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No description available.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science. / ENGLISH ABSTRACT: Undeniably, changes in the environment and dwindling traditional energy resources have resulted in the search for viable, renewable energy alternatives such as biofuels. Cellulose is one of the most abundant polymers on earth and can be converted to simple sugars and fermented to ethanol biofuel fairly easily. Cellulose rich biomass that can serve to supply ethanol biofuel production can be sourced from unexploited agricultural waste. The main drawback to using vegetative tissue as opposed to harvested food stocks from crops results from the structural properties of plant cell walls. Although cellulose is abundant, the contaminating hemicellulose and lignin fibres within the cell wall matrix have a negative impact on the digestibility of the cellulose present. Thus, an important step in creating an effective biofuel production system from agricultural excess is developing crops with improved cell wall polymer characteristics that can be converted to ethanol more efficiently. This project consisted of two parts. Firstly, the aim was to assess lignin production in transgenic sugarcane transformed with a construct aimed at down-regulating the 4- (hydroxyl) cinnamoyl CoA ligase (4CL) gene in the lignin biosynthesis pathway. The second part of the project revolved around discovering the mechanism of impared cell growth caused by expressing the gene encoding cellulose synthase from a marine invertebrate, Ciona savignyi, in the yeast Saccharomyces cerevisiae. Several sugarcane lines that had been previously transformed with a hairpin RNAi construct aimed at down-regulating the 4CL gene in the monolignol biosynthesis pathway were subjected to analysis to determine if lignification had been reduced. Although the presence of the hairpin construct in the genomic DNA had been confirmed for all of the transgenic lines, there was no significant decrease in the lignin levels in any of the transgenic lines. PCR analysis of the mRNA and enzyme assays also confirmed that the 4CL gene was still being expressed. Ongoing work will determine the cause of the unsuccessful down-regulation. Previously, it had been proven that the cellulose synthase gene from C. savignyi could be functionally expressed in S. cerevisiae. However, cellulose production resulted in extremely retarded growth of colonies and cultures, to the point of the apparent death of the cultures. The aim of this part of the project was to determine the mechanism (either metabolic or physical) that causes this effect. To generate enough cell mass to perform metabolic analysis, several strategies to impede cellulose production in transgenic yeast were explored. Attempts to stop cellulose production and induce better growth by introducing Isoxaben (a traditional weed killer that targets cellulose synthases) into the growth medium used for the transgenic yeast proved unsuccessful. To control the expression of the transgene, it was attempted to clone the cellulose synthase gene into an expression system containing an inducible promoter. The cloning exercise proved extremely difficult and multiple attempts with several strategies proved unsuccessful. This process is still ongoing as the growth retarding process induced by cellulose production in yeast remains to be identified. / AFRIKAAANSE OPSOMMING: Dit is onontkenbaar dat veranderinge in die omgewing en minderwordende tradisionele energiebronne veroorsaak dat lewensvatbare en hernubare energiebronne soos biobrandstof gevind moet word. Sellulose is een van die mees volop polimere op aarde en kan redelik maklik omgeskakel word na eenvoudige suikers en gefermenteer word tot etanol-biobrandstof. Sellulose-ryk biomassa wat etanol-biobrandstof kan verskaf, kan herwin word van tot op hede ongebruikte landbou-afval. Die komplekse struktuur van plantselwande is die hoofstruikelblok in die omskakeling van vegetatiewe weefsel tot biobrandstof. Hoewel sellulose volop is, het die kontaminerende hemisellulose- en lignienvesels binne die selwand-matriks ’n negatiewe impak op die verteerbaarheid van die sellulose teenwoordig in die selwand. Daarom is ’n belangrike stap in die ontwikkeling van effektiewe biobrandstof-produksiesisteme vanaf landbou-afval om gewasse te ontwikkel met verbeterde selwandpolimeer-eienskappe wat etanol-produksie kan vergemakilik. Hierdie projek het bestaan uit twee dele. Eerstens was die doel om vas te stel of die lignienproduksie geaffekteer is in transgeniese suikerriet getransformeer met ’n konstruk wat mik om die 4-(hidroksie)-cinnamoyl CoA ligase (4CL) geen te af-reguleer in die lignienbiosintese- padweg. Die tweede deel van die projek het daarop gefokus om die meganisme te ondek wat die belemmerde selgroei veroorsaak, as gevolg van die uitdrukking van die geen wat kodeer vir sellulose-sintase in ’n mariene ongewerwelde, Ciona savignyi, in Saccharomyces cerevisiae. Verskeie suikerriet-lyne, wat voorheen getransformeer is met ’n haarnaald-RNAi-konstruk om die 4CL-geen te af-reguleer in die monolignol-biosintese-padweg, is onderwerp aan analise om vas te stel of lignifikasie verminder is. Hoewel die teenwoordigheid van die haarnaald-konstruk in die genomiese DNA bevestig is vir al die transgeniese lyne, was daar geen beduidende vermindering in die lignienvlakke in die transgeniese lyne nie. PKRanalise van die mRNA en ensiem-aktiwiteitstoetse het ook bevestig dat die 4CL-geen steeds uitgedruk word. Verdere ondersoek sal kan vasstel wat die oorsaak van die onsuksesvolle af-regulering is. Voorheen is bewys dat die sellulose-sintase-geen van C. savignyi funksioneel uitgedruk kon word in Saccharomyces cerevisiae. Egter, selluloseproduksie het die gevolg gehad