Plant cell walls determine cellular shape and provide structural support for the entire plant. Polysaccharides, comprising the major components of the wall, are actively remodelled throughout development. Xyloglucan endotransglucosylase (XET)/hydrolase (XTH, EC 2.4.1.207) cleaves xyloglucan (XyG), the donor substrate, and attaches a portion to another XyG chain, the acceptor substrate. Recently, a novel transglucosylase called mixed-linkage β-glucan (MLG) : XyG endotransglucosylase (MXE) was discovered in horsetails (Equisetum spp.) that could attach a portion of MLG to XyG, resulting in a hetero-polymer product. My aims were to further investigate the nature of this activity, biochemically characterize the enzyme, and explore its physiological role. MXE activity was attributable to an enzyme unlike Equisetum XTHs. MXE had a p1 of 4.1 (XTHs were 6.6-9), a pH optimum of 6.3 (XTHs preferred 5.5), and had higher activity using smaller oligosaccharide acceptor substrates like XXXGol (XTHs were more active using XLLGol). Importantly, the MXE protein was shown to utilize both MLG and XyG as donor substrates, and therefore have both MXE and XET activity. Also, the enzyme was capable of using various glucan oligosaccharides (O) as substrates, including MLGO, XyGO, and cello-O, but not laminari-O. By using a novel ex vivo approach, the proportion of extractable MXE product to XET product was found to increase in older tissues. Transglucosylase products were localized in sclerenchyma and structural parenchyma by in situ assays, implying a strenghening function for MXE. Surprisingly, another novel activity was discovered that could covalently attach cellulose to XyG, and termed cellulose : xyloglucan endotransglucosylase (CXE). This activity was attributed to the MXE enzyme, implying that the protein is a promiscuous endotransglucosylase. The presence of CXE in other plants has not yet been tested. Besides being a novel discovery in plant cell biology, the modification of cellulose has applications in a number of industries.

Research was conducted using algal biomass obtained from the surface of a secondary clarifier at Bridge City Wastewater Treatment Plant and subsequently sent through an electrochemical (EC) batch reactor at various concentrations. The first objective was to achieve maximum cell wall destruction electrochemically using the EC batch reactor and determine the optimal detention time and voltage/current relationship at which this occurred. The second objective was to subject two algal mediums to anaerobic digestion: the algal medium without electrochemical disinfection and the algal medium after disinfection. Every three days, for 12 days, total solids were measured from each apparatus to determine if cell destruction increased, decreased or did not change the consumption rate of algae by anaerobic bacteria. The consumption rate of algae is directly proportional to the production of methane, which can be used as a source of biofuel.

Aspergillus fumigatusis a ubiquitous environmental soil fungus. With recent development and advancement in medical treatments leading to immunosuppression, there has been an increase in incidence in aspergillosis. With the emergence of antifungal resistance isolates and the continued high mortality rate for invasive aspergillosis, the hunt for new antifungal drug targets is critical. Research on A. fumigatus is still in its infancy, partly due to the relatively recent rise of A. fumigatus as a clinically significant pathogen. The cell wall has been demonstrated to be critical for survival of this fungal organism, with interference of cell wall construction leading to cell death or reduced growth. This, coupled with the lack of shared mechanisms in humans, makes targeting cell wall synthesis for antifungal therapy a reasonable possibility. The cell wall of A. fumigatus shares a few similarities to S. cerevisiae. However, major differences exist, including the presence of β-1,3;1,4-glucan in the cell wall of A. fumigatus. In fact, the presence of β-1,3;1,4-glucan was never previously described in fungi before Latge's group reported it a number of years ago. It comprises about 10% of the glucans in the cell wall of A. fumigatus, but its role in the cell wall is unknown. In 2006 and 2009, two papers were published that demonstrated the role of CslF and CslH(Cellulose like synthases) in the production of β-1,3;1,4-glucan of the cell wall in rice and barley, respectively. Taking both protein sequences for these genes, we blasted it against the A. fumigatus database for any possible orthologues. A single orthologue, albeit with weak homology, was identified that named TFT1. We hypothesize that TFT1a plays a direct role in A. fumigatus β-1,3;1,4-glucan synthesis. Through Agrobacterium tumefaciens mediated transformation, an A. fumigatus strain lacking this enzyme (tft1Δ) was generated. From tft1Δ a revertant strain (revtft1) was created where the gene was reintroduced. Immunofluorescence staining with antibodies against β-1,3;1,4-glucan and biochemical quantification both demonstrated complete loss of β-1,3;1,4-glucan within the cell wall of the tft1Δ strain, with recovery detected in revtft1. This strongly suggests that this enzyme does indeed play a role in β-1,3;1,4-glucan synthesis in A. fumigatus. Growth experiments, spore size determination and an in vitro model of virulence also indicated that the loss of TFT1 leads to additional phenotypes. While the precise mechanism for β-1,3;1,4-glucan synthesis is unknown, the results shown herein indicate a pivotal role forTFT1 in its biosynthesis, and resulting phenotypes upon loss of mixed linkage glucan adds some clues to its role in the cell wall of A. fumigatus.

