Methionine mutants of Neurospora crassa were isolated by a filtration concentration technique following ultra-violet irradiation. These and other mutants were classified by the physiological tests of precursor utilisation and heterocaryon complementation. Precursor utilisation data are in agreement with the present outline of methionine synthesis from cysteine but provide little information on the pathway of sulphate reduction and incorporation of sulphur within a carbon skeleton to yield cysteine. Seven cysteine and eight methionine loci are represented on the basis of heterocaryon complementation tests. Four of the cysteine loci and either one or two of the methionine loci, associated with the methylation of homocysteine, have not been reported previously. Two of the eight methionine loci had already been located but five of the remainder were associated with definite chromosome regions. No evidence for the tight clustering of either these or the cysteine loci was obtained. Inter-allelic complementation has been observed at two loci and the fine structure of one of these loci (me-2) has been studied in some detail by analysis of prototroph formation in inter-allelic crosses. The use of "outside markers" has facilitated the demonstration of intra-genic recombination and "conversion". Furthermore, these studies provide evidence for a linear arrangement of mutable sites within the locus with additive prototroph frequencies to yield a site map which can be correlated with a complementation map. The results also show that some alleles are more prone to "conversion" than others.

No description available.

The oxidative pentose phosphate (OPP) cycle is the main route of carbohydrate dissimilation in cyanobacteria in the dark. In heterocysts, the OPP is a supplier of reductant to nitrogenase, thus playing an important role in nitrogen fixation. Molecular genetic analysis of glucose-6-phosphate dehydrogenase (G6PDH) and the other components of the opp cycle have been focused on recently in an effort to understand the function and regulation of the cycle in vegetative cells and in heterocysts of cyanobacteria. This study presents data on nucleotide and amino acid sequences of three enzymes of the OPP cycle (glucose-6-phosphate dehydrogenase, transaldolase and fructose-l ,6-bisphosphatase) of the filamentous, heterocystous strain Anabaena sp. PCC7120. Mutagenesis studies on the transaldolase gene (tal) of Anabaena sp. PCC7120 and the opcA gene of the unicellular strain Synechococcus sp. PCC7942 are also reported. A 4,169 bp region around the zwf gene of Anabaena sp. PCC7120 was sequenced. Three genes are located in the region: fop, tal and zwf, which encode fructose-l,6-bisphosphatase (FBPase), transaldolase and glucose-6-phosphate dehydrogenase (G6PDH), respectively. The fop gene encodes a polypeptide of 349 amino acids. The product of the tal gene consists of 381 amino acids. The zwJ gene encodes a protein of 509 amino acids. Four cysteine residues are present in the enzyme. Two of these cysteines (Cys-187 and Cys-445) are absolutely conserved in cyanobacterial G6PDH. This result reinforces the likelihood of their role in the regulation of enzyme activity. Subclones carrying a range of zwf fragments of Anabaena sp. PCC7120 did not complement the zwJmutant Escherichia coli strain DF214. Western blot analysis showed that Anabaena sp. PCC7120 G6PDH did not express in E. coli DF214. Attempts aimed at production of a deletion/insertion zwf mutant of Anabaena sp. PCC7120 were not successful. The tal gene of Anabaena sp. PCC7120 was mutated to investigate growth and survival of the mutant cells in the presence and absence of combined nitrogen. Transaldolase activity in the tal mutant cells was similar to that in the wild-type cells. The growth rates of the tal mutant cells were not significantly different from that of the wild-type cells. These results imply that more than one copy of the tal gene is present in the Anabaena sp. PCC7120 genome. A gene, designated opcA, downstream from the zwf gene of Synechococcus sp. PCC7942 was mutated to investigate the possible effect of the gene on G6PDH activity. G6PDH activity in the mutant cells was reduced by 98.6%. Western blot analysis showed that the zwf was expressed in the opcA mutant cells, but the active form of the enzyme was a considerable reduced. All the results suggest that the opcA gene affects the assembly of G6PDH or enzyme activity, but possibly not expression of the zwf gene.