dat groei in die transgeniese kolonies en kulture erg gestrem is, tot die punt dat die kulture dood voorgekom het. Die doel van hierdie deel van die projek was om vas te stel wat die meganisme (òf metabolies òf fisies) is wat hierdie verskynsel veroorsaak het. Om genoeg selmassa te genereer om metaboliese analise uit te voer, is verskeie strategieë om selluloseproduksie in transgeniese gis te verhinder, ondersoek. Pogings om selluloseproduksie te stop en om groei te verbeter deur Isoxaben by te voeg in die groeimedium gebruik vir transgeniese gis, was onsuksesvol. Isoxaben is ’n tradisionele onkruiddoder wat sellulose-sintases teiken en inhibeer. Om die uitdrukking van die transgeen te beheer, is ’n poging aangewend om dié sellulose-sintase-geen in ’n uitdrukking-sisteem te kloon met ’n induseerbare promotor. Die kloneringsoefening was uiters moeilik en veelvoudige pogings met verskeie strategieë was onsuksesvol. Hierdie proses moet verder gevoer word aangesien die groeistremmingsmeganisme veroorsaak deur selluloseproduksie in gis nog geïdentifiseer moet word.

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The peptidoglycan layer of Vitreoscilla stercoraria, ATCC 15218, was isolated from intact cells after treatment with sodium lauryl sulfate (SLS) and digestion with Pronase. Amino acid and amino sugar content was analyzed and 67% of the total present was made up of glutamic acid, alanine, diaminopimelic acid (DAP), and glucosamine in a molar ratio of 1:1.7:1:0.7. Electron microscopy of the final peptidoglycan product showed a thin, delicately folded sacculus which exhibited a morphology different from that of the intact vegetative cells. Within these sacculi occurred electron-dense structures which were assayed and found to be poly- 3-hydroxybutyrate (PHB) granules. The final yield of peptidoglycan was 2.9% of the dry weight of the intact vegetative cell.

Cell walls of Bacillus subtilis macrofibers have been biochemically analyzed to determine the contribution of various surface polymers in the twist regulation. Helix hand inversion was induced by a variation in either the growth temperature or the nutritional composition of the culture medium. Initial experiments had demonstrated a fivefold difference in the sensitivity of right- and left-handed forms to muramidases indicating modifications of peptidoglycan as a possible mechanism underlaying inversion. An examination of lysozyme susceptibility of purified cell walls and whole cells derived from the two structural forms, however, exhibited no significant difference suggesting loss of the relevant component(s), perhaps biomechanical in nature, during disintegration of macrofibers. The effect of various twist modulators such as trypsin, ammonium sulfate and D-alanine on the development of helical twist in both switchable and "fixed" mutants were studied. The interaction matrices have established D-alanine as the most potent of right-factors. Intestinal alkaline phosphatase is reported as a newly discovered antagonist to the development of leftward twist. Heat inactivation and protein purification experiments strongly indicated that twist modulation was due to the phosphatase activity rather than minor protease contaminants. The chemical composition of cell walls purified from right- and left-handed structures was determined. No twist correlated differences in the overall content of peptidoglycan, teichoic acid and teichuronic acid were detected. Evidence is presented for the absence of correlation between the extent of ester-linked alanine substitution and twist state. These findings suggest that gross changes in wall composition is perhaps not the mechanism for hand inversion. From the profiles of the wall associated proteins, a 200 Kdal band has been identified whose presence is strongly correlated with the development of leftward twist. This polypeptide was found to be highly sensitive to trypsin; a property it shares with a previously proposed left-twist protein. Preliminary evidence for isolation of left-hand specific polyclonal antibodies is also presented. FJ7, a switchable mutant, was successfully transformed with a plasmid containing the Streptococcus transposon Tn917. A small bank of insertional mutants has been constructed for the isolation of mutants impaired in helix hand inversion.

This thesis studies two particular bacterial outer membrane proteins called OmpC and Wzi, focusing on their expression, purification, crystallization and X-ray structure determination. A series of four naturally occurring OmpC mutants were isolated from a single patient with an E. coli infection of liver cysts. The isolated E. coli strains progressively exhibited increasing breadth of antibiotic resistance in which OmpC was predicted to take a partial role. We carried out an assay in which a strain of E. coli lacking OmpC was used to express the first (antibiotic sensitive) and the last (antibiotic resistant) of the clinical OmpC mutants and drug permeation assessed. Single channel conductance measurements were carried out and the X-ray structures for all the isolates were determined. Protein stability was assessed. With these data we propose that changes in the transverse electric field, not the pore size, underlie the clinically observed resistance to the antibiotics. This is the first demonstration of this strategy for antibiotic resistance. Wzi is a novel outer membrane protein involved in the biosynthesis and translocation mechanism of the K30 antigen from E. coli. The mechanism is a complicated process that requires several proteins including outer and inner membrane proteins. The protein Wzi was expressed, purified and crystallized. Initial crystals were tested and diffracted to 15Å. After optimization, a crystal diffracting to 2.4Å has been obtained.

