Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.

Les extrémités des chromosomes eucaryotes linéaires ont une structure nucléoprotéique particulière, et sont appelées télomères. Étant donnés leur structure et le mécanisme semi-conservatif de la réplication de l’ADN, la longueur des séquences télomériques est instable. Au fil des divisions cellulaires, les réplications successives de l’ADN entraînent une réduction progressive des séquences télomériques. Des télomères courts ne sont plus fonctionnels, ce qui entraîne l’arrêt du cycle cellulaire et de l’instabilité génomique. Il est donc essentiel de prévenir ce raccourcissement. Une enzyme spécialisée rallonge les télomères : la télomérase. La télomérase est une ribonucléoprotéine (RNP) qui maintient les télomères par un mécanisme d’ajout de répétitions de la séquence télomérique. Afin de former un complexe actif, les sous-unités protéiques de l’enzyme doivent s’assembler autour d’un ARN non-codant, nommé Tlc1 chez la levure Saccharomyces cerevisiae. Cependant, le fait que la RNP nécessite plusieurs sous-unités pour son activité implique un assemblage précis et coordonné. Peu de données existent au sujet de l’assemblage de la RNP en un complexe actif, mais il semble qu’un trafic nucléo-cytoplasmique soit requis dans le cycle fonctionnel de l’enzyme. Caractériser le mécanisme d’assemblage de la télomérase permettra de mieux comprendre les phénomènes de régulation de l’activité de l’enzyme, et donc du maintien des télomères chez S. cerevisiae. À cette fin, j’ai d’abord vérifié l’état stoechiométrique de l’enzyme in vivo par des méthodes de FISH sur des molécules individuelles. J’ai ainsi pu montrer que la télomérase ne comportait qu’un seul ARN Tlc1. Ces données in vivo corrèlent avec des données publiées précédemment grâce à des techniques de biochimie, et suggèrent que l’enzyme n’est composée que de complexes individuels contenant une seule copie de chaque sous-unité protéique. Dans le but d’étudier les mécanismes d’assemblage de la télomérase, j’ai aussi développé un système de contrôle de la transcription d’une forme taguée de Tlc1. Cet outil génétique, basé sur les systèmes Cre-Lox et MS2-GFP, permet l’insertion d’un tag MS2 dans le gène TLC1. Ce tag donne la possibilité de suivre des ARN Tlc1 in vivo et en temps réel par microscopie confocale à spinning-disk. Ce système, baptisé CrEMGaT, a permis de montrer que l’insertion du tag dans le gène entraîne l’apparition de Tlc1-MS2, et que ces ARN forment des agrégats nucléaires ayant des caractéristiques similaires aux T-Recs précédemment caractérisés lors d’une collaboration avec le Pr Chartrand. De plus, des résultats préliminaires obtenus avec le CrEMGaT suggèrent que les ARN Tlc1-MS2 finissent leur cycle fonctionnel au cytoplasme. Dans l’ensemble, les données produites et l’outil développé au cours de cette thèse donnent une meilleure idée de l’état d’assemblage de la télomérase. / Abstract : In the eukaryotic kingdom, the extremities of the linear chromosomes have a particular nucleoproteic structure, and are called telomeres. Because of this structure and the semi-conservative nature of DNA replication, telomere length is unstable. DNA replications during consecutive cell divisions leads to a progressive shortening of telomeric sequences. Below a certain threshold, telomeres are not functional, triggering cell-cycle arrest and genomic instability. It is therefore essential to prevent this shortening. A specialized enzyme elongates telomeres: Telomerase. Telomerase is a ribonucleoprotein (RNP) that adds repeats of the telomeric sequence to the end of telomeres. The enzyme formation requires protein subunits to assemble onto a scaffolding ncRNA, Tlc1 in Saccharomyces cerevisiae. The fact that several subunits are needed for RNP activity implies a precise and coordinated assembly occurs. However, data are lacking about telomerase assembly into an active complex, but different observations point towards a nucleo-cytoplasmic trafficking requirement during the enzyme life-cycle. Characteristics about telomerase assembly mechanism would provide useful information in the quest for understanding the phenomena regulating the enzyme activity, and therefore telomere maintenance in S. cerevisiae. Engaged in this quest, I first verified telomerase stoichiometry in vivo. By quantitative single-molecule FISH, the results showed that the enzyme only contains one Tlc1 RNA per RNP in the cell. These in vivo data correlate with previous publications which, based on biochemical experiments, suggested single copies of the different subunits are present in the complex. Taken together, these findings are dismantling a previous dogma that stipulated telomerase is composed of two complexes, and suggest telomerase quaternary arrangement stays simple. Aiming to study telomerase assembly mechanisms, I also developed an inducible genetic system governing the transcription of a tagged version of Tlc1 (i.e. Tlc1-MS2). This system, based on the Cre-Lox and MS2-GFP systems, allows to control the insertion of a MS2 tag into the TLC1 gene. In this system, dubbed CrEMGaT, the genetic insertion is controllable and indeed leads to Tlc1-MS2 appearance. It is then possible to track these tagged RNAs in vivo and in real time with a spinning disk confocal microscope. Furthermore, these RNAs form nuclear aggregates with characteristics of the T-Recs previously described in a collaboration between our lab and Pr Chartrand’s. Finally, preliminary data obtained with the CrEMGaT suggest cytoplasm is the last cellular compartment visited by Tlc1-MS2 RNA. Overall, these data and the system developed during my thesis will give insights into telomerase assembly in vivo.

