Philosophiae Doctor - PhD (Biotechnology)

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Actinobacteria associated with two diverse soil environments and their multicopper oxidase diversity
(University of the Western Cape, 2024) Prins, Alaric; McCullough, Bronwyn Kirby
The Cape Floristic Region (CFR) is a biodiverse region boasting unique plant diversity with a rich concentration of endemic plants. Aspalathus linearis (Rooibos) is an indigenous plant that grows in the Clanwilliam region of the Western Cape and is cultivated for its use as an herbal tea. Emerging peatlands in the CFR have gained increasing attention over recent years through research aiming to understand the microbial diversity associated with these environments. Little is known about the actinobacterial diversity of these regions, and as such, it is necessary to investigate the diversity of the actinobacteria associated with these environments, whilst simultaneously gaining knowledge on whether the associated actinobacteria may produce enzymes of biotechnological interest. Two CFR regions (the Rooibos environment – Clanwillian, and the Springfield emerging peatland environment – Agulhas) were explored through culture-based and genomic screening. Metabarcoding analyses using actinobacterial-specific 16S rRNA gene primers showed that the major taxa contributing to the Rooibos environment were members of the families Mycobacteriaceae, Pseudonocardiaceae, Frankiaceae and Geodermatophilaceae. Members of the families Mycobacteriaceaea, Pseudonocardiaceae, Acidimicrobiaceae and Nocardioiaceae was identified as the major taxa for the Springfield environment. Through selective isolation techniques, actinobacteria from rare (underrepresented) genera were isolated, including members of the genera Dactylosporangium, Actinokineospora, Curtobacterium, Modestobacter, Leifsonia and Actinomadura. The top strains, selected based on exhibiting extracellular multicopper oxidase (MCO) activity through culture-based screening, were subjected to whole genome sequence analysis. These rare genera are also vastly underrepresented among 3 400 bacterial MCO sequences found in the Laccase and Multicopper Oxidase Engineering Database (LccED).
The molecular characterization of trichoplusia ni single nucleocapsid nucleopolyhedrovirus: a study on early regulatory features
(University of the Western Cape, 2003) Wang, Weizhou; Davison, S
With the development of biological insecticides, many research efforts have been made in baculoviruses to investigate fundamental molecular aspects of these viruses, such as the function and regulation of genes, genome organization, mode of entry, DNA replication and virus factors that determine the host range and virulence. Previously, a South African Trichoplusia ni single capsid nuclear polyhedrosis virus (TnSNPV) isolate was partially characterized as a novel baculovirus. During the process of the characterization, a few late genes of the virus were identified. This thesis describes a molecular characterization of the TnSNPV early genes to gain insight into the functional roles of these genes, their unique features and further determination of the placement of TnSNPV in baculovirus phylogeny.
Genome-wide identification and comprehensive analysis of transcriptionsl desert regions
(University of the Western Cape, 2009) Schaefer, UIf; Bajic, Vladimir
The initiation of transcription in mammalian genomes predominatly occurs at 5' promoter regions, however increasingly initiation events have been observed within introns, coding exons and 3' UTRs. Nevertheless there are large segments of mammalian genomes that are not prone to transcription initiation. These locations can be understood to be 'transcription initiation deserts'. It is challenging and useful to demarcate these segments or locations of the genome. The availability of a huge number of transcript data has provided an opportunity to develop a methodology to predict and annotate these genomic segments. A comprehensive collection of data for Homo sapiens ard Mus musculus, consisting of CAGE tags and other evidence for the existence of ffanscription was used to develop a methodology that allows the annotation of locations of mammalian genomes as those that are highly likely to initiate tanscription and those that are unlikely to harbour transcription start sites (TSSs). The algorithm allows the recognition of TSSs with 100% sensitivity, which makes it the superior choice over other existing algorithms for promoter prediction for the task of annotating TSS deserts.
Mitigation of salinity stress using exogenously applied molybdenum in sorghum bicolor
(University of the Western Cape, 2022) Mabiya, Thembeka Confidence; Mulaudzi-Masuku, Takalani
The agricultural sector is the main producer of food throughout the world. However, the constant changes in environmental conditions, such as extreme weather, droughts, and salinity have impacted this sector negatively over the years. These stresses cause nutritional imbalance, delayed seed germination and decreased growth resulting in reductions in crop yield and hence affect food prices. The food and agricultural organization (FAO), reported that the average increase rate in crop production is below the amount required to cater for the growing population. Thus, to meet the food demands, discovery of several strategies to improve crop growth and yield under severe environmental conditions are imperative.