Bacterial cell survival depends on intact peptidoglycan, an extensive cell wall polymer of alternating N-acetylglucosamine and N-acetylmuramic acid residues, cross-linked by short peptides. Peptidoglycan biosynthesis is a viable and validated antimicrobial target; the intracellular, membrane-bound and extracellular synthetic stages provide a multitude of enzymes for interception with inhibitors. The ultimate phase of peptidoglycan biosynthesis occurs on the extracellular face of the cytoplasmic membrane and involves the polymerisation of Lipid II (the monomeric peptidoglycan precursor) by the transglycosylase and transpeptidase activities of the Penicillin-Binding Proteins (PBPs). The work presented in this thesis primarily focused on the biochemical characterisation of the integral membrane proteins Streptococcus pneumoniae PBP1a, PBP2b and PBP2x. These enzymes are clinically relevant; they are essential targets of !-lactam antibiotics and also mediate resistance against this important antimicrobial class. Full-length and truncated forms of the PBPs were cloned, expressed and purified to high levels. Two novel spectrophotometric assays were designed and developed to study the enzymology of the individual transglycosylase and transpeptidase activities of the PBPs with their natural substrate, Lipid II. Preliminary kinetic characterisations of the bifunctional PBP1a transglycosylase activity were performed and assay conditions were optimised to recreate an in vivo environment. PBP1a active site mutants revealed that transglycosylase activity was elevated in the absence of a functional transpeptidase domain. PBP1a and PBP2x exhibited transpeptidase activity with an apparent substrate preference for glycan polymers over Lipid II. PBP2x transpeptidase activity was not detected. The recorded rates of PBP activity were insufficient to support bacterial cell integrity, highlighting a gap in the understanding of PBP requirements. Finally, the PBPs were subjected to crystallisation trials for structural characterisations. This work provides an excellent foundation for the analysis and elucidation of PBP specificities. Future information attained could contribute to the design of novel inhibitors, alleviating the global threat of antibiotic resistance.

Marine picocyanobacteria are the most abundant photosynthetic bacterioplankton occupying a wide range of habitats across the world’s oceans. In order to survive in such diverse habitats, these organisms have developed various mechanisms to respond to specific environmental challenges they might encounter. One such challenge for cyanobacteria is the acquisition and homeostasis of micronutrients such as Zn and Cu, especially for those organisms occupying a variable ecosystem with an erratic nutrient supply. Metallothioneins are metal-binding proteins that potentially participate in such metal homeostasis mechanisms in these marine picocyanobacteria. Metallothioneins are small, cysteine-rich proteins capable of binding multiple metal ions, and have attracted intense scientific interest since their discovery in the 1950s. Over the last decade, they have been reported in every kingdom, from prokaryotic to eukaryotic, from bacteria to plants, from worms to mammals. Eukaryotic metallothioneins have been extensively studied. However, characterisation of bacterial metallothioneins is still rare. This research focused on the coastal cyanobacterium Synechococcus sp. CC9311, which is unusual in possessing four metallothionein genes (sync_0853, sync_1081, sync_2379 and sync_2426) while most marine picocyanobacteria contain only one, or none. Three metallothioneins were comprehensively characterised using a range of analytical and biophysical techniques. Mass spectrometry and nuclear magnetic resonance studies combined with homology modelling led to an unambiguous Zn3Cys8His cluster for BmtA0853, a highly likely Zn4Cys9His2 cluster for BmtA2426, and three possible configurations for BmtA1081. Analysis of gene expression profiles revealed that the four metallothioneins selectively participated in zinc scavenging, zinc homeostasis, cadmium detoxification, or protection from oxidising conditions. Growth of Synechococcus sp. CC9311 under various metal treatments also revealed that this coastal strain has developed a metal intensive physiology compared to the open ocean strain Synechococcus sp. WH8102.

Obesity is one of the biggest public health problems today, leading to type II diabetes, coronary heart disease, hypertension, sterility and cancer (Hashmi et al., 2013). \(C.elegans\) has been used extensively in obesity research to dissect the underlying principles. The overarching goal of my thesis has been to identify molecular signatures linking phenotypic and molecular responses of the nematode \(C.elegans\) to a number of experimental conditions that induce alteration in lipid metabolism. More specifically, I developed experimental procedures in \(C.elegans\) to study the effects of exercise, diet and a \(C.elegans\) antiageing drug valproate. Using a modified lipid assay, I found for the first time that valproate induce lipid accumulation in \(C.elegans\) in a dose dependent manner. In this study obesity is induced in \(C.elegans\) by treating with either cholesterol or sodium valproate. Using microarray technology, expression profiles associated with the induction of obesity are studied. In addition, \(C.elegans\) were treated with monosodium glutamate which is found to decrease lipid mass. Exercise is a recommendation for obese people and hence a \(C.elegans\) model system is developed to study effect of exercise. Effects of valproate on \(C.elegans\) with respect to lipid quantity are studied in detail, with emphasis on insulin signalling pathway. Expression changes associated with insulin mutants are compared with the valproate exposure expression profile to understand how much of the valproate exposure could be explained by insulin signalling. I also found for the first time that valproate cause a global down regulation of nuclear components. A hypothesis on lipid accumulation on valproate exposure is presented. TGF-β pathway is also found to be involved in fat metabolism. Finally I report an interesting phenotype for the first time in which the TGF-β pathway receptor mutant \(sma\)-\(6\) shows paralysis in response to valproate.

