização fenotípica e genética de E. coli isoladas a partir de 9 portadores da DC e 5 pacientes controles, as quais foram submetidas a testes de adesão e invasão celular em células epiteliais, teste de formação de biofilme e identificação de filogrupos da coleção de referência de E. coli (EcoR). Dentre o conjunto de amostras bacterianas estudado, foi encontrada uma amostra isolada de uma paciente com DC que apresentou invasibilidade superior às demais, e foi intensivamente estudada, tendo-se verificado que ela apresenta um perfil de virulência distinto de AIEC. O genoma da amostra em questão foi sequenciado e anotado. Os resultados da caracterização...
FAPESP: 13/04475-3
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Cockram, Charlotte Anne. "Genomic analysis of RecA-DNA interactions during double-strand break repair in Escherichia coli." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/17284.
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Maintaining genomic integrity is crucial for cell survival. In Escherichia coli, Rec-Amediated homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSB) and the SOS response through a series of highly dynamic interactions with the chromosome. A greater understanding of the mechanism of homologous recombination requires quantitative analysis of genomic studies in live cells. The aim of this thesis was to investigate the dynamics of the RecA-DNA interactions in vivo following the induction of a site-specific DSB in the chromosome of E. coli. This DSB is caused by the cleavage of a DNA hairpin by the hairpin-specific endonuclease, SbcCD. The DNA hairpin is formed only on the lagging strand template of replication by a 246 bp-interrupted palindrome. As a result cleavage only occurs on one sister chromosome, leaving one unbroken chromosome to serve as a template for repair by HR. Here, this system has been used as a basis to develop a method that combines chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) and next-generation sequencing (ChIP-Seq) to quantify RecA protein binding during the active repair of a single chromosomal DSB. This study reports that DSB-dependent RecA binding is stimulated in response to the eight base DNA sequence Chi (5’-GCTGGTGG-3’). Increasing the number of Chi sites close to the DSB stimulates more RecA loading to DNA, with ChIP-Seq analysis also revealing a role for subsequent Chi sites in RecA binding during DSBR. If the Chi sites close to the DSB are removed then Chi-dependent RecA binding to DNA can be observed at distances greater than 100 kb from the DSB, suggesting that these subsequent Chi sites can be engaged in DSBR. Through collaboration, these in vivo data were combined with stochastic modeling to determine that, in vivo, Chi is recognised by the RecBCD complex with an efficiency of 20- 35%. The genomic analysis also revealed two unexpected aspects of RecA protein binding. First, ChIP-Seq analyses identified that following a DSB at lacZ there is RecA enrichment detected in the terminus region of the E. coli chromosome. This RecA binding is Chi-dependent, indicating a role for HR. Second, DSB-independent binding was observed at the RNA encoding genes dispersed throughout the chromosome. A temporal analysis of RecA dynamics was also performed. These analyses revealed that RecA binding to DNA near the DSB is extremely dynamic, cycling between periods of high RecA enrichment and periods of low RecA enrichment. This is the first in vivo study of DSB-dependent RecA-DNA distribution and dynamics in recombination proficient E. coli cells.
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Rogers, Ahumada Andrés Eduardo. "Predicción computacional de genes de ARN no codificante pequeño en genomas bacterianos." Tesis, Universidad de Chile, 2012. http://www.repositorio.uchile.cl/handle/2250/111355.