Inter-individual genetic variation and the development of hypertension in a Xhosa African population of Eastern Cape, South Africa
(University of the Western Cape, 2022) Mabhida, Sihle Ephraim; Benjeddou, Mongi
Cardiovascular diseases (CVD) are the leading cause of death globally, accounting for 18.6 million deaths. Hypertension (HTN) drives the global burden of CVD and is a leading cause of cardiovascular-related mortality with 1.4 billion affected adults and 10.4 million deaths globally. This public health condition has been escalating alarmingly in low and middle-income countries. In Sub-Saharan Africa, HTN is a major public health concern with South Africa having the highest prevalence between 27-58%. Accumulative evidence shows that HTN is driven by both modifiable and non-modifiable risk factors.
The interactive effects of salt stress and Fusarium proliferatum infection on maize seedlings
(University of the Western Cape, 2022) Badiwe, Mihlali; Klein, Ashwil
Field crops are often subjected to multiple co-occurring stress factors, and they evolved specific mechanisms to counteract the effects of these stress factors. Most studies explore the fundamental molecular mechanisms involved in plant abiotic and biotic stress interactions. These look at plant responses to individual stressors and not combinations. Studying the effects of individual stress responses is not an adequate approach as plants in nature are challenged by both abiotic and biotic stress factors occurring simultaneously. Modern studies have shown that plants possess the ability to tolerate co-occurring abiotic and biotic stresses through the utilization of tailored responses, which are impossible to understand by direct extrapolation from results of studies examining individual stress factors.
Identification of sub-clinical biomarkers that predict the risk of developing diabetic cardiomyopathy
(University of the Western Cape, 2022) Nxele, Xolisa; Benjeddou, Mongi
Cardiovascular disease (CVD) is the leading cause of death of people with obesity and type 2 diabetes (T2DM). According to a statistical report from the World Health Organization (WHO), approximately 17.9 million people die annually because of CVD and diabetic cardiomyopathy (DCM), a disease of the heart muscle occurring in the absence of coronary artery disease or hypertension. Although not fully elucidated, the pathophysiology of DCM includes myocardial left ventricular hypertrophy, impaired calcium handling, energy metabolism, inflammation, apoptosis and myocardial fibrosis.
Deciphering the determinants of molecular physiological responses to drought and heat stress in sorghum lines contrasting in stress tolerance via a proteomics approach
(University of the Western Cape, 2022) Ali, Ali Elnaeim Elbasheir; Ludidi, Ndiko
Sorghum is an important crop in Sub-Saharan Africa and South Asia. The predicted rise in global temperatures will increase the probability of exposing sorghum to heat waves in combination with drought. Thus, production and availability of sorghum and its products will be negatively affected. Although much progress has been made in identifying molecular processes involved in some crop responses to drought or heat stress, knowledge on such responses in sorghum is limited and in fact does not exist for combined drought and heat stress in sorghum lines that respond differently to these stresses.
Molecular detection and characterisation of RNA viruses of honeybees
(University of the Western Cape, 2009) Topley, Elize Lindsay; Davison, Sean
Honeybees have evolved through the centuries to inhabit most parts of the world except for the extreme Polar Regions. These insects have also been susceptible to pathogens and disease which has always been part of the honeybees’ ecology and has evolved and adapted accordingly. However disease has spread more rapidly into areas where no disease existed before with the transport and moving of hives. Disease has caused massive losses within the honeybee industry in recent history. Using new technology available to scientists, diseases and parasites can be identified and this information used to prevent damage to hives, the livelihood of many crop farmers and beekeepers around the world. Of these diseases honeybee viruses have become of some concern in recent times. Honeybee viruses’ black queen cell virus (BQCV) and acute bee paralysis virus (ABPV) were found to have genomes consisting of 8550 and 9490 nucleotides respectively. The viruses have two open reading frames (ORFs) which encode a non structural protein at the 5’ ORF and a structural protein at the 3’ ORF. Sacbrood virus (SBV) has a different organisation to BQCV and ABPV where it has a single ORF with the structural genes at the 5’ end and the non structural genes at the 3’ end. In an effort to rapidly identify honeybee viruses a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) was developed Primers were designed within the 3’ open reading frame to amplify fragments of 434bp for SBV, 900bp for ABPV and 316bp for BQCV. RNA was extracted from laboratory infected and naturally infected samples. The PCR products were sequenced and found to be that of the appropriate virus. The primers were tested on naturally infected samples with SBV and BQCV being detected. Another well characterised honeybee virus Kashmir bee virus (KBV) was initially added to the multiplex RT-PCR. However inconsistencies with the multiplex PCR led to the sequencing of a 2 kilobase fragment of the KBV Indian (KBV-in) strain. Three overlapping cDNA fragments of KBV were sequenced and aligned with the full length sequence of KBV and a sequenced capsid region of KBV both from North America. Alignment to ABPV was also completed to observe the homology between KBV-in and ABPV. The KBV-in strain was not highly homologous to the North American strains over the region which was sequenced for KBV-in. ABPV was also not highly homologous over the entire 2 Kb region. However over the region where primers were designed for the RT PCR of KBV, ABPV was highly homologous at 80%. This could have led to the inconsistencies when PCR was done. Primer design and correct strain characterisation is needed before primers are designed to detect more than one virus per reaction. Further characterisation and sequencing of this strain is needed in order to make further comparisons. Propagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5’ and 3’ ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus.