With an increasing population suffering from obesity or Diabetes Mellitus (DM), it is more pertinent than ever to understand how physiological changes impact cellular processes. Patients with DM often suffer from obesity, hyperglycemia, altered fatty acids that contribute to vascular dysfunction, and increased risk to ischemia. Caenorhabditis elegans is a model system used to study the conserved insulin signaling pathway, cellular responses in whole organisms and the impact a glucose diet has on oxygen deprivation (anoxia) responses. RNA-sequencing (RNA-Seq) was used to analyze the expression of genes in the anoxia sensitive populations of N2 (wild-type) fed glucose and hyl-2(tm2031), a mutant with altered ceramide metabolism. Comparison of the altered transcripts in the anoxia sensitive populations revealed 199 common transcripts- 192 upregulated and 7 downregulated. One of the gene families that have altered expression in the anoxia sensitive populations encode for Cytochrome P450 (CYP). CYPs are located both in the mitochondria and endoplasmic reticulum (ER), but the CYPs of interest are all predicted to be mainly subcellularly localized to the ER. Here, I determined that knock-down of specific cyp genes, using RNA interference (RNAi), increased anoxia survival in N2 animals fed a standard diet. Anoxia sensitivity of the hyl-2(tm2031) animals was supressed by RNAi of cyp-25A1 or cyp-33C8 genes. These studies provide evidence that the CYP detoxification system impacts oxygen deprivation responses. using hsp-4::GFP animals, a transcriptional reporter for ER unfolded protein response (UPR), I further investigated the impact of cyp knock-down, glucose, and anoxia on ER UPR due to the prediction of CYP-33C8 localization to the ER. Glucose significantly increased ER UPR and cyp knock-down non-significantly increased ER UPR. Measurements of ER UPR due to anoxia were made difficult, but representative images show an increase in ER stress post 9-hour anoxia exposure. This study provides evidence that glucose affects ER stress and that ER stress is involved in oxygen deprivation responses.