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Magíster en Ciencias de la Ingeniería, Mención QuímicaEl objetivo de este estudio es desarrollar un método computacional capaz de predecir con alta especificidad y sensibilidad los genes pequeños de ARN no codificante en genomas bacterianos, identificando las variables involucradas de mayor importancia que deben ser consideradas para su correcta clasificación. El trabajo aquí presentado consistió en investigar y analizar el estado del arte en métodos de predicción computacional de genes pequeños de ARN no codificante en bacterias, recopilar un listado de las variables involucradas y determinar estadísticamente aquellas que diferencian con mayor precisión los genes pequeños de ARN no codificante de secuencias genéticas al azar, comparar distintos métodos de predicción e identificar el que otorgue mejores resultados. Finalmente, se compararon los resultados del método con otros preexistentes y se aplicó el método al genoma completo de Escherichia coli. Los principales resultados obtenidos en este estudio son la identificación de 4 variables que influyen significativamente en la detección correcta de genes pequeños de ARN no codificante. Estas son: Valor z, Valor de partición, EMPI y Porcentaje de bultos, las cuales corresponden al subconjunto de variables con mayor capacidad discriminatoria. Por este motivo se recomienda que futuros métodos predictivos consideren la inclusión de estas 4 variables. Las variables seleccionadas muestran que existe una presión selectiva en la evolución de los genes pequeños de ARN no codificante, la que apunta a aumentar la estabilidad de la molécula al modificar su estructura para disminuir la energía de plegamiento y eliminar subestructuras desestabilizantes no funcionales. El mejor método de clasificación corresponde al Perceptrón Multicapa basado en redes neuronales, con una alta sensibilidad (88,8%) y alta especificidad (85,5%), teniendo una tasa de falsos positivos relativamente baja (14,5%). Con este subconjunto de variables y el método de clasificación, se realizó una predicción sobre el genoma de la bacteria Escherichia coli, generando 1192 predicciones, con un valor de sensibilidad de 30,5% y un valor predictivo positivo de 1,51 % respecto a los genes pequeños de ARN no codificante conocidos. Seleccionando las predicciones cercanas a promotores σ70 o terminadores intrínsecos (independientes del factor ρ), se obtiene un desempeño predictivo similar al logrado por otros autores en la literatura, con el beneficio adicional de requerir la medición de sólo 4 variables y sin la necesidad de información sobre genes homólogos en organismos cercanos filogenéticamente. La contribución de este trabajo consiste en profundizar el conocimiento acerca de las características de los genes de ARN no codificante, al haber estudiado las variables utilizadas previamente en la literatura y definir las 4 más relevantes, las cuales se relacionan directamente con la estructura secundaria y su energía mínima de plegamiento. En segundo lugar se propone un listado de 1192 secuencias del genoma de Escherichia coli y un listado más corto con las 5 más probables de ser genes sARN, estas secuencias pueden ser comprobadas experimentalmente. Estos resultados inciden positivamente en mejorar la calidad de las anotaciones de estos genes en genomas bacterianos, permitiendo mayores avances en estudios de genómica funcional y regulación en redes metabólicas.
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Tomko, Timothy. "Bioprospecting For Genes That Confer Biofuel Tolerance To Escherichia Coli Using A Genomic Library Approach." ScholarWorks @ UVM, 2015. http://scholarworks.uvm.edu/graddis/487.
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Microorganisms are capable of producing advanced biofuels that can be used as 'drop-in' alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms of improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters.
In this study, screened the genomes of two types of bacteria, Pseudomonas aeruginosa and Marinobacter aquaeolei, looking for genes that impart biofuel tolerance when expressed in Escherichia coli. Both of these microbes have adapted in their respective natural environments to contain mechanisms for dealing with environmental stress. For initial work, P. aeruginosa was used to test our experimental design and procedure, and we validated our methods by identifying a gene, ohr from P. aeruginosa, that increased tolerance to the bio-jet fuel precursor limonene in Escherichia coli.
Using genomic DNA from M. aquaeolei, we constructed a transgenic library that we expressed in E. coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance.
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Tomko, Timothy. "Bioprospecting For Genes That Confer Biofuel Tolerance To Escherichia Coli Using A Genomic Library Approach." ScholarWorks @ UVM, 2017. http://scholarworks.uvm.edu/graddis/798.
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Microorganisms are capable of producing advanced biofuels that can be used as ‘drop-in’ alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters.
First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance.
Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP. putida, while others provide tolerance across a wide range of rpoDP. putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries.
This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.
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Khan, Mirzaei Mohammadali. "The efficacy of bacterial viruses against multi-resistant Escherichia coli: from isolation to pharmacology." Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-126328.