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Integral membrane enzymes contribute a unique repertoire to the cell, as they are capable of synthesizing products from substrates of different chemical character at the membrane-water interface. Membrane-embedded enzymes are often responsible for the synthesis of important components of the cellular membrane and contribute to the structural integrity of the cell, maintenance of cellular homeostasis and signal transduction. One of the main focuses of Dr. Filippo Mancia’s laboratory is understanding how enzymes complete these functions by investigating, at an atomic level, the determinants of substrate binding and catalysis within the membrane and at the membrane surface. Here I will present my investigation of two such integral membrane enzyme systems, which are responsible for the synthesis and processing of membrane-embedded molecules in bacteria. Phosphatidylinositol-phosphate Synthase (PIPS) Phosphaitylinositol (PI) is an essential lipid component in mycobacteria, demonstrated by loss of viability when PI is reduced to 50% of wild-type levels. Phosphatidylinositol (PI) is required for the biosynthesis of key components of the cell wall, such as the glycolipids phosphatidylinositol-mannosides, lipomannan and lipoarabinomannan. For these molecules, PI serves as a common lipid anchor to the membrane. In Mycobacterium tuberculosis, the disease causing pathogen of tuberculosis, these glycolipids function as important virulence factors and modulators of the host immune response. Therefore, the enzyme responsible for PI synthesis in this organism is a potential target for the development of anti-tuberculosis drugs. The defining step in phosphatidylinositol biosynthesis is catalyzed by a member of the CDP-alcohol phosphotransferase enzyme family. The enzyme uses CDP-diacylglycerol as the donor substrate, and either inositol in eukaryotes or inositol-phosphate in prokaryotes as the acceptor alcohol of the synthesis reaction. In prokaryotes, phosphatidylinositol-phosphate synthase (PIPS; a member of the CDP-alcohol phosphotransferase family) catalyzes this reaction to yield phosphatidylinositol-phosphate, which is then dephosphorylated to PI by an uncharacterized enzyme. Structures of PIPS from Renibacterium salmoninarum (RsPIPS), with and without bound CDP-diacylglycerol, have revealed the location of the acceptor site as well as molecular determinants of substrate specificity and catalysis of the enzyme. However, RsPIPS has low activity relative to PIPS from Mycobacterium tuberculosis (MtPIPS) and the two share only 40% protein sequence identity. Therefore, these initial structures have limited potential for meaningful homology modeling and drug design. Presented here are the structures of PIPS from Mycobacterium kansasii (MkPIPS), which is 86% identical to MtPIPS, in an apo state to 3.1 Å resolution, in a nucleotide-bound state to 3.5 Å resolution, and in a novel ligand-bound state to 2.6 Å resolution. This work provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens. RodA-PBP2 Complex The cell wall of most gram-negative and gram-positive bacteria (excluding atypical bacteria such as members of Mycoplasmataceae) is composed of peptidoglycan, a mesh of repeating carbohydrates (N-acetylmuramic acid, MurNAc, and N-acetylglucosamine, GlcNAc) cross-linked by small peptides. Peptidoglycan is essential for growth, division and viability of the organism. Any disruption of the biosynthesis of peptidoglycan, whether by genetic mutation, inhibition with antibiotics or degradation by lysozyme, results in bacterial cell lysis. Peptidoglycan helps maintain cell shape and serves as an anchor for accessory proteins and other cell wall components. As essential components of the cell wall, enzymes contributing to the peptidoglycan biosynthetic pathway can be exploited as antibiotic targets. After a hydrophilic peptidoglycan precursor (UDP-MurNAc-pentapeptide) is synthesized in the cytosol, it is attached to the lipid carrier undecaprenyl phosphate (UndP). The lipid-linked precursor (undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide or Lipid I) is modified further to undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (Lipid II) by addition of a GlcNAc moiety. Lipid II is then flipped across the membrane to the periplasm where its sugars are polymerized to form the glycan strands of the peptidoglycan mesh. SEDS proteins, essential for maintaining bacterial processes that determine shape, elongation, cell division and sporulation, are integral membrane enzyme that have been implicated in this process as either Lipid II flippases, glycosyltransferases responsible for sugar polymerization, or both. SEDS proteins are also known to form a functional complex with type b penicillin-binding proteins (PBPs), which are known as transpeptidase enzymes, responsible for the crosslinking of peptides in the formation of the peptidoglycan mesh. Though structures of both RodA (a SEDS protein involved in bacterial growth and elongation) and type b PBPs are available, the interaction between the two proteins and their joint enzymatic activity is poorly characterized. Here, I present the preliminary structural characterization of a RodA-PBP2 protein complex by single-particle cryo-electron microscopy (cryo-EM). We hope this ongoing work will contribute to the understanding of these enzymes and to the development of antibiotics to combat antibiotic resistance.