A proteomic analysis of drought and salt stress responsive proteins of different sorghum varieties
(University of the Western Cape, 2009) Ngara, Rudo; Ndimba, Bongani K.
Sorghum (Sorghum bicolorï, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotie stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI- TOF and MALDI- TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germ in proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. Following 200 mM NaCl and 400 mM sorbitol stress treatments, the expression/abundance of a protein spot similar to a rice wall-associated protein kinase was upregulated in the sorghum secretome in response to both stresses. Amino acid sequence alignment of the matching peptides between these two proteins showed that the sorghum CF spot possesses a protein kinase domain. Therefore, this protein could possibly participate in cell signalling functions, which link the external environment with the cell's cytoplasm. Using whole plant systems, a comparative study of leaf protein expression between two sorghum varieties, AS6 (salt sensitive) and MN1618 (salt tolerant) was conducted. Forty well resolved spots of varying abundances were picked for MS analysis. Of these, 28 were positively identified, representing proteins with functions in carbohydrate metabolism (60.7%), proton transport (17.9%), protein synthesis (7.1%), hydrolytic functions (7.1%), nucleotide metabolism (3.6%) and detoxification (3.6%). Using PDQuest™ Advanced 2D Analysis Software version 8.0.1 (BIO-RAD), a comparative analysis of leaf proteome expression patterns between the two sorghum varieties was conducted. The results indicated proteins with similar expression patterns as well as qualitative and quantitative differences between the two leaf proteomes. The effect of 100 mM NaCI on leaf proteome expression between the two sorghum varieties was also studied. Western blotting analysis of leaf, sheath and root tissues using Hsp70 antibodies showed that this treatment induced Hsp70 expression, a known stress protein, in both varieties. Thereafter, the partially annotated leaf proteome map was used to landmark other salt responsive proteins. Examples of differential expression patterns included glutathione S transferase and hydroxynitrile lyase proteins whose abundances were upregulated in both varieties, while the large subunit of RuBisCo was downregulated in AS6 but upregulated in MN1618. Qualitative spot expression differences in response to salt stress were also observed between the two sorghum varieties but these remained unidentified after both MALDI-TOF and MALDI-TOF-TOF MS, possibly indicating novel and previously uncharacterised sorghum proteins. The results of this study can be used as reference tools by proteomics researchers worldwide as well as a foundation for future studies.
Evaluation of the capacity of hydrogen sulfide to reduce infection of maize
(University of the Western Cape, 2020) Ntloko, A.; Ludidi, N
Maize (Zea mays L.) is grown globally as an important grain crop in South Africa, United States, China and Brazil and plays a major role in the worldwide economy. In South Africa, the grain is utilised for food consumption, livestock feed, for malting purposes and bioethanol production. Maize contains approximately 72% starch, 10% protein, 4% fat and supplying an energy density of 365 Kcal/100 g. The production of grain crops in South Africa is restricted by various factors such as abiotic and biotic stresses. The fungal genus Aspergillus is one of the most important biotic stresses affecting maize in the country. Aspergillus flavus can contaminate a wide range of agricultural commodities either in storage or field. Hydrogen sulfide appears to have a potential in the mechanism of resistance against pathogen attack by Aspergillus flavus.