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The increase of multi-resistant bacteria highlights that the golden era of antibiotics is ending and that alternative treatmentsare urgently needed. Phages have been historically used to treat bacterial infections prior to the discovery of antibiotics and have gained renewed interest in the past decade. Despite the advantages of phage therapy over traditional antibiotic usage, a number of concerns persist over their clinical application centring on their efficacy and safety. This thesis presents four papers that focus on the isolation and characterization of phages that target reference strains and drug-resistant strains of E. coli as well as their infection dynamics and kinetics. In Paper I, six of thirty isolated phages were selected to be characterized for their growth parameters and host range using two commonly used methods. The study showed that the host range (an important selection criteria for phages) of the phages can change based on the assessment method and that the lysis efficiency of phages is host-dependent. The study suggests that standardised methods to assess the host range and lytic activity of phages are required to reduce result variability between research groups. Paper II investigated a rare phage with C3 morphotype from the Podoviridae family and characterised it via genomic, proteomic, morphologic and phylogenetic analysis. The study revealed previously unseen aspects including the formation of a honeycomb structure comprised of phage head during DNA packaging, the possible contractile nature of the tail and the 280 million year co-evolution between the major head protein and the scaffolding protein. Paper III highlights the need to take the immune system into consideration when designing phage therapeutics. In the study, four purified structurally distinct phages (selected from the three main phage families) were exposed to human cells (HT-29 and Caco-2 immortalised intestinal epithelial cell lines and donor-derived peripheral blood mononuclear cells) and the immunogenicity of the phages determined. Phage immunogenicity was shown to vary in a concentration and phage dependent manner with SU63 (a Myoviridae) being the most immunogenic phage and SU32 (a Siphoviridae) the least immunogenic. In the presence of human cells and a suitable host, phages were shown to maintain their killing efficacy as well as the ability to proliferate. Paper IV studies the infection dynamics of an experimental two-phage cocktail against a single bacterial host in vitro and in silico. However, in silico analysis and in vitro analysis produced conflicting results, in which mathematical modelling predicted the complete clearance of bacteria for all treatment scenarios whereas experimental results showed a 1-3log10 reduction in bacterial content. Practical experiments also showed increased anti-bacterial activity when the time between the additions of each phage was varied. This discrepancy suggests that the current mathematical model is unsuitable due to the inability to account for discrete variables such as interference.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
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Plucain, Jessica. "Bases génétiques et écologiques de la diversification adaptative chez Escherichia coli." Phd thesis, Université de Grenoble, 2012. http://tel.archives-ouvertes.fr/tel-00947803.
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Les processus de diversification adaptative, qui sont au cœur de la diversité du monde vivant, ont été étudiés grâce à une stratégie d'évolution expérimentale, initiée par le Pr Richard Lenski en 1988. Douze populations, fondées à partir d'un ancêtre commun d'Escherichia coli, sont propagées indépendamment depuis plus de 55 000 générations par transferts journaliers dans un milieu minimum limité en glucose. Un événement de diversification a émergé après 6500 générations d'évolution dans une seule des douze populations, appelée Ara-2, conduisant à deux lignées cellulaires différenciées, appelées S et L, qui continuent de co-exister depuis notamment grâce à des interactions négatives dépendant de leur fréquence. Deux propriétés confèrent à ce polymorphisme une grande originalité et donc un intérêt d'étude important : sa durée car il s'agit du plus long polymorphisme jamais identifié lors d'expériences d'évolution en laboratoire, et son unicité puisqu'il ne s'est produit qu'une seule fois au sein des douze populations initiées à partir d'un ancêtre commun. L'objectif de ce travail a été d'identifier les mécanismes du maintien au long terme des lignées S et L, ainsi que les bases génétiques de leur émergence. Le maintien du polymorphisme est lié à une forte dynamique des relations écologiques entre S et L, l'une des lignées envahissant systématiquement les niches écologiques de l'autre, qui réagit en conséquence pour éviter l'extinction. L'émergence de la lignée S est due à une succession précise de trois mutations, nécessaires et suffisantes pour établir les phénotypes de la lignée S. Les trois mutations affectent toutes des gènes codant des régulateurs globaux de la transcription, dont deux sont impliqués dans la régulation du métabolisme central. Pour l'un d'entre eux, l'allèle évolué altère les propriétés de liaison à l'ADN de la protéine évoluée. Bien que ce polymorphisme soit unique, ces trois gènes sont pourtant les cibles de la sélection naturelle dans la majorité des autres populations de l'expérience d'évolution. Pour deux d'entre eux, seul l'allèle substitué dans la population Ara-2 confère en fait les phénotypes de la lignée S. Ainsi, l'unicité de cet événement de diversification est liée à une succession d'événements mutationnels très précis, qui affectent par ailleurs les réseaux globaux de l'expression des gènes. Ces modifications graduelles ont ainsi conduit à l'émergence du plus long polymorphisme mis en évidence à ce jour dans des expériences d'évolution en laboratoire.