O uso de fertilizantes minerais nas culturas, inclusive no milho, é uma prática agrícola que provoca danos ambientais e prejuízos econômicos. Uma alternativa promissora, visando melhorar a produtividade e reduzir o uso de fertilizantes, é a utilização de microrganismos benéficos associados às plantas, particularmente as rizobactérias promotoras de crescimento. Essas bactérias vivem na rizosfera e são capazes de colonizar diversos tecidos vegetais, beneficiando o desenvolvimento das plantas através de mecanismos de promoção de crescimento. Na busca por alternativas sustentáveis e mais rentáveis, o presente trabalho teve como objetivo isolar, caracterizar, selecionar e monitorar rizobactérias associadas ao guaranazeiro da Amazônia que possuíssem características promotoras de crescimento vegetal para serem usadas como inoculantes em sementes de milho. Amostras de solo rizosférico de cinco plantas de guaranazeiros foram coletadas e foi realizado o isolamento das rizobactérias. A caracterização molecular foi realizada através do sequenciamento do gene 16S rDNA para análise da diversidade microbiana e identificação das linhagens. Avaliou-se a capacidade das linhagens de produzir ácido indol acético (AIA), fixar nitrogênio atmosférico, solubilizar fosfato inorgânico e de produzir sideróforos. A análise da diversidade microbiana indicou semelhança entre a comunidade bacteriana isolada da rizosfera do guaranazeiro e a do milho encontrada na literatura. Foi observada predominância do filo Proteobacteria, sendo em sua maioria representado pelo gênero Burkholderia. Do total das 101 linhagens obtidas, 89% foram capazes de produzir AIA, 23% fixaram nitrogênio atmosférico, 43% solubilizaram fosfato inorgânico e 24% produziram sideróforos. Cinco linhagens foram selecionadas para o ensaio de promoção de crescimento de milho em casa de vegetação, essas foram identificadas pelo sequenciamento completo do gene 16S rDNA e compuseram os tratamentos como segue: RZ2MS9 - Bacillus sp. (T1), RZ2MS16 - Burkholderia ambifaria (T2) e consórcio (T3) de 5 linhagens (RZ1MS6 - Burkholderia vietnamiensis, RZ1MS11 - Burkholderia sp., RZ2MS9 - Bacillus sp., RZ2MS16 - Burkholderia ambifaria e RZ4MS18 - Delftia acidovorans). As análises estatísticas comprovaram que as linhagens RZ2MS9 (Bacillus sp.) e RZ2MS16 (Burkholderia ambifaria) foram eficientes como promotoras de crescimento em milho, aumentando a altura cerca de 39 e 33%, respectivamente, em relação ao controle, o peso seco da parte aérea cerca de 236 e 114% e do sistema radicular cerca de 248 e 136%, respectivamente, comparado ao controle não inoculado. A linhagem RZ2MS9 (Bacillus sp.) aumentou o conteúdo de Ca nas plantas inoculadas. Para o monitoramento da colonização da bactéria na planta, a linhagem RZ2MS16 (Burkholderia ambifaria) foi transformada com o plasmídio pCM88 e passou a expressar a proteína GFP, sendo possível observar, por microscopia óptica de fluorescência, que, 12 dias após a inoculação na planta, a bactéria encontra-se concentrada no cilindro central da raiz da mesma de onde pode se inserir em algum vaso condutor e colonizar a planta sistematicamente, o que demonstra que a mesma se comporta como endofítica da planta de milho. Assim, fica evidente a importância da exploração de plantas de clima tropical, como o guaranazeiro, como reservatórios de bactérias com enorme potencial biotecnológico. As bactérias estudadas nesse trabalho tem grande potencial para serem utilizadas futuramente como inoculantes. / The use of mineral fertilizers on agricultural crops, including maize, is a practice that causes environmental damage and economical losses. A promising alternative, to improve productivity and reduce fertilizer use is the use of benefical microrganisms associated with plants, particulary the growth-promoting rhizobacteria. These bacteria live in the rhizosphere and are capable of colonizing different plant tissues, benefiting plant growth through mechanisms of growth promotion. In the search for sustainable and more profitable alternative, this study aimed to isolate, characterize, monitor and select rhizobacteria associated with Amazonian guarana that possessed characteristics of plant growth promoters for use as inoculants in maize seeds. Rhizosphere soil samples from five guarana plants were collected and the isolation of rhizobacteria was performed. Molecular characterization was performed by sequencing the 16S rDNA for analysis of microbial diversity and identification of strains. It was evaluated the ability of strains to produce indole acetic acid (IAA), fix atmospheric nitrogen, solubilize inorganic phosphate and produce siderophores. The analysis of microbial diversity indicated similarity between the bacterial community isolated from the rhizosphere of guarana and that found in the literature to maize. It was observed predominance of Proteobacteria phylum, being mostly represented by the genus Burkholderia. Of the total 101 strains obtained, 89% were able to produce IAA, 23% fixed atmospheric nitrogen, 43% solubilized inorganic phosphate and 24% produced siderophores. Five strains were selected for testing growth promotion of maize under greenhouse conditions; these were identified by complete sequencing of the 16S rDNA and compose the treatments as follows: RZ2MS9 - Bacillus sp. (T1), RZ2MS16 - Burkholderia ambifaria (T2) and consortium (T3) of 5 strains (RZ1MS6 - Burkholderia vietnamiensis, RZ1MS11 - Burkholderia sp., RZ2MS9 - Bacillus sp., RZ2MS16 - Burkholderia ambifaria and RZ4MS18 - Delftia acidovorans). Statistical analyzes showed that the strains RZ2MS9 (Bacillus sp.) and RZ2MS16 (Burkholderia ambifaria) were effective as growth promoters in maize, increasing the height about 39 and 33%, respectively, compared to control, shoot dry weight about 236 and 114% and root system about 248 and 136%, respectively, compared to uninoculated control. The strain RZ2MS9 (Bacillus sp.) increased Ca content in inoculated plants. For monitoring of colonization of the bacteria in the plant, the strain RZ2MS16 (Burkholderia ambifaria) was transformed with the plasmid pCM88 and passed to express GFP, being possible to observe by fluorescence microscopy that, 12 days after inoculation on the plant, the bacteria is concentrated in the root central cylinder where the same can be inserted into a vessel conductor and consistently colonize the plant, proving the endophytic life style of this strain during maize interaction. Thus, it is clear the importance of tropical plants, like guarana, as reservoirs of bacteria with great biotechnological potential. The evaluated bacteria accessed in this work have great potential to be used in future as inoculants.

