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    Understanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Wang, Yajie
    Alginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates.
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    Lipid biosynthesis in isolated barley protoplasts : a thesis ... for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1983) Bell, Louise Cynthia
    In most studies of fatty acid and lipid synthesis in plants there has been poor incorporation of radioactive label from acetate into linoleic (18:2) and linolenic (18:3) acids. Consequently the amounts of these fatty acids found in the galactolipids in such studies are much less than their observed endogenous levels. In the present study incorporation of H 14 CO 3 - and (1 14 C) acetate into lipids of barley protoplasts was examined. CO 2 -dependent O 2 evolution rates of the protoplasts were around 180 µmol O 2 /h/mg Chl and intactness was also ascertained by phase contrast microscopy. Incubating proto- plasts with 1mM H 14 CO 3 - or 50 µM (1- 14 C) acetate resulted in 146.2 and 17 nmol/mg Chl being incorporated into lipids respectively after 1 hour. A concentration of 10 mM was optimal for HCO 3 - incorporation and up to 580 nmol/mg Chl was incorporated into lipids at the end of 1 hour. Mg ++i ions used at 2 mM had little effect on HCO 3 - incorporation while PP i appeared to be slightly inhibitory. Acetate assimilation and its incorporation into lipids was markedly affected by pH and pH 5.8 was chosen for the assay medium. In 20 hour incubations 162 nmol acetate/mg Chl was incorporated. About 33% of label from acetate was found in each of palmitic (16:0) and oleic (18:1) acids with less than 9% in each of stearic (18:0), linoleic and linolenic acids. There was little or no incorporation of acetate into DGDG and less than 10% into each of PG, MGDG, PE and U (unknown lipid). Incorporation into PC after 2 1/2 hours was 36.8% then decreased to 8.9%. Acetate incorporation was most significant into U SF (another unknown lipid), being 73.4%. Although acetate was incorporated into a range of glycerolipids, incorporation into constituent 18:2 and 18:3 of these lipids was not significant.
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    Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Chettri, Pranav
    Dothistromin is a virulence factor produced by the fungal pine needle pathogen Dothistroma septosporum. It is similar in structure to a precursor of aflatoxin and sterigmatocystin. Unlike most secondary metabolite genes in fungi, the genes for dothistromin biosynthesis are not clustered but spread over six loci on one chromosome. Another characteristic feature of dothistromin synthesis is that dothistromin is produced mainly during the early exponential growth phase in culture. These unusual features have been proposed to be adaptations for the biological role of dothistromin in the disease process. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of aflatoxin and sterigmatocystin in Aspergillus spp. and to address the question of whether genes in a fragmented cluster can be co-regulated. The availability of the D. septosporum genome facilitated identification of orthologs of the aflatoxin pathway regulatory genes aflR, aflJ and the global regulatory genes veA and laeA. These genes were functionally characterised by knockout and complementation assays and the effects of these mutations on the expression of dothistromin genes and the production of dothistromin were assessed. Inactivation of the DsAflR gene (?DsAflR) resulted in a 104 fold reduction in dothistromin production, but some dothistromin was still made. This contrasted with ?AflR mutants in Aspergillus species that produced no aflatoxin. Expression patterns in ?DsAflR mutants helped to predict the complete set of genes involved in dothistromin biosynthesis. AflJ was proposed to act as a transcriptional co-activator of AflR in Aspergillus spp. Disruption of DsAflJ resulted in a significant decrease in dothistromin production and dothistromin gene expression. Interestingly the expression of DsAflR was not affected by deleting DsAflJ, while conversely DsAflJ transcript levels increased significantly in a DsAflR mutant compared to the wild type. Heterologous complementation with A. parasiticus, A. nidulans and C. fulvum AflJ failed to revert the dothistromin level to wild type suggesting species-specific function of AflJ. VeA is an important regulator of secondary metabolism and development in fungi. Inactivation of the D. septosporum ortholog (DsVeA) resulted in reduced dothistromin production and showed the influence of DsVeA on the expression of other secondary metabolite backbone genes. Asexual sporulation was reduced but mutants were not compromised in pathogenicity. Overall, D. septosporum DsVeA showed functional conservation of the usual role in fungi. LaeA is a global regulator of secondary metabolism and morphogenetic development, first identified in Aspergillus nidulans. Unexpectedly, DsLaeA exhibited an unusual repressive function on the dothistromin pathway and DsLaeA mutants exhibited an extended period of dothistromin production compare to WT in vitro. The mutation of DsLaeA showed varied responses in expression of other secondary metabolite genes and had differences in sporulation and hydrophobicity compared to the wild type.