Genomic and proteomic analysis of drought tolerance in Sorghum (Sorghum bicolor (L.) Moench)
(University of the Western Cape, 2014) Woldesemayat, Adunga,Abdi; Christoffels, Alan; Ndimba, Bongani.K
Drought is the most complex phenomenon that remained to be a potential and historic challenge to human welfare. It affects plant productivity by eliciting perturbations related to a pathway that controls a normal, functionally intact biological process of the plant. Sorghum (Sorghum bicolor (L.) Moench), a drought adapted model cereal grass is a potential target in the modem agricultural research towards understanding the molecular and cellular basis of drought tolerance. This study reports on the genomic and proteomic findings of drought tolerance in sorghum combining the results from in silica and experimental analysis. Pipeline that includes mapping expression data from 92 normalized cDNAs to genomic loci were used to identify drought tolerant genes. Integrative analysis was carried out using sequence similarity search, metabolic pathway, gene expression profiling and orthology relation to investigate genes of interest. Gene structure prediction was conducted using combination of ab initio and extrinsic evidence-driven information employing multi-criteria sources to improve accuracy. Gene ontology was used to cross-validate and to functionally assign and enrich genes. An integrated approach that subtly combines functional ontology based semantic data with expression profiling and biological networks was employed to analyse gene association with plant phenotypes and to identify and genetically dissect complex drought tolerance in sorghum. The gramene database was used to identify genes with direct or indirect association to drought related ontology terms in sorghum. Where direct association for sorghum genes were not available, genes were captured using Ensemble Biomart by transitive association based on the putative functions of sorghum orthologs in closely related species. Ontology mapping represented a direct or transitive association of genes to multiple drought related ontology terms based on sorghum specific genes or orthologs in related species. Correlation of genes to enriched gene ontology (GO)-terms (p-value < 0.05) related to the whole-plant structure was used to determine the extent of gene-phynotype association across-species and environmental stresses.
Cloning, expression and characterization of Novel Lipase and Esterases from Burkholderia multivorans UWC10
(2005) Rashamuse, Konanani J; Cowan, Don A
An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C B-lactames and some peptidases.
New genetic tools to engineer starch production in crops
(University of the Western Cape, 2014) Muteveri, Morleen; Ndimba, Bongani K.
Starch is a major carbohydrate reserve in many plants, providing energy during heterotrophic growth and it is contained in large amounts in staple foods such as potatoes, wheat, maize, rice, sorghum and cassava. Apart from being a major product for use in the food industry, starch is also attracting interest from the biofuels industry as a source of bioethanol. This study reports on the development of genetic tools aimed at increasing starch production in sorghum (Sorghum bicolor L Moench), a crop of key agronomic importance worldwide by exploiting a new discovery of a transcription factor gene that regulates starch accumulation in Arabidopsis thaliana namely LEAFY COTYLEDON I (LECl). Ectopic over expression of this gene in arabidopsis has previously been shown to induce a massive hyper accumulation of starch in vegetative tissues. Therefore, we set out to investigate the function of its orthologous gene counterpart in sorghum with the aim of manipulating starch yield directly. Deduced protein sequence analyses showed that the putative sorghum LEAFY COTYLEDON I gene (SbLEC1) cloned in this study shares an overall high amino acid sequence identity (70 %) with the arabidopsis LEC 1, while the functional central B domain shows an even higher percentage sequence identity (91 %) with the same region of arabidopsis LEC 1. The putative SbLEC1 protein shares 14 out of the 16, signature ammo acids characteristic of the Central B Domain with arabidopsis. Furthermore, the putative SbLEC1 protein was also shown to share a significantly high sequence identity (> 80 %) with other well-characterized LEC1 protein sequences from organisms such as maize, rice, rapeseed as well as other organisms documented in the NCB I database. Similarly, much of the sequence similarity lies within the functional central B domain compared to any other region. Gene expression profiling using semi-quantitative PCR showed that SbLEC1 transcripts accumulated in developing seeds as well as in embryogenic calli tissue and no SbLEC 1 transcripts were detectable in leaf, root or sheath tissue. In order to confirm that the identified transcription factor is a functional ortholog, the full cDNA encoding putative SbLEC 1 transcription factor was identified, isolated and cloned from the sweet sorghum MN 1812 genotype. Plant transformation gene constructs based on the pCAMBIA1305.2 binary vector harbouring the transcription factor gene under the control of different promoter sequences were then assembled and immobilized into Agrobacterium tumefaciens strain LBA4404 in preparation for sorghum and arabidopsis transformation. Transient GUS expression studies showed that the five SbLEC1 gene constructs developed in this study were successfully