In order to develop a stable strain of Bacillus megaterium for Keratinase production, the Keratinase gene (KerA) of Bacillus lichiniformis ATCC 53757 and SPlipA gene from plasmid pHIS1525.SPlipA (Bacillus megaterium origin) were PCR amplified and constructed to give a gene cassette called SPK . Then the gene cassette SPK was cloned into the Integration vector, pMUTIN-GFP+ 6192bps and transformed in Bacillus megaterium ATCC 14945. The chromosomal integration was created using homologous single crossing over mechanism. The strong natural promoter from the chromosomal locus of the SPK not only produced the increased extracellular enzyme, but also functions as a non inducible promoter which does not require any inducer for the production of the enzyme in the new integrant strain. The integrant strain was subjected to feather degradation test and found that it could totally digest the feather meal in complete seven days, resulting in a rich fermentation broth.

Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.

La dynamique mitochondriale est impliquée dans la maintenance et la fonction des mitochondries. Dans les graines sèches tout les processus cellulaires sont arrêtés du fait de la faible teneur en eau des tissues, et la transition développementale que représente la germination requiert la réactivation de la dynamique cellulaire. Une approche de bio-imagerie sur la plante modèle Arabidopsis a été utilisée afin d’étudier la réactivation des mitochondries nécessaire à la germination. La réactivation bioénergétique des mitochondries, mesurée par la présence du potentiel membranaire, intervient dès le début de l’hydratation des tissus. Cependant les mitochondries restent statiques et la dynamique mitochondriale ne reprend que plus tardivement. La réactivation des mitochondries provoque une réorganisation du chondriome impliquant la biogenèse de membranes et une fusion massive menant à la formation de structures réticulaires et périnucléaires, qui permet le mélange des nucléoïdes d’ADNmt précédemment isolés en unités discrètes. La mitophagie, un indicateur de la qualité mitochondriale, est réactivée de manière concomitante à la dynamique, alors qu’elle est réprimée durant la biogenèse des mitochondries. La fin de la germination coïncide avec la fragmentation du chondriome tubulaire, menant au doublement du nombre de mitochondrie et à une redistribution hétérogène des nucléoïdes dans le chondriome, générant une population de mitochondrie adaptée à la croissance des plantules. Cette thèse met en évidence l’imbrication des processus de dynamique mitochondriale, de biogenèse et de contrôle qualité des mitochondries requis pour la germination et pour la transition vers l’autotrophie. / Mitochondrial dynamics underpin their function and maintenance. In dry seeds, all cellular processes are in stasis due to a low water content. Thus, the developmental switch leading to germination necessarily involves a reactivation of cellular dynamics. In order tobetter understand the role played by mitochondrial dynamics during germination we used Arabidopsis as a model for a bioimaging approach to investigate the rapid reactivation of mitochondria that is required in order to provide ATP to support germination. Bioenergetic reactivation, visualised as the presence of a mitochondrial membrane potential, is almost immediate upon rehydration. However, the reactivation of mitochondrial dynamics only occurs after several hours of rehydration. The reactivation of mitochondrialbioenergetics and dynamics lead to a dramatic reorganisation of the chondriome involving massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mtDNA nucleoids. Mitophagy, an indicator of mitochondrial quality, is reactivated concomitant with a reactivation of mitochondrial dynamics, but is repressed at time of mitochondrial biogenesis. The end of germination coincides with fragmentation of the tubular chondriome leading to a doubling of mitochondrialnumber and heterogeneous redistribution of the nucleoids amongst the mitochondria, generating a population of mitochondria tailored to seedling growth. This thesis provides strong evidence for the tight interweaving of mitochondrial dynamics, mitochondrialbiogenesis and mitochondrial quality control that is required to ensure effective germination and the transition to autotrophy.