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    Biosynthesis and metabolism of plant glycosides : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
    (Massey University, 1968) Tapper, Brian Anthony
    The biosynthesis of selected cyanogenic glucosides and glucosinolates was examined in higher plants. Linen flax seedling shoots (linum usitatissimum L.) were used exclusively to study linamarin biosynthesis while prunasin biosynthesis was studies in both peach shoots (Prunus persica Batsch) and cherry laurel shoots (P. laurocerasus L.). Isoprppylglucosinolate and benzylglucosinolate were studied in scurvy grass seedlings (Cochlearia officinalis L.) and garden cress seedling shoots (Lepidum sativum L.) respectively. Altogether 9 isotopically labelled compounds were prepared as part of the study and 14 were administered to plant tissue. The quantity of cyanogenic glucosides was determined by measuring hydrogen cyanide following enzymic hydrolysis. The specific activity or dilution of the labelled compound after incorporation into the glucosides was determined and sued to judge the effectiveness of the administered compound as a precursor of the glucosides. Benzaldehyde from prunasin was measured as its semicarbazone and the isothicyanates. Obtained by enzyme hydrolysis of the glucosinolates, were identified by conversion to thiourea derivatives. Paper and thin layer chromatography and electrophoresis were used to separate non-volatile radioactive compounds. Isobutyraldoxime-U-14C, 2-oximinoisovaleric acid-U-14C, isobutyronitrile-1-14C and 2-hydroxyisobutyronitrile-1-14C were all incorporated into linamarin to extents comparable to that from L-valine-U-14c. By the use of 15N labelled compounds the C-N bond of isobutyraldoxime and 2-oximinoisovaleric acid was shown to remain intact during the cornversjon to linarnarin. Isobutyramide-1-14C and hydrogen cyanide-14c were not significantly incorporated into linamarin. Phenylacetaldoxime-U-14C, 2-oximino-3-phenylpropionic acid-2-14C and phenylacetonitrile-1-14c were converted to prunasin to greater extents than was L-phenylalanine-U-14C. Radioactivity from D,L-mandelonitrile-1-14C and, to a lesser extent, from hydrogen cyanide-14-C was also incorporated into the nitrile moiety of prunasin. Phenylacethydroxamic acid-1-14C was not significantly converted to prunasin. Linen 'lax seedling shoots were examined for both volatile and non-volatile intermediates. Radioactive precursors of linamarin were administered in the presence of other suspected intermediates or inhibitors of linamarin biosynthesis. Both isobutyraldoxime and isobutyronitrile were shown to be formed in the shoots from L-valine-U-14C. A non-volatile compound which accumulated in the presence of a few inhibitors Of linamaran biosynthesis was studied in detail. Treatment with acid under mild conditions yielded isobutyraldehyde while emulsin gave isobutyraldoxime. It was resistant to linamarase. Isobutyronitrile was a product of pyrolysis. The proposed structure for this compound is isobutyraldoxime-0-glucoside. Isobutyraldoxime-U-14C and phenylacetaldoxime-U-14C were both better precursors of the corresponding glucosinolates than were L-valine-U-14C or L-phenylalanine-U-14C. 2-Oximinoisovaleric acid-U-14C was not significantly incorporated into isopropylglucosinolate. It is concluded that aldoximes are intermediates in the biosynthesis of both cyanogenic glycosides and glucosinolates. Other intermediates proposed in cyanogenic glycoside biosynthesis are nitriles and 2-hydroxynitriles in that order, N-Hydroxyamino acids may be intermediates between amino acids and aldoximes. 2-Oximino acids may also be intermediates in cyanoglycoside biosynthesis although it is possible that the observed incorporation was by way of prior non-enzymic conversion to nitriles. The experiments with labelled administered compounds have outlined a pathway of cyanogenic glycoside biosynthesis which may now be profitably studied for confirmation at the enzymic level.
