Bibliography: pages 132-155. / In order for bacteria to survive in their environment they must continuely sense signals such as, presence of host organisms, chemical concentrations, or variationsin other physiological parameters. Many bacteria sense their environment through the use of a two component regulatory systems. These systems usually employ the use of two different proteins, a sensor protein and its cognate response regulator. Some bacteria can survive fluctuations in medium osmolarity through the use of a two component signal transduction system. In Escherichia coli and Salmonella typhimurium this two component system includes the EnvZ sensor protein and its cognate response regulator, OmpR. The two genes that code for these proteins are envZ and ompR genes respectively. The two genes together form the ompB operonrespectively. This operon regulates the expression of two outer membrane proteins, OmpF and OmpC in response to medium osmolarity in E. coli.Erwinia chrysanthemi has been found to be sensitive to desication. Proliferation of soft rot, caused by this organism, has also been associated with irrigation. E.chrysanthemi has also been observed to respond to changes in medium osmolarity. Evidence of an ompB operon was thus sought. Outer membrane proteins were isolated using sodium lauroylsarcosine. Three major outer membrane proteins were isolated, namely Ompl (37.5 kd), Omp2 (35.5 kd) and Omp3 (34.5 kd). Increase in medium osmolarity resulted in an increase in expression of Omp3, while Ompl was suppressed. This lends support to the presence of an ompB like signal transduction system in E. chrysanthemi. Growth temperature was shown to have no effect on the expression of the major OMP. Similarly, culture growth phase had no effect on major OMP expression. However, two induced OMP were present from mid log phase onwards.

One soil sample and various indigenous plant and seaweed samples were used to isolate actinobacteria, with a particular focus on isolating rarer actinobacteria (non-Streptomyces species). A total of 169 putative actinobacterial strains was isolated, of which 42 were selected, based on their morphology, for further identification using a rapid molecular identification method and/or 16S rRNA gene sequence analysis. This includes seven strains isolated from various plant and seaweed samples. A total of 28 non-Streptomyces species was identified, with 23 being isolated from the soil sample, four isolated as plant endophytes (strains SM3BL 1, YPC1, YPC2 and YPL 1), and one isolated as a seaweed epiphyte (Y2UE1). Two Streptomyces species were isolated as endophytes (with strain SC1 isolated from a seaweed sample, and YMH1 isolated a plant species). Forty-two strains, including all non-Streptomyces species, were screened for their antibacterial activity against Mycobacterium aurum A+. Six strains showing promising antibacterial activity were selected for antibiotic extraction. The strains were also investigated for their antibiotic biosynthetic potential by PCR screening for the genes for ansamycin, glycopeptide and Type II (aromatic) polyketide antibiotics. Amplification of the AHBA synthase gene (ansamycin biosynthesis) was achieved for strains SE22 and YPC1. Strains SE22 and YMH1 were positive for the presence of the KSα-KSβ gene pair, with strain SE22 potentially producing a Type II (aromatic) polyketide. The gene product of isolate YMH1 was identified as a spore pigment gene. Antibiotic extraction was successful for strains SE22 and YM55, with numerous active compounds isolated from strain SE22, and one active compound isolated from strain YM55. Fifteen strains were selected for full characterisation based on their isolation source, their identification to non-Streptomyces genera and/or their antibacterial activity. These included four Micromonospora strains, three Kribbella strains, three Streptomyces strains, one Actinomadura strain, one Kineococcus strain, one Nocardia strain, one Nonomuraea strain and one Verrucosispora strain. Two previously isolated Microbispora strains were also characterised. The 17 strains were subjecte d to 16S rRNA gene sequence analysis to determine their closest phylogenetic relatives. The use of gyrB gene based phylogeny was also investigated for the two Microbispora isolates, resulting in a more stable phylogenetic tree. However, differences observed between the 16S rRNA and gyrB gene tree topologies suggest that horizontal gene transfer has occurred within the genus. The 17 isolates were distinguished from their closest phylogenetic neighbours through morphological and physiological characteristics. It is likely that the majority of the isolates are novel, although 16 isolates will require DNA-DNA hybridisation studies to determine if they are new species. Actinomadura strain YPC2 may be proposed as a novel species without the need for DNA-DNA hybridisation.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterised by behavioural and social impairments. ASD shows evidence of a genetic aetiology, with a large body of research linking ASD to polymorphisms in several different genes and gene families, including those involved in circadian rhythm generation and melatonin biosynthesis. Sleep disorders are highly comorbid with ASD in both children and adults, and range from sleep onset delay, phase shift and sleep disruption. These parasomnias can have a significant impact on the quality of life for persons with ASD and their families, and sleep deprivation can feed into the behavioural deficits in ASD. Melatonin supplementation is often prescribed to assist in alleviating the above mentioned sleep dysfunction. Melatonin is a hormone in the circadian clock system, and is a biochemical signal for darkness to synchronise peripheral cells to the master oscillator. Clinical trials reported that melatonin supplementation at night assists in sleep initiation. However both the mode of action of supplemental melatonin, as well as whether melatonin deficiency is common in ASD, remains unclear. Furthermore, any research on ASD is often hamstrung by the heterogeneous nature of the disorder, necessitating clear phenotyping. This study examines single nucleotide polymorphisms (SNPs) in the gene acetylserotonin methyl transferase (ASMT), which encodes an enzyme in melatonin biosynthesis, in a South African ASD cohort (n=28) and controls (n=6). All participants completed and Autism Diagnostic Observation Schedule-2 assessment that allowed partitioning of the ASD individuals into ASD endophenotypes, to reduce phenotyping heterogeneity. This study found SNPs previously associated with ASD in the promoter and intronic region. Additionally, this study found novel SNPs, and a SNP in a putative transcription factor binding site not previously associated with ASD. The associations found between SNPs and ASD endophenotypes, together with the positions of the SNPs, suggest a potential link between ASMT polymorphisms and ASD symptom severity. Further research, using language assessment tools as well as quantitative measures of melatonin and sleep disruption, may establish the role of melatonin in language impairment in ASD.

