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Despite their beneficial effects, concerns have been raised about the role of antimicrobial growth promoters (AGP) in the emergence of antibiotic resistant bacteria. This study evaluated the effects of approved AGP on the emergence of antibiotic resistance in commensal E. coli and foodborne pathogen Salmonella. A survey of antibiotic resistance levels in commercial broiler chicken farms in the Fraser Valley (B.C.) and an experimental feeding trial were conducted from May 2004 to February 2005 and May to November 2005, respectively. The latter examined the effects of ten AGP formulations (bambermycin, penicillin, salinomycin, bacitracin, combination of salinomycin and bacitracin, chlortetracycline, virginiamycin 11ppm, virginiamycin 22ppm, monensin and narasin) on bird performance as well. Multiple antibiotic resistant commensal E. coli and Salmonella carrying virulence genes were found at commercial broiler chicken farms and therefore may serve as reservoirs for these genes. There was no significant difference between feed formulations on the phenotypic or genotypic characteristics of the isolates, except for tetracycline resistance gene tet(B). In the experimental feeding trial, broiler chickens were fed a diet including or excluding AGP. Birds were sampled prior to and weekly during feeding of the control and the AGPP containing diets. Although not detected on day 0, E. coli increased after day 7 to more than 9.9 log10 CFU/g in ceca. Multi-drug resistant E. coli were isolated from birds fed the ten AGP containing diets as well as the control diet. Except for penicillin, none of the AGP containing diets significantly improved bird performance compared to the control diet (P>0.05). Good management practices can significantly improve broiler chickens performance and decrease the mortality rate. / Land and Food Systems, Faculty of / Graduate

Using proteolytic probes, the pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was analyzed for its native topography in the cytoplasmic membrane. Before analyses could be performed, the isolation of transhydrogenase-enriched ISO (inside-out) cytoplasmic membrane vesicles was accomplished by modification of the procedure followed by Clarke (Clarke, D. M. and Bragg, P. D. (1985) Eur. J. Biochem. 149, 517-523) in purifying the enzyme from overexpressing E.coli JM83pDC21 cells. Two major changes were made. One was that the solubilization of the bacterial membrane and subsequent purification steps were omitted. The other was the separation of outer membranes from the cytoplasmic membrane preparation by sucrose gradient density centrifugation. This was essential owing to the contaminating presence of a 30 kD protein in the outer membrane of the original preparation. Transhydrogenase-enriched RSO (right-side-out) membrane vesicles were isolated by a different procedure using lysozyme-mediated breakage of E.coli spheroplasts and subsequent vesicular reformation. To identify possible transhydrogenase fragments arising from proteolytic cleavage, anti-E.coli transhydrogenase polyclonal antibodies were generated in rabbits. Two sets of polyclonal antibodies were produced. One set cross-reacted with both the α (52 kD) and β (48 kD) subunits of the transhydrogenase. The other reacted with the α subunit only. Trypsin and proteinase K were the main proteolytic probes used against both ISO and RSO cytoplasmic membrane vesicles, although chymotrypsin was also used in preliminary experiments with ISO membrane vesicles. Identification of fragments resulting from proteolytic cleavage of the enzyme was obtained using anti-transhydrogenase antibodies and by N-terminal sequencing and/or C-terminal sequencing. In some of these experiments, isolation of the proteolytic fragments was necessary prior to analysis. This was done using a number of different methods. The particular methods applied, which included column chromatography strategies and elution procedures from SDS-Polyacrylamide gels, depended on the type of analysis carried out. The analyses indicated that the α subunit has at least a 41 kD sequence extending from its N-terminus which is exposed to the cytoplasmic side of the membrane. This sequence may contain an active site of the enzyme. This is suggested by the binding of this fragment to a NAD-affinity column. The membrane-imbedded region of the α subunit anchoring the 41 kD region predicted by hydropathy plotting (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) could