A system was developed for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis the scrX gene, contained within the sucrose catabolic regulon, was replaced by the A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, iscX gene cluster, resulting in duplicate genomic copies of these genes, one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in null growth only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted for HscBA could be partially reversed by culturing cells under conditions of low oxygen. These results are interpreted to indicate that HscBA and IscA could have functions related to the protection or repair of the primary IscS/IscU machinery when grown under aerobic conditions. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or display a partial or complete dominant-negative growth phenotype were also identified using this system. Inactivation of the IscR repressor protein resulted in a slow growth phenotype that could be specifically attributed to the elevated expression of an intact [Fe-S] cluster biosynthetic system. This system was also used to investigate the extent to which the two [Fe-S] biosynthetic systems in A. vinelandii, Nif and Isc, can perform overlapping functions. Under normal laboratory growth conditions, no cross-talk between the two systems could be detected. However, elevated expression of Isc components as a consequence of inactivation of the IscR repressor protein results in a modest ability of the Isc [Fe-S] protein maturation components to replace the function of Nif-specific [Fe-S] protein maturation components. Similarly, when expressed at very high levels the Nif-specific [Fe-S] protein maturation components could functionally replace the Isc components. Oxygen levels were also found to affect the ability of the Nif and Isc systems to perform common functions. Nevertheless, the lack of significant reciprocal cross-talk between the Nif and Isc systems when they are produced only at levels necessary to satisfy their respective physiological functions, indicates a high level of target specificity with respect to [Fe-S] protein maturation. / Ph. D.

A stable biofilm of Penicillium chrysogenum in a slab geometry was characterized with respect to penicillin production at various sugar and oxygen concentrations by studies using a novel bench scale bioreactor. The biofilm was submerged in aerated liquid media, mounted vertically in a tube and oriented so that the liquid media and sparged gas bubbles flow uniformly over the two slab faces. The conditions necessary for definite oxygen and sugar limitations were found by operating under various bulk nutrient concentrations. The periodic determination of total biofilm volume (rate of growth) and dry cell weight provided additional information. Definite oxygen mass transfer limitations were found to exist in the biofilm. These limitations could be overcome by increasing the oxygen supply rate to the biofilm. / Master of Science

The gram-negative bacterial pathogen Brucella abortus is a zoonotic pathogen causing brucellosis in a variety of animal species including humans. The loss of the O-side chain in the lipopolysaccharide of the outer membrane decreases Brucella virulence. To understand the genetics of O-side chain biosynthesis and its relationship to virulence, studies were initiated to characterize specific O-side chain mutants. B. abortus rough mutant strain RA2 was derived by transposon (Tn5) mutagenesis of smooth B. abortus 2308. The chromosomal region of strain RA2 with the Tn5 and flanking chromosomal region was cloned into the sequencing vector pGEM-3Z to create a suicide plasmid pNW-2. The plasmid pNW-2, or a derivative of it (pNW-3), in which Tn5 was replaced with a Kank gene, were electroporated into wild type smooth B. abortus 2308 in order to assess the phenotypic conversion from smooth to rough. The electroporation parameters such as cell growth stage, pulse field strength and pulse length were optimized. It was determined that using late log phase cells (approximately 70-77 Klett units), 10 ms and 13 KV/cm were the best conditions for achieving transformation by pNW-2 or pNW3. Kanamycin resistant and ampicillin sensitive Brucella were screened for double reciprocal crossovers between the suicide plasmids (pNW-2 and pNW-3) and Brucella chromosomal DNA. The recombinants were checked for their O-side chain by crystal violet uptake and immunoblotting with monoclonal antibody specific for the O-side chain. The locations of Tn5 and the flanking region in the genome of these recombinants were characterized by Southern blot using either a Tn5 probe or a flanking region probe. An analysis of KanR colonies showed that none of the recombinants were rough. The B. abortus DNA in pNW-2 was sequenced and compared with other genes. No Significant homology was found between the Brucella DNA in pNW-2 and gene sequences in the gene bank. Analysis of the recombinants suggests no linkage between the Tn5 element in strain RA2 and the rough phenotype. / Master of Science