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Aal, Owaif Hasan. "Regulation of transcription of the Escherichia coli Group 2 capsule gene clusters." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/regulation-of-transcription-of-the-escherichia-coli-group-2-capsule-gene-clusters(70a8508a-9b50-49bb-ba3f-418ab4b485e0).html.
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Capsular polysaccharides are expressed by many bacteria and in the case of pathogens are believed to act as essential virulence factors conferring resistance to host defenses. Escherichia coli strains that express Group 2 capsules are associated with a number of infections including urinary tract infections, and life-threatening infections such as neonatal meningitis and septicemia. The expression of Group 2 capsular polysaccharide is controlled by two temperature regulated promoters PR1 and PR3 that allow the transcription at 37°C but not at 20°C. The 5' untranslated regions of these promoters are involved in the regulation of transcription by interacting with global regulators. In this study, PR1-lacZ and PR3-lacZ transcriptional fusions and transposon mutagenesis was used to identify new regulators of Group 2 E. coli capsule expression. The cAMP-CRP complex was identified to be a new transcriptional regulator of E. coli Group 2 capsular polysaccharide expression. Deletion of cyaAor crp gene led to significant decrease in the transcriptional activity of PR1 and PR3. It was found that PR1 activity is affected by different carbon sources, glucose reduces the transcriptional activity of PR1, while the addition of glycerol increases PR1 activity. The results showed that the cAMP-CRP complex indirectly activates the transcription at PR1 while it directly upregulates the transcription from PR3 by binding to the CRP binding site at +240 bp relative to the transcription start site. Site-directed mutagenesis of the CRP binding site abolished the binding capability of CRP and led to significant decrease in the transcription from PR3. This regulation of transcription at both PR1 and PR3 promoters by the cAMP-CRP complex is growth phase dependent, the transcription from both promoters at mid-log phase is not affected by CRP but at late exponential or stationary phase CRP is required for maximal transcription from these promoters. In addition, this study demonstrated that the transcription from the promoter on the antisense strand of kpsF, occurring mainly at stationary phase, is not regulated by RpoS and does not regulate the transcription from PR1. In conclusion, this study identified a new regulator of E. coli Group 2 capsule gene expression and added more to the current model of the complex regulation of E. coli capsular polysaccharide expression. By understanding capsule expression, it is hoped to detect therapeutic targets in pathogenic E. coli to block capsular polysaccharide expression in the future.
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Alghoribi, Majed. "Molecular epidemiology, virulence potential and antibiotic susceptibility of the major lineages of uropathogenic Escherichia coli." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/molecular-epidemiology-virulence-potential-and-antibiotic-susceptibility-of-the-major-lineages-of-uropathogenic-escherichia-coli(f1feac7d-0d26-4b6a-b240-f7da26fb1afa).html.
