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The environment of the gastrointestinal (GI) tract is an extremely complex system made up of not only host cells, but also many beneficial microbes. Disruption of this environment can often lead to disorders and health issues. To help balance this system, probiotics have often been administered to both humans and animals, such as livestock. This study aimed to determine what beneficial effects a novel strain of Enterobacter cloacae, strain JD6301, could offer to a host in the presence of an enteric infection with E. coli O157:H7. Upon administration of JD6301, supplemented animals had overall less E. coli present in the colon and caecum. Moreover, these animals shed more E. coli than control groups. Supplemented animals also had increased concentrations of serum triglycerides one day prior to challenge. Together, these data suggest that Enterobacter cloacae JD6301 could perform as a novel probiotic providing energy and protection to the host.

A mating between Escherichia coli 4318 (thi-leu-Las-Hfr) and E. coli A-1 (Met-Las+ F-) resulted in the formation of phototrophic recombinantshaving L-asparaginase activities at three distinct levels. The physiology of L-asparaginase synthesis in these recombinants is described.

The Pho regulon is controlled by the PstSCAB transporter, PhoU, and the two-component proteins, PhoB and PhoR. PhoU is a negative regulator of the Pho regulon under phosphate-replete conditions. How PhoU functions is unknown. Many PhoU homologues are found widely throughout prokaryotic domains. There are several conserved amino acid residues in the PhoU protein. It is hypothesized that these residues play an important role in the function of PhoU. To test this hypothesis, several site directed mutations in the phoU gene have been produced with single amino acid changes in conserved residues. After testing these mutants, it was found that some of the mutants abolished repression of the Pho regulon while other mutants had little or no effect. Further study of these mutants and their phenotypes will reveal more about how PhoU functions and help to better understand bacterial signaling in general.

Les aminoacyl-ARNt synthetases (aaRS) sont des enzymes clés dans le mécanisme de biosynthèse des protéines. Chacun des membres de cette famille d'enzymes reconnaît et lie les éléments d'identités propres à chaque acide ribonucléique de transfert (ARNt) permettant ainsi leur aminoacylation par un acide aminé spécifique. Une fois l'ARNt correctement chargé, celui-ci peut lier un facteur protéique (EF-Tu pour les aminoacyl-ARNt élongateurs) qui le conduit au ribosome pour la biosynthèse des protéines. Plus particulièrement la glutamyl-ARNt synthetase (GluRS) d'Escherichia coli est une protéine capable d'estérifier l'ARNtGIU par un résidu glutamate. Cette enzyme monomérique est subdivisée en 5 domaines structuraux, chacun capable de lier une portion de l'ARNt qui lui est propre. La région formant la boucle d'anticodon de l'ARNtG'u est reconnue par les domaines 4 et 5 séparés par une charnière, et situés en C-terminal de l'enzyme. L'étude d'une GluRS tronquée, délétée de son domaine 5, a montré l'importance de ce domaine sur l'activité catalytique de cette enzyme. Afin de comprendre les mécanismes mis en place entre ces deux derniers domaines lors de la liaison du substrat ARNtGly, des variants protéiques visant à altérer les interactions existant entre les domaines 4 et 5 et la charnière ont été synthétisés, et leur constantes catalytiques mesurées. L'étude du simple variant, substitué au niveau du second résidu chargé de la charnière de la GluRS de E. coli a montré l'importance de ce dernier dans les interactions mises en place entre le domaine 5 et la charnière. Par ailleurs l'altération des constantes cinétiques (en comparaison avec les valeurs trouvées pour l'enzyme sauvage), de double et triple variants, substitués au niveau des résidus chargés trouvés sur la charnière séparant les domaines 4 et 5, ont révélé l'importance de certain de ces résidus dans les interactions existant entre ces deux domaines.