not be detected by our methods. Susceptible tryptic cleavage sites along the 41 kD region were identified by partial proteolysis and may reflect areas in the subunit's tertiary or quaternary structure that are exposed to the surrounding medium. Major cleavage sites were at arg₁₅, Iys₂₂₇, Iys₂₆₄, arg₂₆₈, Iys₂₇₅, arg₃₅₅, and arg₃₆₁. There do not appear to be significant portions of the subunit protruding into the periplasm as neither trypsin nor proteinase K had any effect on the subunit in RSO-oriented membrane vesicles. Proteinase K experiments with ISO and RSO membrane vesicles suggest that a 20 kD portion of the β subunit is protected from cleavage and is imbedded in the membrane. The identity of this fragment could not be confirmed. Hydropathy analysis of the transhydrogenase gene-derived amino acid sequence (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) suggests that this could be a sequence extending from the N-terminus of the β subunit. This is a hydrophobic sequence containing 7 possible transmembranous helices and having a theoretical molecular weight in the range of 20 kD. The proteinase K results also indicate that the rest of the β subunit is exposed to the cytoplasmic side of themembrane rather than the periplasmic side. The results obtained here are consistent with hydropathy predictions made with regard to this subunit. In addition, two different experiments indicate that an α-α subunit interaction may be present in the oligomeric structure of the membrane-bound enzyme (Hou, C, Potier, M. and Bragg, P. D. (1990), Biochim. Biophys. Acta 1018, 61-66). Substrates of the enzyme did not appear to affect the transhydrogenase's general conformation upon binding as detected by experiments using partial tryptic proteolysis. Partial trypsinolysis also revealed that selective detergent extraction of transhydrogenase-enriched ISO vesicles with Triton X-100 and sodium cholate did not affect the overall conformation of the membrane-bound enzyme despite greatly reducing the enzymatic activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Previous work in many laboratories has established that the addition of L-valine to a culture of Escherichia coli K-12 growing exponentially on minimal medium causes inhibition resulting in persistent linear growth. The inhibition can be removed by addition of isoleucine. Growth of other strains of E. coli e.g. E. coli B and two mutants of K-12 (E. coli AB1020 and AB1005) was not affected by addition of valine. E. coli LL5, another mutant of K-12, showed no inhibition of growth by valine added in concentrations up to 1 x 10ˉ⁴M. However, the addition of 1 x 10ˉ³M valine caused a decrease in the logarithmic growth rate, and 1 x 10ˉ²M valine caused persistent linear growth. The inhibition of growth by valine in LL5 was antagonized by L-isoleucine (as is the case with wild-type E. coli K-12), but not by α-ketobutyrate, threonine or pyruvate. Kinetic studies showed that the maximal inhibition of acetohydroxy acid synthetase (AHAS) by 1.5 x 10ˉ³ M valine was 84% for E. coli K-12, 55% for E. coli B, 75% for E. coli AB1005, 77% for E. coli AB1020 and only 20% for E. coli LL5. It is proposed that the persistence of linear growth of K-12 in valine-containing medium is the result of incomplete (84%) feed-back inhibition of AHAS. Analysis of the data for valine inhibition of AHAS was carried out by the method of Levitsky and Koshland (1969) using Hill Plots. E. coli strains K-12, AB1005 and AB10 20 showed "positive cooperativity" between inhibitor (valine) binding sites at low valine concentrations, and "negative cooperativity" between inhibitor binding sites at high valine concentrations. The AHAS of E. coli strains E and LL5, however, showed only negative cooperativity between binding sites for inhibitor, which could be the mechanism for incomplete inhibition of AHAS by valine. Preliminary kinetic analyses using Michaelis-Menten plots were carried out with strains K-12 and LL5. The levels of threonine deaminase (TD) and AHAS were also examined in four of the E. coli strains, K-12, B, AB1020 and LL5. AHAS was repressed and TD derepressed in E. coli K-12 grown (linearly) with 10ˉ³M valine. In E. coli strains B and AB1020, growth with 10ˉ³M valine had no effect on the levels of AHAS or (derepressed) TD. In E. coli strain LL5, growth with 10ˉ³M valine did not change the AHAS level but caused significant derepression of TD. Sensitivity of growth to valine has been correlated with three properties