<p>Cell division in the Gram-positive bacterium <i>Streptomyces coelicolor</i> starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of <i>S. coelicolor</i> exploit two types of cell division with strikingly different outcomes depending on the developmental stage. </p><p>The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery.</p><p>By isolation and characterization of a non-sporulating <i>ftsZ</i> mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The <i>ftsZ17</i>(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation.</p><p>An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings. </p><p>The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of <i>ftsZ</i> expression and the turnover of the FtsZ protein. </p><p><i>S. coelicolor</i> is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like <i>Escherichia coli</i>. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves. </p>

Since its characterization, the definitive shortcoming of the bacterial luciferase (lux) bioluminescent reporter system has been its inability to express at a functional level in the eukaryotic cellular background. While recent developments have allowed for lux function in the lower eukaryote Saccharomyces cerevisiae, they have not provided for autonomous function in higher eukaryotes capable of serving as human biomedical proxies. Here it is reported for the first time that, through a process of poly-bicistronic expression of human codon-optimized lux genes, it is possible to autonomously produce a bioluminescent signal directly from mammalian cells. The low background of the bioluminescent signal, along with its characteristic lack of substrate amendment required for bioluminescent production, makes a mammalian-based lux reporter system ideal for real-time monitoring of cell culture or murine model systems. The delectability of a lux-based system provides for a functionally equivalent process to monitoring firefly luciferase-expressing cells under cell culture or subcutaneous imaging conditions without the well-documented uncertainties stemming from additional substrate introduction. However, the relatively blue-shifted emission wavelength of the lux reporter system, along with its low quantum yield, has been shown to reduce its effectiveness for use during deep tissue imaging of animal subjects. Despite these disadvantages, it has been demonstrated that a human cell line expressing the human codon-optimized lux genes can function as a biosensor for determination of human bioavailability of toxic compounds and that, by regulating the production of the luxC and luxE genes, the lux system can be employed as the first mammalian, real-time, fully autonomous bioreporter. These cell lines provide unique and efficient models for the detection and monitoring of human-relevant compounds of interest. The limiting reagent for bioluminescent production in the mammalian cellular background has been determined to be the cytosolic availability of the FMNH2 co-substrate and, in light of this evidence, directions for future optimization have been characterized and evaluated in respect to their ability to increase bioluminescent yield under these conditions.

Shiga toxin-producing Escherichia coli (STEC) has been recognized as an important foodborne pathogen. Of this group, O91 is one of the common serogroups frequently isolated from patients and food in some countries, with O91:H21 being previously implicated in hemolytic uremic syndrome (HUS). Cattle are principle reservoirs for STEC, and studies examining STEC shedding in cattle often include experimental inoculation of strains of interest using antibiotic resistance markers for identifiable recovery. However, indigenous fecal microbes exhibiting similar resistance patterns can confound such studies. Such was the case in a study by our group when attempting to characterize shedding patterns of O91:H21 in calves, leading us to seek other, more effective, markers. Among our strategies was the development of a chromosomally integrated bioluminescence marker via transposon mutagenesis using a luxCDABE cassette from Photorhabdus luminescens and a plasmid borne GFP marker via transformation of the pGFP vector. The luxCDABE marker was inserted on host chromosome at a site that was 27 nucleotides before the stop codon of gene yihL and confirmed to have little impact on important virulence genes and growth rate with a very high stability. In contrast, plasmid borne GFP marker showed poor stability without the application of appropriate antibiotic selection pressure. For calves receiving luxCDABE-marked O91:H21, the fecal counts of the organismranged from 1.2 x 10 3 to 1.3 x 10 4CFU/g at two days post inoculation and decreased to 5.8 to 8.7 x 10 2 CFU/g or undetectable level after two weeks.Intestinal contents sampled from various positions at day 14 post inoculation indicated that cecum and descending colon may be the primary localization sites of this O91:H21 strain. Compared to antibiotic resistance markers, the use of bioluminescence markers does not require the restricted pre-inoculation screening of animals. The enumeration of luxCDABE-marked O91:H21 from feces and intestinal contents was easily accomplished and confirmed reliable by M-PCR analysis under the presence of indigenous bacteria which cannot be eliminated by antibiotic-supplemented selective plates. Therefore, the chromosomal integrated luxCDABE marker may be a better model for the study of STEC colonization and shedding in cattle.