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Uropathogenic E. coli (UPEC) is the most frequent cause of urinary tract infection (UTI), being responsible for up to 85% of community acquired and 40% of nosocomial cases. UPEC strains harbour various virulence factors that contribute to their ability to cause disease. The high prevalence across the globe of multidrug resistant UPEC is a significant threat to therapy. Virulent and resistant UPEC strains have been recognised as belonging to major lineages and we have only recently begun to understand the factors contributing to their successful global dissemination. Work in this thesis was carried out to identify the population structure of E. coli isolates recovered from urosepsis and biliary sepsis, to reveal any differences in genetic background. A total of 100 isolates from the blood and urine of 50 patients presenting with urosepsis and 27 isolates from cases of biliary sepsis were subjected to genotypic and phenotypic analysis, including MLST, virulence gene detection and antibiogram and metabolic profiling. Urosepsis paired isolates showed identical genotypes and antimicrobial resistance profiles. However, several pairs of isolates showed discrepant metabolic activity profiles suggesting niche specific regulation of metabolism. Members of the ST131 clone were significantly associated with antibiotic resistance and ST38 isolates were associated with the highest level of metabolic activity. An in vivo infection model was used to investigate the virulence potential of isolates from the major UPEC lineages. Galleria mellonella larvae inoculated with ST69 and ST127 isolates showed significantly higher mortality rates than those infected with other strains. However, one isolate of ST127 (strain EC18) was avirulent and comparative genomic analyses with a single virulent ST127 strain revealed an IS1 mediated deletion in the O-antigen cluster in strain EC18, which is likely to explain the lack of virulence in the larvae and demonstrates the importance of this cell surface molecule in the model system. Finally, a total of 202 UPEC isolates were recovered from community and hospital urine samples from a tertiary care hospital in Riyadh, Saudi Arabia. Molecular epidemiological investigation of the strains was carried out to examine the overall UPEC population structure, for the first time in any part of Saudi Arabia. The most common lineages were ST131 (17.3%), ST73 (11.4%), ST38 (7.4%), ST69 (7.4%) and ST10 (6.4%). The findings highlight the successful spread of multidrug resistant, CTX-M positive ST38, ST131 and ST405 UPEC in Saudi Arabia. The high proportion (35%) of ESBL producing E. coli isolates is a particular concern and is driving frequent prescription of carbapenem antibiotics. A total of four isolates of ST38 were positive for aggR, which is a virulence marker of enteroaggregative E. coli (EAEC); ST38 strains that cause UTI but have an EAEC genetic background are becoming recognised as novel UPEC and this clonal group warrants further study.
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Updegrove, Taylor Blanton. "Binding properties of Hfq to RNA and genomic DNA and the functional implications." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/41075.
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The bacterial RNA binding protein Hfq is a key component for bacterial sRNA mediated riboregulation of mRNA expression. A kinetic and thermodynamic analysis of Hfq binding to its sRNA targets DsrA, RprA, and OxyS, and to its mRNA target rpoS was carried out. The ability of Hfq to significantly enhance the stability of the DsrA-rpoS and RprA-rpoS complex was demonstrated, and the entire untranslated leader region of rpoS was shown to be important for Hfq binding and in Hfq facilitated sRNA-mRNA duplex formation. Hfq was not shown to enhance OxyS binding to rpoS. DsrA and OxyS were shown to bind mostly to the proximal surface region of Hfq, while RprA bound to both proximal and distal surface regions. The rpoS leader region was shown to possess at least two distinct Hfq binding sites, with one site binding the proximal region and the other to the distal region of Hfq. These sites were shown to be important for Hfq to stimulate DsrA-rpoS binding. The outer-circumference region and the C-terminal tail of Hfq does not play a major role in binding DsrA, RprA, OxyS and rpoS, and in stimulating DsrA-rpoS binding. Evidence was obtained implicating Hfq to bind DsrA, RprA, OxyS, and oligo rA18 in a 1:1 protein to RNA stoichiometry. Binding properties of Hfq to E. coli genomic DNA were examined. Double stranded DNA was shown to bind mostly on the distal surface region and the C-terminal tail of Hfq with an affinity 10 fold less than Hfq targeted RNA. Single stranded DNA binds Hfq more tightly than double stranded DNA and binding seems to be sequence specific. Evidence indicates Hfq binds certain sequences of the E. coli genome.
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Tsilibaris, Virginie. "A la recherche de la fonction des systèmes toxine-antitoxine chromosomiques d'E. coli K12." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210519.