transformed into arabidopsis (Ws ecotype) and sorghum (variety MN1812) callus and cell suspension cultures. The transformed tissues thus represent essential tools that are useful to evaluate the effect of over expressing the putative SbLEC1 protein. Transient GUS expression assays also further revealed differences in efficiency among promoters in driving transgene expression. Transient GUS activity was highest for the maize ubiquitin promoter (MUbi1), followed by the sorghum LEC1 promoter (SLECP), the arabidopsis LEC1 promoter (ALECP) and lastly the maize alcohol dehydrogenase promoter (MAdh1). The ability of the putative SbLEC 1 gene to complement the arabidopsis lecI mutation was also investigated and our findings were not conclusive as they only revealed partial complementation. A detailed comparison of SbLECI full cDNA sequences isolated and cloned from twenty-eight different F2 population plants from different sorghum varieties revealed the existence of sequence variation within the SbLEC 1 gene, which appeared to be allelic. The allelic variation was further shown to affect the amino acid composition of the putative SbLEC 1 protein. Heterologous protein expression studies of the SbLECI gene using an E. coli system showed that the predicted 29.16 kDa putative SbLEC 1 protein could be expressed in vitro both as an development of an efficient tissue culture protocol is a prerequisite for plant genetic engineering, this study also reports on the evaluation of thirteen sorghum genotypes from different genetic backgrounds for their in vitro culture response. A tissue culture protocol for three previously unexplored sorghum genotypes namely Agricol white, AS4 and MNI812 was established. The effect of plant genotype, explant and medium composition on in vitro culture response was highly significant (95 % Cl) in this study. Taken together, the findings in our study demonstrate efforts to draw a baseline foundation for the development of molecular technologies that can be used to increase starch production in sweet sorghum as a water efficient and sustainable feedstock for biofuel production.
Respiratory and photosynthetic C and N metabolism of nodulated Lupin roots during phosphorus deficiency
(University of the Western Cape, 2010) Le Roux, Marcellous R; Valentine, AJ
Growth of symbiotic legume hosts is P limited, because of the high energetic requirements associated with N2 fixation. Attempts to overcome P deficiency in soils where legumes are grown involve addition of P-based fertilisers. However, these are produced from fmite, non-renewable resources that could be exhausted in the next 50-80 years. For this and other prudent reasons, viable alternatives are sought that include producing genetically enhanced plants with better P use efficiency (PUE). There exist some inter- and intraspecific genetic variation for associated traits of PUE in various legumes and these will have to be exploited to realize the development of P efficient cultivars. With the advent of sophisticated molecular tools, good progress has been made to understand the molecular response of some common physiological and morphological functions observed under LP. The research aims here were to investigate the energy costs and the alternative metabolic routes associated with C and N metabolism under LP in legumes, which is very scant in literature. We also investigated the recovery responses of nodulated roots upon P alleviation. Consequently, improvement strategies to produce legume varieties for better adaptation in poor P soils are envisaged. We have demonstrated varying degrees of sensitivity between the amide and ureide legume systems being investigated under short-term LP. The species-specific responses were ascribed to differences related to the agro-climatic origins, nodule morphologies and the type of N containing export product of the different legume types. These different responses also underscore possible different regulatory mechanisms under LP. Lupins were probed further, because of its apparent tolerance to P deficiency. Lupin nodules had between 3 to 5-fold higher Pj concentrations compared with soybeans under LP and HP, respectively. The maintenance of Pj levels, as oppose to a decline in the total P pool, is discussed in relation to its role in maintaining N2 fixation in lupins. Under LP, an effective Pj recycling mechanism in nodules is proposed to occur via the induction of the PEPc- MDH-ME route. This route also enhanced the capacity of root nodules to procure high malate concentrations that are used to fuel bacteroid respiration and N2 fixation. Two distinctly different cMDH proteins, one corresponding to HP and another corresponding to LP, were identified. The high malate concentrations reported here are speculated to have arisen through LP-induced cMDH. Metabolically available Pj decline developed gradually as P deficiency progressed. This coincided with a 15% decline in the %Ndfa. Moreover, under prolonged P deficiency the disproportionate synthesis of organic acids, most notably malate, that occurred at the expense of amino acids was proposed to account for this decline. The recovery in response to alleviation from LP involved alterations in the allocation of respiratory costs to growth and nutrient acquisition. Under LP, smaller nodules were formed and nodule metabolism revolved around accentuating PUE. Thus, there is considerable potential for improvement of P efficiency in legumes through manipulation of root: shoot partitioning.