UDP-galactopyranose mutase (UGM) is a flavoenzyme which catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDPGalf). UDP-Galf is the active precursor of Galf residues. Glycoconjugates of Galf residues are found in the cell wall of bacteria and on the cell surface of higher eukaryotes. Galf residues have not been found in humans and the fact that they are essential for the growth of pathogenic bacteria makes UGM a potential antibacterial target. In the present study, crystal structures of UGM from Deinocococcus radiodurans (drUGM) in complex with substrate (UDP-Galp) were determined. UDP-Galp is buried in the active site and bound in a U-shaped conformation. The binding mode and active site interactions of UDP-Galp are consistent with the previous biochemical and mechanistic studies. The mobile loops in the substrate complex structures exist in a closed conformation and Arg198 on one of the mobile loops stabilizes the phosphate groups of the substrate. The anomeric carbon of galactose is 2.8 Å from the N5 of FAD (in the reduced complex) favorable to form FAD-galactosyl adduct. In addition to substrate complex structures, the crystal structures of drUGM in complex with UDP, UMP, and UDP-Glc have been determined. The mobile loops in all these complexes exist in a closed conformation. Inhibitors for UGM were identified by ligand-based and structure-based methods. The phosphonate analog of UDP-Galp (GCP) showed only weak inhibition against various bacterial UGMs. The structure of drUGM in complex with GCP provided a basis for its inhibitory activity. Poor stabilization of the phosphate groups by conserved arginines (Arg198 and Arg305) and altered sugar binding mode account for its activity. Novel indole-based (LQ1, LQ6 and LQ10) inhibitors of UGM were identified through structure-based virtual screening (SBVS) of a chemical library. Inhibition studies also allowed the identification of an active site aspartic acid that plays role in inhibitor binding. The structural studies on drUGM provided a basis for understanding substrate binding to UGM. In vitro enzyme inhibition studies allowed the identification of novel indole-based inhibitors. The structural and inhibition studies reported here enhance the understanding of UGM-ligand interactions and will assist in the development of more potent inhibitors of UGM.

he influence of two levels of yeast cell wall on phase one laying hen performance was investigated in this study. A total of 75 Lohmann W-36 replacement pullets, 17-weeks-old, were distributed among 75 laying hen cages (1 bird per pen). A total of 3 treatments were sequentially assigned to pens and each treatment had 25 replicates. Feeds were prepared according to the management guide for those birds and a single basal diet was divided into three treatments. First was the control basal diet only with no feed additives. The second was the basal diet supplemented with 250 ppm of yeast cell wall (YCW 250) and the third treatment was the basal diet supplemented with 500 ppm of yeast cell wall (YCW 500). Individual birds per cage served as the experimental unit for this study. Feed and water were offered ad libitum. Data were collected when birds were 21 weeks old and hen day egg production was > 90%. Treatment YCW 250 resulted in significantly higher egg weight than the control and YCW 500 treatment in the first and second production period and was higher than YCW 500 in the third and fourth production period. Feed consumed per dozen eggs was significantly lower in treatment YCW 500 versus treatment YCW 250, but not significantly lower than the control for the first production period and all treatments were not different from each other for the rest of the study. Average feed consumed per bird per day, and monthly cumulative egg production was not different between treatments. Period feed conversion ratios were lower for the second and fourth period (P = 0.15 and 0.18 respectively). There was no treatment effect on interior egg quality except for yolk color for the YCW 500 treatment in the fourth period which had significantly higher Roche color scores than the YCW 250 treatment. Specific gravity, egg shell thickness, egg shell weight, and percent shell weight were significantly higher in hens fed YCW 500 versus YCW 250. Egg shell breaking force was significantly higher in hens fed YCW 250 versus the control group. Overall, feeding a diet supplemented with yeast cell wall improved laying hen performance and 250 ppm YCW had the most significant influences, particularly with respect to increasing egg weight in early production laying hens.

