Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. …

The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, …

Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion …

Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated by an enzyme referred to as cyanide oxygenase (CNO), previously shown to require components in both high (H) (>30 kDa) and low (L) (<10 kDa) molecular weight cell fractions. In this study, tetrahydrobiopterin (H4biopterin) was identified as a cofactor in fraction L, thus making CNO appear as a pterin- dependent hydroxylase. CNO was purified 150-fold (specific activity 0.9 U/mg) and quantitatively converted cyanide to formate and ammonia as reaction products. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted. CNO was found to be an aggregate of known enzymes that included NADH oxidase (Nox), NADH peroxidase (Npx), cyanide dihydratase (CynD) and carbonic anhydrase (CA). A complex multi-step reaction mechanism is proposed in which Nox generates hydrogen peroxide which in turn is utilized by Npx to catalyze the oxygenation of cyanide to formamide accompanied by the consumption of one and two molar equivalents of oxygen and NADH, respectively. The further hydrolysis of formamide to ammonia and formate is thought to be mediated by CynD. The …

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 …

The soil-nematode Caenorhabditis elegans survives oxygen deprivation (anoxia < 0.001 kPa of O2, 0% O2) by entering into a state of suspended animation during which cell cycle progression at interphase, prophase and metaphase stage of mitosis is arrested. I conducted cell biological characterization of embryos exposed to various anoxia exposure times, to demonstrate the requirement and functional role of spindle checkpoint gene san-1 during brief anoxia exposure. I conducted a synthetic lethal screen, which has identified genetic interactions between san-1, other spindle checkpoint genes, and the kinetochore gene hcp-1. Furthermore, I investigated the genetic and cellular mechanisms involved in anoxia-induced prophase arrest, a hallmark of which includes chromosomes docked at the nuclear membrane. First, I conducted in vivo analysis of embryos carried inside the uterus of an adult and exposed to anoxic conditions. These studies demonstrated that anoxia exposure prevents nuclear envelope breakdown (NEBD) in prophase blastomeres. Second, I exposed C. elegans embryos to other conditions of mitotic stress such as microtubule depolymerizing agent nocodazole and mitochondrial inhibitor sodium azide. Results demonstrate that NEBD and chromosome docking are independent of microtubule function. Additionally, unlike anoxia, exposure to sodium azide causes chromosome docking in prophase blastomeres but severely affects embryonic viability. …

The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.

Invasive pulmonary aspergillosis is a life-threatening fungal infection commonly observed in immunocompromised patients and has a mortality rate approaching 100% once the disease is disseminated. Aspergillus fumigatus is the most common pathogen. Early diagnosis improves the prognosis but is very difficult since most signs and symptoms are nonspecific. Antifungal therapy, usually based on sterol biosynthesis inhibitors, is also of limited efficacy. In my attempts to discover a diagnostic sterol marker for aspergillosis, I observed that A. fumigatus incorporates large amounts of cholesterol from serum-containing medium. This observation suggested the hypothesis that exogenous cholesterol from the host can be imported by A. fumigatus and used as a substitute for ergosterol in the cell membrane. This proposed mechanism would reduce the efficacy of antifungal drugs that act as sterol biosynthesis inhibitors. Experiments to test this hypothesis were designed to determine the effects of serum-free and serum-containing medium on growth of A. fumigatus in the presence and absence of azole antifungal agents. The results showed a marked increase in growth in the presence of human serum. Cultures in media containing cholesterol but no serum also showed enhanced growth, a result indicating that a non-cholesterol component of serum is not primarily responsible for the …

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.

Flamingo species generate tremendous interest whether they are small captive groups or wild populations numbering in the thousands. Genetic pedigrees are invaluable for maintaining maximum genetic diversity in captive, as well as wild, populations. However, presently there is a general lack of genetic data for flamingo populations. Microsatellites are loci composed of 2-6 base pair tandem repeats, scattered throughout higher eukaryotic genomes, often exhibiting high levels of polymorphism and heterozygosity. These loci are thus important genetic markers for identity, parentage and population studies. Here, six microsatellite loci were isolated from a microsatellite-enriched Caribbean flamingo partial genomic library. Two are compound complex repeats and four are perfect trinucleotide repeats. Each locus was amplified from Caribbean, African greater, Chilean and lesser flamingo genomic DNAs. Heterozygosity frequencies were calculated for Caribbean (range 0.12-0.90) and African greater flamingos (range 0.23-0.94) loci. All six microsatellite loci were found to be in Hardy-Weinberg equilibrium and linkage disequilibrium analyses did not suggest linkage for any pair of two greater flamingo subspecies (African and Caribbean) loci. At least five of the loci also exhibit polymorphism in Chilean and lesser flamingos, but due to small sample numbers, relevant allele/heterozygosity frequency calculations could not be estimated. Nucleotide sequence comparisons of …

Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, …

Polyunsaturated fatty acids are major structural components of plant chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases (designated FAD2 and FAD3) desaturate 75% of the fatty acids in the endoplasmic reticulum. The w -6 fatty acid desaturase (FAD2) may be responsible for cold acclimation response, since polyunsaturated phospholipids are important in helping maintain plant viability at lowered temperatures. To study regulation of FAD2 gene expression in cotton, a FAD2 gene was isolated from two genomic libraries using an Arabidopsis FAD2 hybridization probe and a cotton FAD2 5¢ -flanking region gene-specific probe, respectively. A cotton FAD2 gene was found to be in two overlapping genomic clones by physical mapping and DNA sequencing. The cloned DNA fragments are identical in size to cotton FAD2 genomic DNA fragments shown by genomic blot hybridization. The cotton FAD2 coding region has 1,155 bp with no introns and would encode a putative polypeptide of 384 amino acids. The cotton FAD2 enzyme has a high identity of 75% with other plant FAD2 enzymes. The enzyme has three histidine-rich motifs that are conserved in all plant membrane desaturases. These histidine boxes may be the iron-binding domains for reduction of oxygen during desaturation. To confirm that this FAD2 …