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    The biosynthesis of dothistromin : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy
    (Massey University, 1975) Shaw, G John
    This thesis is concerned with the biosynthesis of dothistromin (2,3,3a,12a tetrahydro-2,3a,4,6,9 pentahydroxy-anthra[2,3-b]furo[3,2-d]furan-5,10 dione) by the fungus Dothistroma pini. The biosynthesis of related secondary metabolites is reviewed and as a working hypothesis it is proposed that dothistromin is solely acetate-derived. In the preliminary phases of the investigation strains of the organism giving high yields of the metabolite were sought and isolated from natural sources. Some growth media were tested for their ability to support growth, promote sporogenesis and sustain high yields of dothistromin. A medium containing malt and dried whole yeast was chosen. The growth characteristics of the organism in this medium were studied and the temporal relationship between growth and pigment production for a variety of cultural conditions was found. The findings of these experiments suggested times when it would be favourable to add possible precursors. Incorporation studies with [1-14C]-sodium acetate revealed that dothistromin incorporated isotope from this precursor and disclosed/that the lipids heavily incorporated the label. Subsequent experiments were concerned with examining the effects that precursor concentration, time of precursor addition and time of metabolite harvesting had on the isotope enrichment and yield of dothistromin. It was found that the optimisation of these two parameters were mutually exclusive processes and compromise conditions which were compatible with obtaining both reasonably good enrichments and yields of dothistromin had to be selected. Initially attempts were made to determine the distribution of isotope in dothistromin, which had incorporated isotopically labelled acetate, by chemical degradation. Potassium tertiary-butoxide/water cleavage of the anthraquinone ring of the pentamethyl derivative of dothistromin labelled by [1-14C]-acetate yielded 1,4-dimethoxybenzene which had a molar specific radioactivity that was 0.33 times that of the starting material. This finding was consistent with the formation of dothistromin from nine molecules of acetate. Subsequently 13C-NMR techniques were used to determine the distribution of isotope in dothistromin derived from [1-13C] and [2-13C]-acetates. Pulsed Fourier transform 13C-NMR spectra of the monoethyl acetal derivative of dothistromin were obtained using broad-band proton decoupling and off-resonance proton decoupling. By comparison with the 13C-NMR spectra of a number of model compounds the resonances in the spectrum of the dothistromin derivative were assigned in most cases to specific carbon atoms and in a few instances to two or three alternatives. The 13C-NMR spectra of the dothistromin derivatives which had been enriched by isotope from the carbon-13 labelled acetates showed nine resonances with intensities enhanced by enrichment from the carboxyl carbon of acetate, eight from the methyl carbon and one resonance of uncertain origin. The distribution of isotope in the anthraquinone moiety of the molecule was consistent with its formation from a polyketide precursor but this was not proven because of the equivocal assignment of some of the NMR signals. The distribution of isotope in the furo-furan ring moiety and its relation to the distribution in the anthraquinone part was the same as that reported by others for corresponding structures in aflatoxin B1, and sterigmatocystin.
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    Metabolism and translocation of linamarin in cassava (Manihot Esculenta Crantz) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, New Zealand
    (Massey University, 1977) Bediako, Badu Michael Kofi
    The metabolism of linamarin in cassava (Manihot esculenta Crantz) has been investigated. Information on the biosynthetic pathway, synthetic sites, translocation and turnover of the cyanoglucoside has been obtained by precursor administrations to various parts of cassava plants grown under partially controlled conditions in the glasshouse. Three volatile 14C-labelled precursors of linamarin isobutyronitrile, isobutyraldoxime and 2-hydroxyisobutyronitrile were prepared, purified and administered to cassava leaves by a new technique in which the leaves were allowed to take up precursor vapour in an enclosed glass chamber. The incorporation of these precursors, and of L-valine administered by solution uptake, was consistent with a pattern of linamarin biosynthesis in cassava involving the reaction sequence through valine, isobutyraldoxime, isobutyronitrile and 2-hydroxyisobutyronitrile established for other plants. The solution administration of L-[U- 14C] valine to various organs of the plant indicated that the leaves and the shoot apex synthesised linamarin more efficiently than the woody stem and the roots and tubers. More detailed investigations of leaf biosynthesis showed much higher incorporation of 14C-valine into linamarin by the petioles and midribs (45-62% 14C incorporation by petioles and 20% by midribs) than the leaf blades (2%). There was no direct relationship between endogenous linamarin content (which was higher in the blades than the petioles) and the apparent ability to synthesise linamarin from exogenous valine. However, the low ability of the blade tissue to incorporate valine into linamarin could be due to more active competing pathways removing the exogenously administered valine. In further investigations with tuber peels and the edible cores, similar competing pathways have been implicated for an apparently low biosynthetic efficiency of linamarin. The translocation of linamarin was demonstrated by specifically labelling 14C-linamarin in attached leaves with 2-hydroxy[1-14C] isobutyronitrile vapour and following the change in labelled linamarin content in the leaf and the distribution of linamarin to other parts of the plant. In both non-tuberous and tuberous plants there was a rapid loss of 14C-linamarin due to translocation from the fully expanded leaves up to 69 hours after synthesis. However a residual component of the 14C-linamarin (25-37% of that initially synthesised) remained in the leaves. A compartmentation of synthesised linamarin in cassava leaf tissues into a readily mobile and partially immobile fraction would account for these observations. In senescing leaves a continuous loss of both 14C-labelled and endogenous linamarin occurred leaving almost no residual component although this was attributed to both translocation and turnover. Translocated linamarin was distributed to all parts of the plant but the general pattern of translocate flow differed between non-tuberous and tuberous plants. An apical direction of linamarin distribution existed in the non-tuberous plants while tuber-directed linamarin translocation prevailed in the tuberous plants. Leaf senescence apparently enhances linamarin translocation to the tubers. There was little turnover of freshly synthesised 14C-linamarin in detached leaves and tuber tissues over a period of 1 to 3 days. However the low recoveries of 14C-linamarin in the whole plant translocation experiments suggest that active turnover may be occurring during translocation or in certain sink tissues.
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    Polymerisation and export of alginate in Pseudomanas aeruginosa : functional assignment and catalytic mechanism of Alg8/44 : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Microbiology
    (Massey University, 2007) Remminghorst, Uwe
    Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.