of the organism: 1) Feed-back sensitivity of acetohydroxy acid synthetase, to L-valine; 2) The level of acetohydroxy acid synthetase in the cell; 3) The level of biosynthetic threonine deaminase - the regulatory enzyme for isoleucine biosynthesis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Cell duplication in Escherichia coli involves complex events, coordinated with chromosome replication. Because of the importance of chromosomes in perpetuating the normal cell cycle the initiation of their replication must be coordinated with cellular division. Following initiation, the cell must replicate and segregate its chromosomes, create a site necessary for septation and divide. These events could be coordinated by either; (1) biochemical reactions involving diffusible enzymes, or (2) multienzyme complexes which are localized at the site of DNA replication and cell division. In the latter case, the cyclic events of replication, segregation and cell division may be coordinated by physical-chemica1 or biochemical means. In any case, physical association implies pleiotropic effects. To test this hypothesis, cell division of the initiator mutant of E. coli , isolated by Kohiyama (1968) was studied. The temperature-sensitive initiator mutant E. coli CR 34T83 (ts DnaA) grew normally at 30 C, and at the restrictive temperature (42 C). The DNA replication as measured by radioactive precursor uptake, stopped after approximately 40 minutes and was equivalent to completion of rounds of replication started. Measurement of ribo- and deoxyribonucleotide triphosphate pools by thin-layer chromatography at 30 C and 42 C indicated residual DNA synthesis was not due to a limitation in the DNA precursors. Using a combination of density and differential radioactive labelling for the starts and ends of chromosomes, a preferred place for reinitiation of new replication cycles was shown. It was shown that DNA replication at 42 C terminated at a fixed region of the chromosome, and was identical to the 150 μg/ml chloramphenicol sensitive step involved in the process of initiation of chromosome replication in E. coli. A cessation of cellular division was noted by measurement of cell growth by Coulter Counter, at a shift from 30 C to 42 C, resulting in filamentous growth. Upon a return to 30 C, the cells resume division after approximately 15 - 20 min. The pleiotropic behaviour, that is, the cessation of cell division and initiation of DNA replication was a result of a point mutation in the gene DnaA, coding for a membrane bound protein involved in initiation. This mutation was mapped by transduction and was located at the isoleucine-valine region of the E. coli map. When this gene was transduced to different strains of E. coli K(12) the same pleiotropy was observed. This pleiotropy could be uncoupled, however, at 30 C by inhibitors of DNA synthesis or initiation. During recovery at 30 C from growth under 42 C, expression of cell division was proportional to cell equivalents generated at the restrictive temperature. RNA and protein synthesis, for 10 minutes during the recovery period, was obligatory for initiation of new rounds of replication, but not for the expression of cell division. A cell division "potential" protein was present under the restrictive growth condition. This "potential" was made at a derepressed rate and underwent a rapid degradation if kept at 42 C. At any given time, when returning from 42 C to 30 C, this "potential" allowed expression cell division based on DNA/mass or normal cell equivalents generated at 42 C. The half-life for decay of the division "potential" was estimated to be 1.4 minutes. The results were interpreted, in terms of an enzyme complex, which is common to the initiation of DNA replication and cellular division. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity on glycoproteins. The scarce supply of sialic acid hinders its advancement in basic research, diagnostic development and therapeutic production. In this work, the recombinant E. coli BRL04 (pBRL89) producing sialic acid was studied by some batch and fed batch runs of high cell density cultivation using a 3-L fermentor. Some cultivation conditions including carbon source, induction time, dissolved oxygen were optimized and different feeding strategies were compared to enhance sialic acid production. The results may be helpful to the further scale-up of sialic acid production and the production of other recombinant proteins by high cell density cultivation of E. coli.

