<p>Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures<i> </i>and growing<i> </i>plants<i>. </i>The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.</p><p>A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.</p><p>Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.</p><p>Expression of the bacterial UGDH in <i>E. coli</i> resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. </p><p>Overexpression of UGD1, one of four <i>A. thaliana</i> UGDH genes, in <i>A. thaliana,</i> resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.</p>

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated. A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb. Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis. Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

Encapsulated Escherichia coli are responsible for a number of life threatening infections of man. These range from urinary tract infections to septicemia and neonatal meningitis. A common property of these E. coli strains is the expression of a polysaccharide capsule or K antigen. The expression of a capsule is an essential virulence factor protecting the bacterium from host defenses. Like many virulence factors capsule gene expression is regulated by temperature, such that at 37 0C inside the host the capsule is expressed whereas at 20 0C it is not. The project used the K5 capsule gene cluster as a model system to study in detail the regulation of capsule gene expression. Expression of E. coli K5 gene cluster is regulated at the transcriptional level by two convergent promoters PR1 and PR3. The temperature regulation-dependent expression is in part controlled at the level of transcription by complex regulatory network involving the regulators SlyA, H-NS and IHF acting at PR1 and PR3. A large 5’ untranslated region (5’ UTR) is involved in transcriptional regulation by interacting with global regulator proteins. In this study, a combination of lacZ reporter gene fusions, 5’ RACE analysis and site-direct mutagenesis at promoter functional elements were used to investigate the promoter. These studies identified that the PR1 promoter was more complex than initially thought and contains, in addition to previously characterized PR1-1 promoter at +1, three additional tandem promoters PR1-2, PR1-3 and PR1-4 transcribing in the same direction from the site +133, +142 and +182, respectively. In order to analyse the contribution for the transcription from PR1 among these multiple promoters, these multiple tandem promoters’ activities were measured by β-galactosidase assay and Real-time quantitative reverse PCR assay. We determined that PR1-2 and PR1-3 are two cryptic promoters with very low transcription activity while PR1-1 and PR1-4 are the major promoters that contributed evenly to the total transcripts into kps operon in the mid-exponential phase. Furthermore, we demonstrated that the promoter PR1-1 and PR1-4 are tightly coupled and the activity of PR1-4 can be co-ordinately reduced by disrupted PR1-1.Different minimal PR1-lacZ promoter fusions were also transformed into strains with mutations in the genes that encode these regulatory proteins (IHF, SlyA and H-NS) and the transcription activity was examined by β-galactosidase assay at both 37 0C and 20 0C. IHF is required indirectly for maximum transcription at PR1-1 promoter but directly represses transcription from PR1-4 due to binding at +160 region at 37 0C. Global regulator H-NS represses the transcription at both 37 0C and 20 0C at PR1 and plays an important role for transcriptional temperature regulation at PR1 region. The anti-repressor SlyA activates transcription at PR1-1 at 37 0C. This study identified for the first time growth phase dependent expression from the PR1 promoter. Also, this study discovered different temporal patterns of promoter PR1-1 and PR1-4 transcription was coordinated with bacterial growth cycle. Overall this study will be helpful to decipher the complex regulation of capsule gene expression in E. coli.