UDP-galactopyranose mutase (UGM) is a flavoenzyme which catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDPGalf). UDP-Galf is the active precursor of Galf residues. Glycoconjugates of Galf residues are found in the cell wall of bacteria and on the cell surface of higher eukaryotes. Galf residues have not been found in humans and the fact that they are essential for the growth of pathogenic bacteria makes UGM a potential antibacterial target. In the present study, crystal structures of UGM from Deinocococcus radiodurans (drUGM) in complex with substrate (UDP-Galp) were determined. UDP-Galp is buried in the active site and bound in a U-shaped conformation. The binding mode and active site interactions of UDP-Galp are consistent with the previous biochemical and mechanistic studies. The mobile loops in the substrate complex structures exist in a closed conformation and Arg198 on one of the mobile loops stabilizes the phosphate groups of the substrate. The anomeric carbon of galactose is 2.8 Å from the N5 of FAD (in the reduced complex) favorable to form FAD-galactosyl adduct. In addition to substrate complex structures, the crystal structures of drUGM in complex with UDP, UMP, and UDP-Glc have been determined. The mobile loops in all these complexes exist in a closed conformation. Inhibitors for UGM were identified by ligand-based and structure-based methods. The phosphonate analog of UDP-Galp (GCP) showed only weak inhibition against various bacterial UGMs. The structure of drUGM in complex with GCP provided a basis for its inhibitory activity. Poor stabilization of the phosphate groups by conserved arginines (Arg198 and Arg305) and altered sugar binding mode account for its activity. Novel indole-based (LQ1, LQ6 and LQ10) inhibitors of UGM were identified through structure-based virtual screening (SBVS) of a chemical library. Inhibition studies also allowed the identification of an active site aspartic acid that plays role in inhibitor binding. The structural studies on drUGM provided a basis for understanding substrate binding to UGM. In vitro enzyme inhibition studies allowed the identification of novel indole-based inhibitors. The structural and inhibition studies reported here enhance the understanding of UGM-ligand interactions and will assist in the development of more potent inhibitors of UGM.

The cell wall is essential for fungal survival in natural environments. Galactofuranose (Galf) decorates certain carbohydrates and lipids of Aspergillus cell wall, is absent in humans and appears to play a role in fungal cell wall maturation. Previous studies in our lab showed that deletion of any of three sequential-acting genes (ugeA, ugmA, and ugtA) of Galf pathway caused substantially reduced growth and spore production. Two genes upstream of the Galf pathway, galD and galE are essential for galactose metabolism in many systems including the budding yeast, Saccharomyces cerevisiae. Interestingly, characterization of galD and galE in A. nidulans using cell and molecular techniques showed that unlike yeast, neither of these genes was essential for growth at physiological pH 7.5. Nevertheless for each case, their expressions were up-regulated by growth on galactose, revealing the relative complexity of galactose metabolism in A. nidulans. Our study also showed that repression of the three sequentially acting Galf pathway genes by conditional promoters phenocopied previously characterized deletion morphology. Using anti-Galf (L10) we also showed that deletion and repression of these genes caused no Galf in the hyphal wall. Gene deletion or repression also increased sensitivity to the wall-targeting drug, caspofungin. Related results from qPCR showed that deletion or repression of ugmA increased gene expression of α-glucan synthase agsB and decreased that of β-glucan synthase fksA. Therefore, Galf is non-essential but important for many aspects of Aspergillus growth, sporulation, and wall maturation. Aspergillosis, the most common airborne systemic fungal disease, is typically caused by Aspergillus fumigatus. Several A. fumigatus UgmA (AfUgmA) mutants with altered enzyme activity due to single amino acid changes were used to assess their effect on growth and wall composition in A. nidulans. Wild type AfugmA complemented the phenotypic defects in an A. nidulans ugmAΔ strain, consistent with these two genes being homologous. The AfUgmA crystal structure has been solved, and the in vitro enzymatic effects of specific mutations in the enzyme active site have been published. AfUgmA mutated strains with reduced activity in vitro impaired A. nidulans growth in a manner substantially similar to gene deletion and gene down-regulation. Site directed mutagenesis showed that AfUgmA residues R182 and R327 were critical for Galf generation both in vivo and in vitro. This supports previous results showing that UgmA is essential for Galf biosynthesis. Using fluorescent latex beads, we showed that reduction of wall Galf increased hyphal surface adhesion. Consistent with qPCR studies, immunofluorescence and ELISA results showed that loss or absence of Galf increased wall α-glucan but reduced wall β -glucan. Galf is important for wall surface integrity and for maintaining dynamic co-ordination with other pathways. To begin to assess this dynamic co-ordination, Tandem Affinity Purification (TAP) tagging combined with LC-MS/MS was used to identify the interacting partners of UgmA. Our results showed that UgmA interacted with proteins that are involved in cytoskeleton generation, osmotic adaptation, and cell signalling pathway. Further study will help us to understand the dynamic coordination of Galf biosynthesis pathway with other wall carbohydrate polymers for Aspergillus wall formation. In summary, my thesis results have clearly shown that Galf plays important roles in Aspergillus growth, and wall surface integrity. We also showed that Galf deficient strains are hypersensitive to wall-targeting drugs, indicating that Galf biosynthesis pathway could be potential target for combination therapy. The Galf pathway also maintained a dynamic co-ordination with alpha-glucan and beta-glucan carbohydrate pathways. Future study may include developing an inhibitor against UgmA and exploring the relationship of Galf pathway with alpha-glucan and beta-glucan carbohydrate pathways.