Multilocus Sequence-Typing (MLST) is a phylogenetic technique based on the detection of differences in multiple conserved housekeeping genes. Together with powerful evaluation software, MLST provides an extensive classification scheme for highly diverse species. However, despite the increasing use of MLST as a trusted epidemiological tool, the population structure of UPEC has been poorly studied using this technique, as most of the previous studies conducted have been limited either by bias towards certain characteristics, such as antimicrobial resistance and serogroup, or included a limited number of strains. Such studies can give a false impression of the population structure due to overrepresentation of certain Sequence types (STs).In this thesis, MLST was applied to 300 E. coli isolates collected from in the North West of England between June 2007 and June 2009. Firstly, the prevalence, diversity, epidemiological relationships and phylogenetic origins of the identified STs were determined. Secondly, possible associations of key UPEC STs with other genotypic and phenotypic profiles were assessed. Thirdly, as ST131 was recently reported as one of the most successful UPEC clones, an extensive examination of isolates of this clone was carried out involving identification of multiple drug resistant subclones and attempts were made to recognise putative predictor markers for identification of the ST131 clone.MLST analysis of the studied population revealed a consistent profile of STs that occurred repeatedly in the collection. It consisted primarily of ST73 (16%) followed by ST131 (13.3%), ST69 (9%), ST95 (6.3%), ST10 (4.3%), ST127 (3.6%), ST14 (2.6%) and ST405 (1.6%) some of the STs (ST127 and ST80) in the panel have never been reported as remarkable uropathogens.The broad range of virulence factor (VF) genes screened here allowed the recognition of VF patterns significantly associated with different STs. Most notably, ST127, which, based on phylogenetic analysis, appears to be a newly evolved clone, gave the highest virulence score. This virulent genotype may permit survival of ST127 isolates in the population long enough for them to gain antibiotic resistance. In contrast, multidrug resistant isolates of the ST131 clone were defined by a low virulence score and distinctive VF profiles.Metabolic reactions have been conventionally used for the classification of bacteria into families and species. Interestingly, in the assessment of the metabolic activity of different STs, members of the ST131 clone showed a high metabolic capacity compared to those of other STs, which may compensate for the low virulence capacity and explain the virulence reported for members of this ST. In contrast, ST127 showed the lowest metabolic capacity, even though it held the highest VF-score among the commonly detected STs. Multivariate logistic regression analysis demonstrated that ST131 is best described by its fluoroquinolone resistance and possession of PAI, the ibeA gene and expression of DR antigen-specific adhesins, whereas the O25b-CTX-M-15 ST131 sub-clone was only differentiated from the rest of the ST131 clone members by the production of Extended spectrum Beta-lactamase (ESBL) enzymes.

