Purine and pyrimidine bases are the central precursors of DNA and RNA and theirintracellular concentration is balanced by three pathways- de novo, salvage and catabolicpathways. Uracil catabolism pathway has been found in several bacteria and in some fungi(including yeast). Seven genes, URC1-7 have been found to be involved in this novelpathway. There are two “unknown genes” in the yeast Lachancea (Saccharomyces) kluyveri,namelyURC1 and URC4, which play a central role in this pathway and their exact functionremains a mystery.In this project, two S. kluyveri genes, URC1&URC4, were over-expressed in the bacterialsystem and successfully purified. Our preliminary functional assay showed that uridinemonophosphate (UMP) is a likely substrate for Urc1p at pH7, 25ºC. It was shown clearly thatboth uracil and uridine were not the substrate for Urc1p. We tried to phosphorylatechemically synthesized ribosylurea using Drosophila melanogaster deoxyribonucleosidekinase and compared the activity between phosphorylated and non- phosphorylated RU atdifferent conditions. Phosphorylated ribosylurea seemed to be a likely substrate for Urc4p atpH7, 37ºC. Keywords: Uridine monophosphate (UMP), ribosylurea (RU), uracil catabolism.

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Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

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Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, and phenylpyruvate could be used as sole carbon sources by wild type B. cenocepacia K56-2 but not by a PA catabolism defective mutant. These intermediates also induced a PA-inducible reporter system. Furthermore, proteomic analysis utilizing iTRAQ were used to study the protein expression of B. cenocepacia K56-2 grown in the amino acid-rich SCFM. Our results showed the over-expression of several proteins involved in amino acid and carbohydrate transport and metabolism. Interestingly, our results also showed the over-expression of flagellin and membrane efflux protein which are involved in the virulence of B. cenocepacia.

Hydroxyproline (trans-4-hydroxy-L-proline (4-L-Hyp)) can be used by certain microorganisms as a source of carbon and nitrogen. The nitrogen fixing bacterium, Sinorhizobium meliloti carries a cluster (hyp cluster) of 14 genes responsible for the transport and degradation of this amino acid in the cell. The biological functions of several gene products in the hyp cluster are still unknown. So far, it is known that the conversion of trans-4-hydroxy-proline to α-ketoglutarate, one of the intermediate of the TCA cycle, occurs in four enzymatic reactions. The whole hyp cluster is up regulated in the presence of 4-hydroxy-proline in the media. Previous studies have shown several other 4-hydroxy-proline-inducible genes. One of these genes, smc04388, has been annotated as a putative omega amino transaminase. The role of this omega transaminase in the main catabolic pathway of 4-hydroxy-proline has not been investigated. In order to address this, two mutant strains; a single smc04388 mutant and a double smc04388 hypD mutant were created. Growth curves of these mutants in minimal media showed that the Smc04388 protein is not required for the growth of S. meliloti in the presence of trans-4-hydroxy-L-proline as the sole carbon and nitrogen source. The Smc04388 protein was overexpressed as a Strep-tagged and purified from S. meliloti. The purified enzyme showed amino transaminase activity with pyruvate and α-methylbenzylamine. In addition, an enzymatic reaction using the product of the second enzyme of the 4-hydroxy-proline pathway, Δ1-pyrroline-4-hydroxy-2-carboxylate, was carried out to test the activity of Smc04388 with this compound. Mass spectrometry analysis of this reaction mixture revealed the formation of L-alanine from pyruvate and Δ1-pyrroline-4-hydroxy-2-carboxylate, suggesting the utilization of this compound as an amino donor by the Smc04388 transaminase. It was also shown that transamination activity in cell extracts increase in the absence of Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase, the enzyme that catalyzes the conversion of this compound in the 4-hydroxy-proline pathway. These results confirmed the hypothesis that the Smc04388 omega amino transaminase is involved in a secondary pathway related to the known catabolic pathway of 4-hydroxy-proline in bacteria. Further understanding of this secondary pathway will contribute to the study of the metabolism of 4-hydroxy-proline in bacteria. In addition to this, the complete characterization of the Smc04388 omega amino transaminase could have practical application in the pharmaceutical industry. / Thesis / Master of Science (MSc)

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