Bibliography: pages 109-118. / The relationship of various tobacco necrosis viruses, isolated from various crops and other sources in South Africa was determined. An isolate from avocado was chosen for partial characterisation to confirm that it was a member of the Necrovirus group of plant viruses. TNV was detected in potatoes that exhibited abnormal necrotic foliar symptoms and a D-type TNV was isolated and identified from the Up-to-Date potato variety. Immunoelectroblotting assay grouped the TNV isolates studied into two serotypes (A and D). This result was confirmed by NA hybridization with probes derived to the coat protein of an A- (TNVWheat) and a D-type (TNV-Papaya green lesion) isolate. RT-PCR with A and D specific primers did not amplify the coat protein of three A- and D-type TNV isolates which appears to indicate that the detection by PCR with specific primers is too selective to be used for a general test, unless degenerate primers to a more conserved region of the coat protein gene are used. Sequence analysis of the coat protein gene was used to determine the phylogenetic relationship amongst nine TNV isolates examined and also by comparison to three isolates for which the coat protein gene had already been sequenced. Sequence analysis showed high degree of homology amongst the A-type isolates and the D-type isolates, with approximately 45 % homology between the two TNV types.

Bibliography: pages 122-135. / A programme was undertaken to obtain high lysine producing bacteria by mutation of selected wild type strains. Overproduction of glutamic acid under suitable physiological conditions, viz biotin limitation, was chosen as a good indication of the potential of wild type bacteria for improvement in lysine production by mutation. Brevibacterium lactofermentum ATCC 13869 was found to produce the highest amount of glutamic acid under the conditions used. Homoserine and leucine auxotrophic mutants were obtained from this organism and tested for ability to produce lysine. The combination of homnserine and leucine auxotrophy was found to be most effective in overcoming several of the control mechanisms present in the lysine biosynthetic pathway. Lysine production was increased approximately forty fold over the wild type B. lactofermentum. Lysine analogue resistant strains were obtained by further mutation, and lysine production was increased by 20%. The activity and properties of aspartate kinase, a key enzyme in biosynthesis and control of lysine production, was determined to elucidate the nature of the anafogue resistance. Although resistance to feed-back control by lysine and threonine was not responsible for the improvement in lysine production, a considerably higher enzyme activity was found. As a result of the enzyme study a possible novel regulatory system in the lysine biosynthetic pathway of B.lactofermentum ATCC 13869, and the mutants derived from it, was indicated. Environmental optimization studies were undertaken on potentially suitable mutants in order to increase lysine production still further. Fermentation media were improved and a series of fermentations were conducted under precisely controlled conditions in 12 and 20 litre laboratory scale fermenters. The most successful attempt incorporated incremetal feeding of yeast extract and glucose to an S-(2-aminoethyl)-L-cysteine resistant double auxotrophic mutant. A yield of 32 mg/mQ L-lysine.HCI was obtained after 73 hours.

Climate change is a pressing reality in the current era. Changing environmental conditions and limited water availability are associated with the loss of arable land in areas where farming has traditionally thrived. Thus, linked to climate change, is the risk of a global food shortage. Resurrection plants are phenomenal in that they are able to survive extended periods of drought in a state of anhydrobiosis and then resume full metabolism upon rehydration. These plants serve as models to scientists and genetic engineers who hope to replicate, to a degree, the 'resurrection phenomenon' in drought sensitive crop species. The ability of resurrection plants to survive drought needs to be