Histidine is an important amino acid involved in enzyme catalysis and metal binding in many proteins. Bacteria, fungi and plants synthesise histidine via the 10 step histidine pathway, whereas histidine is an essential amino acid required in the diet of animals. The synthesis of histidine is energetically expensive with about 41 ATP needed to produce one histidine. As a result the histidine biosynthetic pathway is tightly regulated and some microorganisms can consume excess histidine as a nitrogen and carbon source via a histidine utilisation (hut) pathway. The absence of a histidine biosynthetic pathway in mammals makes it a good target for the development of antimicrobial and antifungal drugs as well as herbicides. In addition to an overall regulation of the histidine synthesis by charged histidyl-tRNA and histidyl-tKNA synthetase, the histidine pathway is regulated in various ways by the first enzyme of the pathway ATP-phosphoribosyltransferase (HisG). The enzyme catalyses the condensation of ATP and phosphoribosylpyrophosphate (PRPP) to form phosphoribosyl-ATP (PR-ATP) and is feedback regulated by histidine, the end product of the pathway. Furthermore, HisG is inhibited by its product PR-ATP and also by AMP and ppGpp, reflecting the overall energy status of the cell. Inhibition of HisG coincides with a change in aggregation state, in which the active dimers form inactive hexamers. Some bacterial genomes contain a hisG gene, which lacks about 80 residues at the C-terminus, but have an additional gene, hisZ. This short form of HisG itself is inactive and forms an active complex with HisZ. This thesis describes the solution of the 3-dimensional structures of the hexameric forms of bacterial HisGs by X-ray crystallography. The structure of the Escherichia coli HisG was determined in the presence of the inhibitors AMP and PR- ATP. Additionally, a histidine-bound structure of HisG from the thermophilic bacterium Methanobacteriim thermoautotrophicum has been obtained. The monomer of HisG comprises of three a/p domains. The first two domains show a periplasmic protein-like fold with the active site located in a cleft between the two domains. The C-terminal domain, similar to Pn signal transduction proteins, binds histidine and is mainly involved in the formation of the hexamers. Additionally the structure of HisG represents a new type of phosphoribosyltransferase. The structure of the binary complex with AMP has allowed the identification of the PRPP binding site and explains the competitive inhibition by AMP with respect to both substrates. Furthermore the AMP binary structure and the PR-ATP-complexed structure identified the binding site of the ATP purine ring. The histidine-bound structure explains the histidine inhibition by disruption of the PRPP binding site on the dimer interface by elongation of the dimer and hence the hexanier. A mode of inhibition common to all the inhibitors investigated is the formation of hexamers upon inhibitor binding which buries the active sites in the inside of the hexamer and makes the sites less accessible to the substrates. Mutational and kinetic analysis showed that the extra C-terminal domain in the long form of HisG is not essential for activity. Subsequently, it was demonstrated that the naturally short form of HisG can be activated by thermal energy. This suggests a tense conformation for the short form of the enzyme which can be relaxed and activated by forming an oligomeric complex with HisZ. The structure of an unidentified protein in the histidine utilisation (hut) pathway from Pseudomonas aeruginosa, PAS104, was determined to better than 1 A resolution. Solution of the structure was accomplished by a unique approach combining SIRAS phasing with direct methods (Sayre equation). PAS104 is an all-beta protein with a bicupin fold. One of the P-barrels is open and accommodates a small molecule binding site. A different conformation of PAS 104 was obtained from a second structure solved using a different crystal form. Comparison of the structures and interactions in the active site identifies glutamate or derivatives (intermediates in the hut pathway) as possible substrates, which can induce a conformational change and may give PAS104 a regulatory function.