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Les systèmes toxine-antitoxines (TA) sont abondants dans la majorité des génomes bactériens séquencés à ce jour. Ces systèmes codent une toxine stable qui inhibe soit la transcription, soit la traduction, et une antitoxine qui contrecarre l’effet de la toxine par formation d’un complexe avec celle-ci. L’antitoxine est instable suite à sa dégradation continue par les protéases ATP-dépendantes. Afin de maintenir un ratio antitoxine :toxine constant en condition normale de croissance, l’expression des systèmes TA est régulée négativement au niveau transcriptionnel par le complexe toxine-antitoxine.Au début de notre travail, cinq systèmes TA étaient identifiés dans le chromosome d’E. coli. Il avait été montré par notre laboratoire que parmi ces systèmes, seul yefM-yoeB était activé en condition de surproduction de la protéase ATP-dépendante Lon. Ce résultat était surprenant puisque Lon était connue pour dégrader également l’antitoxine RelB du système chromosomique relBE. Un des objectifs de notre travail était de comprendre les mécanismes sous-jacents à cette spécificité. Nous avons montré que l’antitoxine YefM était dégradée à la fois par Lon et les protéases ClpAP et ClpXP. Nous avons également montré qu’en condition de surproduction de Lon, YefM était fortement instable (t1/2~ 10 min. vs 60 min en condition normale). Cette instabilité accrue permet donc l’activation du système yefM-yoeB, c’est-à-dire la libération de la toxine YoeB du complexe qu’elle forme avec YefM. Nous avons également avons montré que le t1/2 de RelB n’était pas affecté par la surproduction de Lon, ce qui explique pourquoi le système relBE n’est pas activé dans ces conditions. Notre hypothèse était qu’un cofacteur soit nécessaire à la dégradation de RelB par Lon et que celui-ci serait limitant dans nos conditions expérimentales. Le crible génétique que nous avons réalisé n’a cependant pas permis d’identifier de cofacteur de dégradation ni de régulateur transcriptionnel en trans du système relBE.
Un deuxième volet de notre travail de thèse a consisté en l’étude de la fonction des systèmes TA chromosomiques. L’hypothèse prévalente au début de notre travail était que les systèmes TA soient intégrés dans les voies adaptatives de réponses au stress. Cependant, le résultat de leur activation était controversé. L’hypothèse du groupe de Gerdes était que leur activation mène à un état bactériostatique réversible alors que le groupe d’Engelberg-Kulka montrait que le système mazEF était un système de mort programmée. Afin d’éclaircir le rôle des cinq systèmes TA dans la physiologie d’E. coli, nous avons testé l’effet de nombreux stress sur la croissance et la viabilité de souches sauvages et de souches délétées de ces systèmes. Aucune des conditions que nous avons testées n’a entraîné une diminution de la viabilité excluant de manière définitive l’hypothèse de la mort programmée. De plus, l’inhibition de croissance causée par ces différents stress s’est avérée être indépendante des cinq systèmes, de même que la phase de récupération suivant les différents stress. Enfin, nos expériences de compétition ont clairement démontré que les cinq systèmes ne procuraient aucun avantage sélectif aux bactéries dans des conditions de compétition en carence nutritive. Les systèmes TA étudiés dans ce travail ne jouent donc aucun rôle dans l’adaptation aux stress que nous avons testé puisqu’ils n’améliorent ni l’aptitude (fitness), ni la compétitivité des bactéries dans ces conditions.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Muthukumarasamy, Uthayakumar Verfasser], Karsten Peter Steffen [Akademischer Betreuer] [Hiller, and Susanne [Akademischer Betreuer] Häussler. "The genomic and transcriptomic landscape of clinical Escherichia coli and Pseudomonas aeruginosa isolates / Uthayakumar Muthukumarasamy ; Karsten Hiller, Susanne Häussler." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1186251476/34.
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Segura, Audrey. "Portage animal des Escherichia coli entérohémorragiques : colonisation et interaction avec le microbiote digestif animal." Thesis, Université Clermont Auvergne (2017-2020), 2018. http://www.theses.fr/2018CLFAC002/document.