studied on a molecular level if it is to be implemented in transgenic crops. Currently, the molecular mechanisms of desiccation tolerance are only somewhat understood, and considerable investigation is still required. Xerophyta humilis is a monocotyledonous resurrection plant in which one of the responses to extreme water loss is the upregulation of several Late Embryogenesis Abundant (LEA) genes. The protein products of these genes, called LEA proteins, are known to be correlated with abiotic stress tolerance in plants, invertebrates and microorganisms. However, the precise molecular mode(s) of action of LEA proteins are still poorly understood. In this study, a group LEA_4, LEA protein, which we have termed XhLEA3-2, shown to be transcriptionally upregulated during desiccation of the resurrection plant X. humilis, has been characterized. A bioinformatic, predictive analysis was performed to detect any LEA-like characteristics of XhLEA3-2. Recombinant XhLEA3-2 was produced in Escherichia coli, purified, and used to generate XhLEA3-2 specific antibodies for expression analyses. The ability of XhLEA3-2 to function as a molecular chaperone was assessed using a lactate dehydrogenase (LDH) enzyme stability assay. Transgenic expression of XhLEA3-2 in E. coli and tobacco was also investigated. In summary, this thesis demonstrates that XhLEA3-2: has typical LEA protein properties according to bioinformatic analyses, has two close homologs in X. viscosa, is present in dry X. humilis leaf tissue, has homologs present in dry X. viscosa leaf tissue, has some molecular chaperone activity, can protect E. coli from desiccation but not from osmotic stress, and can be transiently expressed in tobacco.

The sessile nature of plants demands that they respond appropriately to changes in their environment (stresses) in order to survive. Critical to survival is the maintenance of water and ion homeostasis. The mechanisms by which plants achieve this are poorly understood. Traditionally plant stress responses were thought to be communicated by five classical plant hormones - auxin, cytokine, gibberellic acid, absisic acid and ethylene. Nowadays a plethora of other molecules are known to fulfil this function including nitric oxide, salicylic acid, jasmonic acid, brassinosteroids and peptide hormones. Plant natriuretic peptides have been proposed to be peptide hormones involved in maintaining water and ion homeostasis in plants. Evidence for this has been provided by studies of plant responses to exogenous natriuretic peptide treatment, however a demonstration of their function in planta remains outstanding. This study was undertaken to gain insight into the mechanisms regulating water and ion homeostasis in Arabdopsis by examining second messenger responses to stresses that perturb water and ion homeostasis; characterization of an Arabidopsis thaliana plant natriuretic peptide (atpnp-a) mutant and transcriptome analysis of AtPNP-A, in order to establish whether AtPNP-A plays a role in maintaining water and ion homeostasis in planta. Results indicated that recombinant AtPNP-A induces second messenger responses reminiscent of the response to NaCl, suggesting that AtPNP-A may play a signalling role in response to disturbances in water and ion homeostasis. In support of this, characterization of an atpnp-a mutant revealed that AtPNP-A is likely to be involved in processes that require adjustments to water and ion homeostasis including cell expansion, stomatal opening and NaCl and osmotic stress responses, consistent with reported responses to natriuretic peptide treatment. Furthermore, the atpnp-a mutant revealed a role for AtPNP-A in the defence response. Evidence to support this came from the computational analysis of AtPNP-A expression which correlates with genes involved in the defence response. Additionally, the transcriptome response to recombinant AtPNP-A treatment further implicated the involvement of AtPNP-A in the defence response. Therefore AtPNP-A is hypothesized to play a role in growth, abiotic and biotic stress responses that enables the plant to mount an integrated response to the environment.