Escherichia coli was grown in the presence of amino acid analogues or in the absence of required amino acids. The tRNAs. were isolated and characterized. Numerous changes were observed in the total tRNA acceptance for particular amino acids although in no instance did these changes occur for amino acids corresponding to the adverse growth condition. The isoacceptor patterns for particular labelled aminoacyl-tRNAs were determined on the anion exchanger RPC-5. Novel isoacceptor tRNAs were observed under several growth conditions. Significant changes in tRNA isoacceptor distributions were noted. In certain instances it appeared that changes in total amino acid acceptance could be explained in terms of the increased or decreased synthesis of particular tRNA isoacceptors while for other tRNAs it seemed that changes occurred in the synthesis of all isoacceptors for a particular amino acid such that the relative amounts of isoacceptors remained constant even when total amino acid acceptance changed considerably. E. coli was grown over a wide temperature range, 17°C to 44°C, and the tRNA isolated and characterized. Novel tRNA isoacceptors were observed at both high and low growth temperatures for most but not all tRNAs. It was shown that the same isoacceptors could be formed at both extremes of temperature. Preliminary results suggest that the novel isoacceptors are formed as the result of a temperature aggravation of a nutritional problem at extremes of growth temperature. One of the novel tRNA isoacceptors formed under a variety of adverse growth conditions, tRNA3[sup Val] , was purified and partially characterized. The results are consistent with tRNA3[sup Val] being an undermodified precursor of the major isoacceptor tRNA₁[sup Val]. E. coli str[sup D] was grown and the tRNA isolated and characterized. Major differences in the amino acid acceptances for several tRNAs were observed. These changes were accomplished without any significant changes in the relative isoacceptor distributions as determined by RPC-5 chromatography. Gel electrophoretic analysis was performed on tRNA from cells grown at extremes of growth temperature. Significant differences were observed in the 5S region; there was an accumulation of material in cells grown at low temperature and a decrease of material in cells grown at high temperature. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Doctorado en Ciencias de la Ingeniería, Mención Química / El estudio de péptidos como moléculas terapéuticas ha generado gran interés en la industria farmacéutica debido a sus características de alta especificidad y actividad biológica y baja toxicidad. Igualmente, la exploración de nuevos blancos terapéuticos al interior de la célula es bastante atractiva ya que, posibilita la modulación e intervención de procesos intracelulares. En este contexto, disponer de un sistema de expresión recombinante de péptidos en un huésped como Escherichia coli, permitirá potenciar el descubrimiento y desarrollo de nuevos fármacos, especialmente en el estudio de péptidos terapéuticos con actividad biológica intracelular y que requieran unión a péptidos de penetración celular como transportador, ya que el tamaño del péptido a sintetizar podría no ser adecuado para su síntesis química en forma eficiente y sustentable. El trabajo realizado consiste en el diseño de un sistema de expresión bacteriana de péptidos potencialmente terapéuticos, con blanco intracelular y que utilicen al péptido de penetración celular Penetratin como transportador, que permita la producción de diversos péptidos con el fin de estudiar su potencial terapéutico. El sistema fue evaluado mediante la expresión de los péptidos p53pAnt y PNC27, los cuales han mostrado tener actividad biológica antitumoral in vitro. Se diseñó el vector pET31HT que permite la expresión de los péptidos fusionados a la proteína cetoesteroide isomerasa (KSI) en una forma tal que puedan ser posteriormente separados y obtenidos con su secuencia original mediante corte con Trombina. Utilizando técnicas de ingeniería genética se construyó el vector de expresión diseñado. Para la implementación del sistema, se clonó las secuencias nucleotídicas correspondientes a los péptidos p53pAnt y PNC27 en el vector pET31HT. Se logró la producción de los péptidos fusionados en células transformadas con las construcciones pET31HT-p53pAnt y pET31HT-PNC27. La expresión se obtuvo en forma de cuerpos de inclusión y se determinó que los niveles de expresión de proteína recombinante se mantienen constantes y alrededor del 20% del peso seco celular, para distintas condiciones de inducción. Los péptidos fusionados fueron purificados desde la fracción de proteína insoluble por cromatografía de afinidad en condiciones desnaturantes. Mediante una estrategia que incluye precipitación por dilución y solubilización de las muestras en un buffer específico, se logró la separación del péptido p53pAnt de la proteína KSI en un proceso de proteólisis altamente eficiente. Sin embargo, no se logró separar al péptido PNC27 desde la proteína de fusión probablemente debido a la rigidez impuesta por sus aminoácidos iniciales a la región de reconocimiento de la proteasa. El péptido p53pAnt fue purificado desde el producto de digestión, según la estrategia diseñada, obteniéndose un péptido altamente puro. Las características del sistema, en conjunto con el proceso de purificación desarrollado, permiten la síntesis biológica de péptidos con una productividad mayor a 30 mg/g de peso seco celular y con una pureza de al menos un 95%. Con esto, el sistema diseñado es apropiado para su uso en la producción de péptidos para investigación científica.