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Crohn’s disease (CD) is an inflammatory bowel disease characterised by chronic inflammation with a complex pathophysiology involving host, environmental, and microbial factors. The intestinal microbiota is an important regulator of inflammation within the intestine, and a disruption of the interplay between gut bacteria and host immunity is a key factor in CD development. Intestinal inflammation itself is known to cause changes to the intestinal physiology that affect the ability of various bacteria to survive. Additionally, certain environmental risk factors for CD such as antibiotics are also known for their ability to impact the intestinal microbiota. CD is associated with various changes in the intestinal microbiome including increased colonisation with a group of bacteria known as adherent-invasive Escherichia coli (AIEC). The purpose of this study is to investigate how AIEC interact with antibiotics and intestinal inflammation in vivo. Multiple classes of antibiotics were found to increase the colonisation of AIEC and to increase its persistence. These antibiotics caused a loss diversity in the intestinal microbiome, but this did not explain the increased infectivity of AIEC. Antibiotic-induced inflammation was found to produce metabolites that benefitted AIEC growth in the intestine and similar results were found with chemically-induced inflammation. These results show that AIEC can benefit from both antibiotics and other sources of inflammation through inflammation-derived metabolites, which contributes to a greater understanding of the interactions between AIEC and CD. / Thesis / Doctor of Philosophy (PhD) / Crohn’s disease is a type of inflammatory bowel disease that affects many young adults in Canada. It causes a wide range of symptoms including nausea, pain, and diarrhea. While the disease can be treated with surgery and medications, it is considered incurable and affects most individuals for life. The exact cause of Crohn’s disease is not known, but it is thought to be caused by a combination of factors including genetics, environmental exposures, and changes in the number and types of bacterial species in the intestine. Intestinal bacteria play an important role in preventing inflammation in the intestine. An unusual strain of bacteria called adherent-invasive E. coli is found more commonly in Crohn’s disease patients than in healthy individuals. This strain does not cause the disease on its own, but it may interact with other environmental factors that are also associated with Crohn’s disease, such as taking antibiotics. Antibiotic use is a risk factor for developing Crohn’s disease later in life and antibiotics have previously been shown to promote the growth of other E. coli strains in the intestine. In a mouse model of Crohn’s disease, we found that antibiotics made mice more vulnerable to infection with this E. coli strain. This increased vulnerability was because the antibiotics caused inflammation, and we also found that other sources of inflammation benefitted this E. coli strain. These findings help us understand how gut bacteria and other Crohn’s disease risk factors might interact to cause the disease.

PspE, encoded by the last gene of the phage shock protein operon, is one of the nine proteins of Escherichia coli that contain a rhodanese homology domain. PspE is synthesized as a precursor with a 19-amino acid signal sequence and secreted to the periplasm. Mature PspE is the smallest rhodanese of E. coli (85 amino acids) and catalyzes the transfer of sulfur from thiosulfate to cyanide forming thiocyanate and sulfite. Cation exchange chromatography of a freeze-thaw extract of a PspE-overexpressing strain yielded two major peaks of active, homogeneous PspE. The two peaks contained two forms of PspE (PspE1 and PspE2) of distinct size and/or charge that were distinguished by native polyacrylamide gel electrophoresis and gel chromatography. PspE2 was converted to the more compact PspE1 by treatment with thiosulfate, which suggested that PspE1 is the persulfide form. One equivalent of cyanizable sulfur was associated with PspE1, with much less present in PspE2. Consistent with the conclusion that the single active site cysteine of PspE1 contains a persulfide sulfur was the observation that this form was much more tolerant to chemical inactivation by thiol-specific modifying reagent DTNB (5,5′-dithiobis(2-nitrobenzoic acid)). Rhodanese activity was subject to inhibition by anions (sulfite, sulfate, chloride, phosphate and arsenate), suggesting PspE has a cationic site for substrate binding. Kinetic analysis revealed that PspE employs a double-displacement mechanism, as is the case for other rhodaneses. The Kms for SSO32- and CN- were 3.0 and 43 mM, respectively. PspE exhibited a kcat of 72 s-1. To aid in understanding the physiological role of PspE, a strain with a pspE gene disruption was constructed. Comparison of rhodanese activity in extracts of wild-type and mutant strains revealed that PspE is a major contributor of rhodanese activity in E. coli. The pspE mutant displayed no obvious growth defect or auxotrophies, and was capable of molybdopterin biosynthesis, indicating that pspE is not essential for production of sulfur-containing amino acid or cofactors. Growth of wild-type and mutant strains deficient in pspE and other sulfurtransferase paralogs in medium with cyanide or cadmium was compared. The results indicated that neither PspE nor other E. coli rhodanese paralogs play roles in cyanide or cadmium detoxification. The physiological role of PspE remains to be determined. / Master of Science