The ability of various fermentation products to induce sporulation was in order to design a sporulation induction medium for Clostridium acetobutylicum P262. Of acetic acid, butyric acetone and butanol, was found to be most effective at induction. Induction was more efficient at low pH values under certain conditions. The heat resistance of mature spores was determined and the optlmal temperature for spore quantification was shown to be 75·C. acetobutylicum mutants m5 06 were by transposon mutagenesis using the conjugative transposon Tn925::Tn917, not transposon Tn925 as previously thought [Babb. B.L. 1990. B.Sc. (Honours) thesis, University of Cape Town. South Africa]. The spore development and the fermentation profiles of mutants were in batch over a period of 60h. Mutant m5 was shown to be oligosporogenous with majority of cells blocked at sporulation stage H. It was deficient in acetone and butanol production. Mutant 06 proceeded to sporulation stage VII and produced acetone and butanol at levels similar to that of the wild type strain. Mutants 06 appeared to contain two respectively from Southern hybridization experiments using a probe for left transposon junction. However, when a probe to right transposon junction was used, the chromosomal deoxyribonucleic acid (DNA) of mutant m5 was shown to contain approximately eight junction sites. The cause for the anomalous hybridization pattern was non-specific restriction enzyme activity nor a result of independent transposition of transposon Tn917.

Includes bibliographical references. / As part of an antibiotic-screening programme, an actinomycete, Streptomyces polyantibioticus SPRT, was isolated from soil collected from the banks of the Umgeni River, KwaZulu-Natal Province, South Africa. It exhibited antibiosis against M. tuberculosis H37RvT, prompting interest in its antibiotic production. An antibiotic produced by S. polyantibioticus SPRT was isolated and its structure determined by nuclear magnetic resonance (NMR) and X-ray crystallography to be 2,5- diphenyloxazole (DPO). Of great interest is the independent confirmation of the antibiotic activity of DPO and extension of the data to show activity against non-replicating persistent cells of M. tuberculosis. It seems likely that 2,5-DPO is synthesized non-ribosomally by S. polyantibioticus SPRT. It is proposed that DPO is synthesised from the starting units of benzoic acid and -hydroxyphenylalanine or phenylalanine, undergoing peptide bond formation followed by cyclization and decarboxylation to form DPO.

Crimean-­Congo Hemorrhagic Fever (CCHF) is a highly infectious, life threatening disease, caused by the Crimean-­Congo Hemorrhagic Fever Virus (CCHFV), a nairovirus that forms part of the Bunyaviridae family. CCHFV has a case fatality rate of approximately 40%. Current diagnostic methods for CCHFV involve the use of live virus antigen, requiring biosafety level 4 (BSL4) conditions for safe handling. The development of a safer diagnostic reagent for detection of this disease is therefore desirable. This project involves the development of a recombinant CCHFV nucleocapsid protein (NP). The nucleocapsid (NP) protein was expressed in Nicotiana benthamiana and purified using a 6x histidine-­tag. The protein was then reacted against serum samples collected from confirmed CCHFV patients to determine its ability to detect IgG antibodies against CCHFV in human sera.