Pseudomonas fluorescens does not appear to regulate the enzymes of de novo pyrimidine biosynthesis at the level of gene expression. Little or no apparent repression of pyr gene expression is observed upon addition of exogenous pyrimidines to the growth medium. The Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) were sized down and cloned into the broad host range plasmid, pKT230. Upon introduction into a P.fluorescenspyrB mutant strain, ATCase showed repression in response to exogenously fed pyrimidine compounds. Thus, it was possible to bring about changes in pyrimidine nucleotide pool levels and in transcriptional regulation of gene expression at the same time.

Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references. Also available via the World Wide Web.

Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008. / Committee Chair: Hud, Nicholas V.; Committee Member: Fox, Ronald F.; Committee Member: Lynn, David G.; Committee Member: Powers, James C.; Committee Member: Wartell, Roger M.; Committee Member: Williams, Loren D.

No description available.

The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.

No apparent repression of pyr gene expression in Pseudomonas aeruginosa is observed upon addition of exogenous pyrimidines to the growth medium. Upon introduction of the subcloned Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) into a P. aeruginosa pyrB mutant strain, repression was observed in response to exogenously fed pyrimidine compounds. The results proved that it is possible to bring about changes in pyrimidine nucleotide pool levels and changes in transcriptional regulation of gene expression as a result. Thus, the lack of regulatory control in P. aeruginosa pyr gene expression is not due to an inability to take up and incorporate pyrimidine compounds into metabolic pools, or to an inability of the RNA polymerase to respond to regulatory sequences in the DNA but is probably due to a lack of specific regulatory signals in the promoter of the genes themselves.

Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.

The origin of the first RNA polymers is central to most current theories regarding the origin of life. However, difficulties associated with the prebiotic formation of RNA have lead many researchers to conclude that simpler polymers, or proto-RNAs, preceded RNA. These earlier polymers would have been replaced by RNA over the course of evolution. A remaining difficulty for this theory is that the de novo synthesis of a feasible proto-RNA has not yet been demonstrated by plausible prebiotic reactions. This thesis focuses on two problems associated with prebiotic proto-RNA synthesis: The formation of nucleosides and the necessity of reversible backbone linkages for error correction in nucleic acid polymers. "The Nucleoside Problem", or the lack of success in forming pyrimidine nucleosides by plausible prebiotic reactions, represents a significant stumbling block to the RNA world hypothesis. Nearly four decades ago Orgel and coworkers demonstrated that the purine nucleosides adenosine and inosine are synthesized by heating and drying their respective bases and ribose in the presence of magnesium, but these reaction conditions do not yield the pyrimidine nucleosides uridine or cytidine from their respective bases. In this thesis a potential solution to The Nucleoside Problem is hypothesized based upon a proposed chemical mechanism for nucleoside formation. This hypothesis is supported by the successful synthesis of 2-pyrimidinone nucleosides by a plausible prebiotic reaction in good yield, demonstrating that pyrimidine nucleosides could have been available in the prebiotic chemical inventory, but that uridine and cytidine were likely not abundant. Reversible backbone linkages are necessary to provide a mechanism for error correction in non-enzymatic template-directed syntheses of proto-RNAs. In this thesis, acetals are explored as low-energy, reversible linkage groups for nucleosides in polymers. The synthesis of glyoxylate-acetal nucleic acids (gaNAs) through simple heating-drying reactions from neutral aqueous solutions is demonstrated, and these linkages are shown to be hydrolytically stable under a considerable range of solution conditions. Computational models demonstrate that the glyoxylate linkage is an excellent electronic and isosteric replacement for phosphate. Molecular dynamics simulations also indicate that a gaNA duplex would have structural properties that closely match a phosphate-linked RNA helix, suggesting the possibility for cross-pairing between gaNAs and RNAs, allowing for sequence transfer and genetic continuity through the evolution from proto-RNAs to RNA. The principles illustrated in this thesis by 2-pyrimidinone nucleoside and gaNA synthesis can be extended to other prebiotic condensation reactions. Factors affecting condensation yield, such as thermodynamics, kinetics, reactant solubility, and salt effects, are summarized herein.

Pseudomonas aeruginosa is a Gram-negative rod, aerobic, non-fermenting, oxidase positive, pigment producing, and nutritionally versatile bacterium. Infections by P. aeruginosa are the most important cause of morbidity and mortality in immunocompromised patients, given virulence factor production that suppresses antibiotic therapy and promotes persistent infection. This research is the first comprehensive report of the pyrimidine biosynthetic pathway for all phases of growth in minimal and rich media coupled with the evaluation of virulence factor production of P. aeruginosa in comparison to four other bacterial species (Pseudomonas putida, Pseudomonas fluorescens, Burkholderia cepacia, and Escherichia coli wild-type strains). Cellular growth and passing genetic information to the next generation depend on the synthesis of purines and pyrimidines, the precursors of DNA and RNA. The pyrimidine biosynthetic pathway is essential and found in most organisms, with the exception of a few parasites that depend upon the pyrimidine salvage pathway for growth. Both the pyrimidine biosynthetic and salvage enzymes are targets for chemotherapeutic agents. In our laboratory, research on pyrimidine auxotrophic mutants showed the role of the pyrimidine biosynthetic pathway and its intermediates on P. aeruginosa metabolism and impaired virulence factors production. The present research shows that pyrimidine enzymes are active in all phases of growth, including the production of two forms of ATCase in the late log phase in P. aeruginosa. This finding may be explained by the displacement of the inactive PyrC' by the active PyrC or PyrC2 to form a new and larger pyrBC encoded ATCase. Pseudomonas aeruginosa wild-type appears to produce by far the most virulence factors, haemolysin, iron chelation, rhamnolipid, adherence, and three types of motility (swimming, swarming, and twitching) investigated in this study, when compared to the other four wild-type strains. Growth analysis was carried out as typically done in minimal medium but also in rich medium to simulate conditions in the blood and lung tissues of humans as P. aeruginosa infections develop.