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Les Escherichia coli entérohémorragiques (EHEC) sont des E. coli producteurs de Shiga-toxines (STEC) représentant le quatrième agent responsable de toxi-infections alimentaires en Europe. La contamination par ces pathogènes résulte principalement de l’ingestion de produits alimentaires contaminés par les fèces de bovins, dont le tube digestif apparait comme le principal réservoir naturel des EHEC. Ces pathogènes survivent dans le tractus digestif du ruminant, qui est porteur sain, et semblent bien adaptés à l’ensemble de cet écosystème complexe. Réduire le portage animal est une stratégie de choix afin de limiter les toxi-infections humaines à EHEC. L’objectif de cette thèse était d’approfondir les connaissances sur la physiologie et l’écologie des EHEC dans le tube digestif du bovin, une étape primordiale pour proposer, à terme, différentes stratégies visant à limiter le portage. L’analyse du transcriptome de la souche EHEC O157:H7 de référence EDL933 a permis l’identification de voies métaboliques utilisées par les EHEC dans différents compartiments du tube digestif de l’animal. Certains sucres, dont ceux issus de la couche de mucus intestinal, et acides aminés ainsi que l’éthanolamine semblent représenter des substrats importants pour la survie des EHEC tout au long du tube digestif du bovin. Cette étude transcriptomique a également mis en évidence l’activation, par la souche EHEC, de nombreux systèmes de résistance à différents stress rencontrés dans le tube digestif bovin, dont les systèmes toxines/anti-toxines. L’activation de ces systèmes et la capacité à former des biofilms ont également été observées chez une souche STEC O157:H7 d’origine bovine, la souche MC2, dans des conditions mimant une persistance dans l’environnement. La caractérisation génomique et phénotypique permet de considérer cette souche comme pathogène et des études réalisées in vitro et in vivo ont indiqué que la souche MC2 était capable de persister dans le tube digestif du bovin mais aussi dans l’environnement de l’élevage. L’inoculation expérimentale de bovins par la souche MC2 a permis de mettre au point le premier modèle animal reproductible de portage et d’excrétion des STEC O157:H7 décrit en France. Ce modèle pourra être utilisé pour tester in vivo l’effet d’additifs alimentaires, tels que les probiotiques, afin de réduire le portage et l’excrétion de souches EHEC par les bovins, et donc limiter la contamination de l’HommeEnterohaemorrhagic Escherichia coli (EHEC) are Shiga-toxin producing E. coli (STEC) which represent the fourth pathogen leading to foodborne illness in Europe. Contamination by these pathogens results mainly from the ingestion of food contaminated by feces of bovine, for which the digestive tract appears as the main natural reservoir of EHEC. These pathogens survive in the digestive tract of ruminants, which is healthy carriers, and seem well-adapted to this complex ecosystem. Reducing animal carriage is a strategy of choice to limit EHEC human infections. The aim of this thesis was to increase our knowledge on the physiology and ecology of EHEC in the digestive tract of bovine, a key step to propose, ultimately, different strategies to limit the carriage. Transcriptome analysis of the EHEC O157:H7 reference strain EDL933 allowed the identification of metabolic pathways used by EHEC in different compartments of the digestive tract of the animal. Some carbohydrates, including those from the intestinal mucus layer, and amino acids as well as ethanolamine appear to be important substrates for the survival of EHEC throughout the bovine digestive tract. This transcriptomic study also revealed the activation, by the EHEC strain, of several stress resistance systems encountered in the bovine digestive tract, including toxin/anti-toxin systems. The activation of these systems and the ability to form biofilms have also been observed in a bovine STEC O157:H7 strain, MC2 strain, under conditions mimicking persistence in the environment. Genomic and phenotypic characterization allows this strain to be considered as pathogenic and in vitro and in vivo studies indicated that the MC2 strain was able to persist in the bovine digestive tract but also in the farm environment. The experimental inoculation of bovines with the MC2 strain led to the development, for the first time in France, of a reproducible animal model of carriage and excretion of STEC O157:H7. This model could be used to test in vivo the effect of food additives, such as probiotics, in order to reduce the carriage and excretion of EHEC strains by bovines, and thus limit the contamination of humans