The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six …

The Medicago truncatula NIP gene is located on M. truncatula Linkage Group 1. Informative recombinants showed crossovers that localize the NIP gene between markers 146O17 and 23C16D. Marker 164N9 co-segregates with the NIP gene, and the location of marker 164N9 is between markers 146O17 and 23C16D. Based upon data from the Medicago genome sequencing project, a subset of the model legume Medicago truncatula bacterial artificial chromosomes (BACs) were used to create a physical map on the DNA in this genetic internal. BACs near the potential NIP gene location near marker 164N9 were identified, and used in experiments to predict the physical map by a BAC-by-BAC strategy. Using marker 164N9 as a center point, and chromosome walking outward, the physical map toward markers 146O17 and 23C16D was built. The chromosome walk consisted of a virtual walk, made with existing sequence of BACs from the Medicago genome project, hybridizations to filters containing BAC DNA, and PCR reactions to confirm that predicted overlapping BACs contained DNA that yielded similar PCR products. In addition, the primers which are made for physical mapping via PCR could be good genetic markers helpful in discovering the location of the NIP gene. As a result of efforts repotted …

Campylobacter organisms are the most commonly reported bacterial causes of foodborne infection in the world, with Campylobacter jejuni and Campylobacter coli responsible for over 99% of reported infections. Traditionally, Campylobacter species detection is an arduous process, requiring a special incubation environment as well as specific growth media for an extended growth period. The development of a rapid and reliable diagnostic tool for the detection of Campylobacter species would be a valuable aid to the medical diagnostic decision process, especially to rule out Campylobacter infection during the enteric pre-surgical time period. Improved patient outcomes would result if this rapid assay could reduce the number of enteric surgeries. Assays performed during this dissertation project have demonstrated that both SYBR® green and hydrolysis probe assays targeting an 84 nucleotide portion of cadF, a fibronectin-binding gene of Campylobacter jejuni and Campylobacter coli, were able to detect from 101 to 108 copies of organism from stool specimens, did not detect nonspecific targets, and exhibited a coefficient of variation (CV) of 1.1% or less. Analytical validation of sensitivity, specificity and precision, successfully performed in these studies, warrants additional clinical validation of these assays.

Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP …

A cotton PATE cDNA clone has a 1.7-kb insert with an coding region for 410 amino acids, lacking codons for the three N-terminal amino acids. The predicted amino acid sequence of the PATE preprotein has a characteristic stromal-targeting domain and a 63% identity to the Arabidopsis FatB1 thioesterase sequence. A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-ACP thioesterase (FatB1) gene. The gene spans 3.6 kb with six exons and five introns. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein is identified as a FatB thioesterase from its deduced amino acid sequence similarity to those of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential promoter/enhancer elements including basic helix-loop-helix elements (E box). Alkaline blot hybridization of cotton genomic DNA suggests the presence at least two FatB1 thioesterase genes in cotton. Four plasmid constructs for both constitutive and seed-specific anti-sense RNA suppression and gene-transgene co- suppression of PATE gene expression were successfully generated. Two overlapping cotton genomic clones were found to encompass a Δ-12 fatty acid desaturase (FAD2-3) …

Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was …

Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic …

The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a …

Cloning and molecular characterization of new genes constitutes a useful approach in studying the symbiotic interactions between the model plant Medicago truncatula and Synorhizobium meliloti. Large numbers of expressed sequence tags (ESTs) available for Medicago truncatula, along with numerous cDNA, oligonucleotides, and Affimetrix DNA microarray chips, represent useful tools for gene discovery. In an attempt to identify a new gene that might be involved in the process of nodulation in Medicago truncatula, preliminary data reported by Fedorova et al. (2002), who identified 340 putative gene products or tentative consensus sequences (TCs) expressed only in nodules, was used. This research was focused on TC33166 (TC103185), which has 3 ESTs in the TC, and whose strongest BLASTX hit of TC103185 is the incomplete root hair elongation (IRE) protein kinase-like protein (NP_192429) from Arabidopsis thaliana. The Arabidopsis IRE gene is required for normal root hair growth, and a role in apical growth was suggested (Oyama et al., 2002). Infection thread growth can be looked at as an inward growth of the root hair. Thus, TC103185 was a good candidate for identifying a gene that may be involved in early events of nodulation. MtIRE (GenBank accession AC122727) is organized in 17 exons and 16 …

Methods to determine genetic diversity and relatedness within populations are essential tools for proper wildlife management. Today the approach of choice is polymerase chain reaction-based microsatellite analysis. Seven new polymorphic loci were isolated from a microsatellite-enriched Caribbean flamingo genomic library and used to characterize survey populations of Caribbean and African greater flamingos. In addition, four of these loci were used to verify parentage relationships within a captive-breeding population of African greater flamingos. Parentage predictions based upon gamekeeper observations of breeding and nesting did not always agree with genetic-based parentage analyses of the nine suggested family groups. Four family groups were supported (groups I, II, III and VI) by there results. However, an analysis of the remaining five suggested groups, with a total of eight offspring/dam and eight offspring/sire suggested relationships, yielded seven exclusions of the suggested dam and six exclusions of the suggested sire. This put the overall suggested dam exclusion rate at 35% and exclusion rate for suggested sires at 29%. Although the keeper observation data for our family groups must be considered a variable of concern at this time, these findings are certainly suggestive that more carefully controlled studies may reveal that flamingos are not monogamous as long …

Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all …

This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.