Upregulation of intracellular and secreted acid phosphatases (APases) is a universal response of orthophosphate-starved (-Pi) plants. APases hydrolize Pi from a broad spectrum of phosphomonoesters at an acidic pH. Plant APases belong to a relatively large multigene family whose specific functions in Pi metabolism are poorly understood. This study focuses on the identification and characterization of cell wall (CW) localized purple acid APases (PAPs) upregulated by -Pi Arabidopsis thaliana. Three glycosylated PAP isozymes secreted into the CW of -Pi Arabidopsis suspension cells were purified and identified by peptide mass fingerprinting using mass spectrometry (MALDI-TOF MS) and N-terminal microsequencing as AtPAP12 (At2g27190; subunit size 60-kDa), AtPAP25 (At4g36350; subunit size 55-kDa) and AtPAP26 (At5g34850; subunit size 55-kDa). Both AtPAP12 and AtPAP26 were previously shown to be upregulated and secreted by –Pi Arabidopsis to scavenge Pi from extracellular organic-P. However, the novel AtPAP25 has never been suggested to be involved in the plant Pi-starvation response. Biochemical characterization of AtPAP25 revealed a monomeric 55 kDa protein. Similar to other PAPs it was purple-in-solution and insensitive to tartrate. Glycoprofiling via LC MS/MS revealed highly complex NXS/T glycosylation motifs at Asn172, Asn367 and Asn424. I hypothesize that these motifs play a role in AtPAP25 targeting and function. Kinetic characterization revealed a broad pH optimum centered at 5.6 and inhibition of activity by several common APase inhibitors. AtPAP25 exhibited broad substrate selectivity, low Vmax, and a Km (phosphoenolpyruvate) value of 0.52 mM. Immunoblot and semi-quantitative RT-PCR transcript analysis indicated that AtPAP25 is exclusively synthesized under –Pi conditions. Deduced amino acid sequences were compared using multiple sequence alignment and phylogenetic analysis. Growth of atpap25 T-DNA insertion mutant knockout seedlings was completely arrested when transferred to a soluble Pi deficient organic-P containing soil mix, pointing to a potential regulatory function of AtPAP25 during nutritional Pi stress. Overall, this research is helping to shed light on the functional importance of specific PAP isozymes in facilitating plant acclimation to nutritional Pi deficiency. This is important because there is an urgent need to engineer Pi-efficient transgenic crops to minimize the huge input of expensive, non-renewable, and polluting Pi fertilizers in agriculture. / Thesis (Master, Biology) -- Queen's University, 2012-09-10 08:28:21.631

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23958号 / 農博第2507号 / 新制||農||1092(附属図書館) / 学位論文||R4||N5393(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 伊福 健太郎, 教授 梅澤 俊明, 准教授 小林 優 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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