All of the glp genes of Escherichia coli are subject to negative regulation by the glpR-encoded repressor (GlpR). Comparison of the repressor binding affinity for consensus and altered consensus operator regions showed that positions 3, 4, 5, 7 and 8 bp removed from the center of operator symmetry are most important for repressor binding. Cooperative binding of repressors to tandem operators was demonstrated. Cooperativity was maximal when two 20-base pair operators were directly repeated and decreased with the deletion of 2 base pairs or the addition of 4 base pairs between the operators. Cooperative binding was eliminated by a 6 base pair insertion between tandem operators. / Master of Science

As a first step in characterizing the genes involved in O-side chain synthesis in <i>Brucella abortus</i> strain 2308, a portion of the genomic DNA was cloned from a rough mutant created by Tn5 (KnR) mutagenesis. This mutant was rough based on the lack of reactivity by either whole cells or extracted LPS to an O-side chain monoclonal antibody (BRU-38). A 30 kb <i>Xba</i>I genomic fragment (including Tn5) from the rough strain was subcloned into a sequencing vector to create pJM6. When <i>B. abortus</i> 2308 was electroporated with pJM6, KnR clones were unable to react with BRU-38; a Southern analysis of these clones revealed Tn5 in the 30 kb <i>Xba</i>I genomic fragment. Various regions of the 30kb fragment were subcloned and tested for their ability to complement specific <i>rfa</i> and <i>rfb</i> mutants of <i>Escherichia coli</i> and <i>Salmonella typhimurium</i>. One particular DNA fragment complemented an <i>rfbD</i> mutation in <i>E. coli</i> as judged by agglutination with <i>E. coli</i> anti-O (0:85) serum. The same DNA fragment failed to cause <i>E. coli rfbD</i> to react with either BRU-38 or <i>B. abortus</i> anti-O polyclonal antisera. The <i>B. abortus</i> 30 kb <i>Xba</i>I fragment contains a gene which has been identified by comple-mentation as containing the equivalent of the <i>rfbD</i> gene encoding dTDP-rhamnose synthetase in <i>E. coli</i>. Since <i>Brucella</i> is not known to have rhamnose in its core this enzyme may have a different function in <i>Brucella</i> LPS synthesis. / Master of Science

The biosynthesis of virginiamycin S₁, a macrocyclic peptidolactone antibiotic, was studied by growing a strain of Streptomyces virginiae in a complex medium and observing the incorporation of radiolabeled compounds into the antibiotic. These studies have established several of the biosynthetic precursors of virginiamycin S₁. L-(U-14C)-Proline and L-(U-14C)-threonine were effectively incorporated into the respective amino acid components in the antibiotic. N-Methyl-L-phenylalanine was shown to arise from L-(U-14C)-phenylalanine and L-(methyl-14C)-methionine. L-(U-14C)-Phenylalanine was also efficiently incorporated into L-phenylglycine. The origin of the remaining three components was less clear. A small amount of L-(U-14C)-threonine was observed in D-α-aminobutyric acid. A biosynthetic pathway is known between these two amino acids which suggests that L-threonine may be the biosynthetic precursor of D-α-aminobutyric acid. Both L-(U-14C)-aspartic acid and L-(U-14C)-lysine were incorporated into 4-oxo-L-pipecolic acid and 3-hydroxypicolinic acid. A biosynthetic pathway was hypothesized to explain these results. / Master of Science

Yes / The use of methylmalonyl-CoA epimerase (MCEE) to improve stereoselectivity in crotonase-mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl-CoA carboxylase reductase and carboxymethylproline synthase in a three-enzyme one-pot sequential synthesis of functionalised C-5 carboxyalkylprolines starting from crotonyl-CoA and carbon dioxide. / Biotechnology and Biological Sciences Research Council, The Wellcome Trust, and CONACyT and FIDERH (Mexico, RGC) The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Boehringer Ingelheim, the Canada Foundation for Innovation, the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust (092809/Z/10/Z).

Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype. In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Δpgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Δpgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive. / Master of Science

Vitamin C (ascorbic acid) is one of the most essential organic compounds required by the human body for normal metabolic function. Unfortunately, this valuable nutrient is not produced in the human body but most plants and animal can produce this molecule. Although ascorbic acid was not isolated until the early part of the twentieth century, it was known that eating limes and other citrus fruits could ward off the affects of scurvy as early as the 1500's. Ascorbate serves many critical functions in plants as well as the human body. In both, it works as a cofactor in the production of hydroxyproline-rich compounds and helps protect molecules such as proteins, lipids and fatty acids from oxidation. Although the biochemical pathway in animals has been known since the 1950's (Jackel et al., 1950), the exact process by which ascorbic acid is made in plants has eluded scientists. It was shown in 1963 that the inversion of the hexose carbon chain, which occurs in the animal pathway, is not a possible mode of synthesis in plants (Loewus, 1963). As an alternative, a non-inversion pathway was proposed, which achieves ascorbic acid using D-mannose and L-galactose as intermediates, referred to as the Smirnoff-Wheeler pathway (Wheeler et al., 1998). It was shown that transforming lettuce (cv. Grand Rapids and Black Seeded Simpson) and tobacco (cv. Xanthi) with the terminal enzyme in the animal biosynthetic pathway (GLO; L-gulono-gamma-lactone oxidase) increases the ascorbic acid content between 4 and 7 fold. It was also shown through feeding studies that wild type tobacco plants had elevated ascorbate levels when fed the animal precursor (Jain and Nessler, 2000). These data suggest that at least part of the animal pathway could be present in plants, along with the Smirnoff-Wheeler (1998) pathway. To further investigate this discovery, wild type and ascorbic acid-deficient Arabidopsis thaliana were transformed with the glo. Homozygous lines of these transformants were generated and the ascorbic acid levels were compared to the untransformed wild type and mutant plants. Although the wild type plants containing glo did not show a significant increase in ascorbic acid production, all five of the vtc mutant lines had an increased ascorbic acid content relative to wild type level. These data suggest that an alternative pathway is present in plants that does not require many of the steps in the published Smirnoff-Wheeler (1998) pathway to produce ascorbic acid. / Master of Science

This dissertation comprises three parts and is presented in two chapters. Chapter 1 concerns Arthrobacter, a bacterium with an intriguing growth cycle. Whereas most bacteria exist as either a rod or coccus, this bacterium shares the rod/coccus lifestyle. It therefore seemed important to examine the growth regulatory pathways from the rod and coccus. The committed step, that catalyzed by aspartate transcarbamoylase (ATCase), in the pyrimidine biosynthetic pathway was chosen. The ATCase in Arthrobacter is like the well known Pseudomonas enzyme except that it has an active dihydroorotase (DHOase) associated. Included in Chapter 1 is the description of a microorganism, Burkholderia cepacia, whose ATCase has characteristics that are at once reminiscent of bacteria, mammals, and fungi. It differs in size or aggregation based on environmental conditions. In addition, it has an active DHOase associated with the ATCase, like Arthrobacter. B. cepacia is important both medically and for bioremediation. Since B. cepacia is resistant to most antibiotics, its unique ATCase is a prime target for inhibition. Whereas the first chapter deals with the de novo pathway to making pyrimidines, which is found mainly in the lag and log phase, Chapter 2 addresses the salvage pathway, which comes more into play during the stationary phase. This section focuses on the isolation, identification, and grouping of a number of natural soil bacteria from various soil locations. These organisms are important agriculturally, medically, and industrially. Addition of these soil isolates to poor soils has been found to improve the soil. In a previous study by D.A. Beck, the salvage schemes for a number of laboratory strains of microorganisms were determined. Nine separate classes of salvage were designated by determining the salvage enzymes present. In this study emphasis has been placed on soil bacteria, which had not previously been analyzed. A number of species of soil bacteria were identified using the MIDI. The salvage enzymes were then determined for these organisms and a comparison of these isolates to the previous study was performed in order to group the new organisms into 19 salvage schemes, that is 10 more than in the previous study.