The observation that malonyl-CoA is involved in the synthesis of HMG-CoA by a pigeon liver fatty acid synthesising system, has led to uncertainty concerning the origin of the acetoacetyl intermediate for isoprenoid synthesis. A study was undertaken of the utilisation of malonyl-CoA for this purpose. Initial experiments with crude preparations of rat liver, yeast and the latex of Hevea brasiliensis indicated that malonyl-CoA was incorporated into isoprenoid compounds, including mevalonic acid. However, when avidin was added to incubations, fatty acid synthesis from acetate or acetyl-CoA was inhibited while the incorporation into isoprenoids was not affected. This indicated that a biotin dependent carboxylation of acetyl-CoA is not involved in the synthesis of isoprenoid compounds, and that malonyl-CoA was decarboxylated before the incorporation of acetyl-CoA as such. Experiments in which the incorporation of [2-¹⁴C] malonyl-CoA was compared to the incorporation of [1,3-¹⁴C] malonyl-CoA did not resolve this question, but degradation of HMG-CoA and Ergosterol indicated that the incorporation of radioactivity was that to be expected from acetyl-CoA. In accord with these observations, the malonyl-CoA decarboxylase activity could account for the incorporation of malonyl-CoA observed. When this was reduced by isolation conditions in which mitochondrial integrity was preserved, the incorporation into mevalonate was reduced. An examination of the purification of the pigeon liver fatty acid synthetase showed that HMG-CoA synthesising activity did not copurify with fatty acid synthesising activity. It was concluded that, in the systems studied, the acetoacetyl intermediate for isoprenoid synthesis arises by the condensation of two molecules of acetyl-CoA without the intervention of malonyl-CoA.

Streptomyces are Gram-positive bacteria, usually found living within the soil and they are saphrophytes. Among this class of bacteria are some plant pathogenic species, which cause infection of the roots or the tubers of some plants. The model Streptomycete plant pathogen is Streptomyces scabies; this infects root crops, such as potato or radish and is a known cause of scab disease. Most Streptomyces species are producers of secondary metabolites, many of which possess important biological activities, such as antibacterial, iron-chelating, anticancer or immunosuppressant. One group of these secondary metabolites are called siderophores. These are small organic molecules, which can chelate ferric iron. The iron in the environment is mainly present as iron (III) hydroxide, which is not very water soluble and cannot, therefore, be taken up directly by microorganisms. Some bacteria solve this problem through production of siderophores. The siderophores are released into the environment by the microorganisms to chelate iron (III) from the environment and transport it into the cell across the cell membrane. Iron is required for many life processes. Analysis of the Streptomyces scabies genome sequence resulted in the identification of gene clusters predicted to direct the biosynthesis of known siderophores, e.g. desferrioxamines and pyochelin, as well as, potentially novel siderophores. A gene inactivation and comparative metabolic profiling approach has been employed to identify the metabolic products of these gene clusters. A PCR-targeting method was used to replace part of or whole genes in the S. scabies 87.22 putative secondary metabolite gene clusters. An internal fragment of the scabichelin biosynthetic gene scab85471 and the putative S. scabies desC gene were deleted using this method. The scabichelin and desC gene mutants were subsequently analysed by LC-MS allowing confirmation of the function of the genes investigated. Production of scabichelin by S. scabies 87.22 wild type was analysed by comparing it with the authentic standard. The chemical and genetic complementation of the Δ desC mutant was carried out to establish the involvement of the desC gene in the biosynthesis of desferrioxamines. The S. scabies 87.22 cryptic tetronate biosynthetic gene cluster predicted to encode a novel agglomerin-like product, which could potentially be involved in plant pathogenicity was also investigated. The expression of the gene cluster was first analysed using reverse transcriptase PCR (RT-PCR) which was carried out on the total RNA isolated from the wild type S. scabies. Following this, an attempt was made to disrupt the scab63021 gene, a putative transcriptional activator of the cryptic tetronate-like cluster in the S. scabies genome. Transcriptional analysis of the wild type S. scabies and the putative Δscab63021 mutant genomes did not show any difference in the expression of the tetronate genes between the wild type strain and the Δ scab63021 mutant.

In order to examine the physiology of verotoxin (VT) production in vitro, it was necessary to develop an assay to enable specific measurement of VTl and VT2 expression. Initial efforts focussed on quantifying VTl and VT2 messenger RNA synthesis. RNA extraction and electrophoresis was consistently achieved, however, subsequent probing of Northern filters was not successful. A gene fusion between the slt-ll operon and the transposon vector TnphoA, such that expression of the phoA gene was brought under the control of the 5/MI promoter, was then developed. The presence and site of insertion of TnphoA within the s/MI operon was confirmed by restriction and sequence analysis, and a single copy fusion derivative within the VT2-producing E. coli strain E32511 was then obtained by exchange recombination. Use of the resulting single copy fusion derivative (E32511 SLF22/1) and a plasmid encoded slt-\. fïnphoA fusion (pSC105) demonstrated significant differences in the synthesis, secretion and localisation abilities of VTl and VT2 during growth under aerobic, anaerobic and iron limiting conditions. These characteristics could have important implications on the relative abilities of the two toxins to cause disease in vivo.