Bioactive actinobacteria associated with two South African medicinal plants, Aloe ferox and Sutherlandia frutescens
(University of Western Cape, 2021) King, Maria Catharina; Kirby-McCullough, Bronwyn
Actinobacteria, a Gram-positive phylum of bacteria found in both terrestrial and aquatic environments, are well-known producers of antibiotics and other bioactive compounds. The isolation of actinobacteria from unique environments has resulted in the discovery of new antibiotic compounds that can be used by the pharmaceutical industry. In this study, the fynbos biome was identified as one of these unique habitats due to its rich plant diversity that hosts over 8500 different plant species, including many medicinal plants. In this study two medicinal plants from the fynbos biome were identified as unique environments for the discovery of bioactive actinobacteria, Aloe ferox (Cape aloe) and Sutherlandia frutescens (cancer bush).
Massively-Parallel Computational Identification of Novel Broad Spectrum Antivirals to Combat Coronavirus Infection
(University of the Western Cape, 2015) Berry, Michael; Gamieldien, Junaid
Given the significant disease burden caused by human coronaviruses, the discovery of an effective antiviral strategy is paramount, however there is still no effective therapy to combat infection. This thesis details the in silica exploration of ligand libraries to identify candidate lead compounds that, based on multiple criteria, have a high probability of inhibiting the 3 chymotrypsin-like protease (3CUro) of human coronaviruses. Atomistic models of the 3CUro were obtained from the Protein Data Bank or theoretical models were successfully generated by homology modelling. These structures served the basis of both structure- and ligand-based drug design studies. Consensus molecular docking and pharmacophore modelling protocols were adapted to explore the ZINC Drugs-Now dataset in a high throughput virtual screening strategy to identify ligands which computationally bound to the active site of the 3CUro . Molecular dynamics was further utilized to confirm the binding mode and interactions observed in the static structure- and ligand-based techniques were correct via analysis of various parameters in a IOns simulation. Molecular docking and pharmacophore models identified a total of 19 ligands which displayed the potential to computationally bind to all 3CUro included in the study. Strategies employed to identify these lead compounds also indicated that a known inhibitor of the SARS-Co V 3CUro also has potential as a broad spectrum lead compound. Further analysis by molecular dynamic simulations largely confirmed the binding mode and ligand orientations identified by the former techniques. The comprehensive approach used in this study improves the probability of identifying experimental actives and represents a cost effective pipeline for the often expensive and time consuming process of lead discovery. These identified lead compounds represent an ideal starting point for assays to confirm in vitro activity, where experimentally confirmed actives will be proceeded to subsequent studies on lead optimization.
The C-economy, nutritional benefits and symbiotic performance of dual inoculated Phaseolus vulgaris (L.) plants, under variable nutrient conditions
(University of the Western Cape, 2010) Mortimer, Peter E; Valentine, AJ
The tripartite symbiosis between Phaseolus vulgaris, arbuscular-mycorrhiza and the nodule bacteria, Rhizobia have been the focus of many studies ranging over a number of decades, however these studies have failed to answer certain questions relating the role of the symbionts in regard to host nutrition and the subsequent influence of these symbionts on the host C- economy. There is little doubt over the synergistic benefits involved in the dual inoculation of bean plants, as well as the resultant C-costs of maintaining the 2 symbionts, yet the specific contribution of the individual symbionts to the hosts overall nutrient and C-economy remain to be clarified. Thus the aim of this thesis is to help clarify these points by determining the symbiont induced photosynthetic, respiratory and nutritional changes taking place in the host. This was achieved by a series of experiments in which nodulated bean plants were split into two categories-those with and without AM colonized roots. These plants were then exposed to a range of growing conditions, including hi and low P, and a series of N treatments, ranging from zero N through to 3 mM NH/. Under these differing nutrient conditions growth, photosynthetic, respiratory, nutrient and amino acid responses were monitored, thus allowing for the determination of the symbionts influence on the host and the hosts reliance on the respective symbionts. Host reliance was noted most strongly under nutrient limiting conditions. Under low P treatment AM was the dominant symbiont as far as host C was concerned, allowing for the early establishment of the AM, thus ensuring the uptake of P for both host and nodule development. High P affected AM colonization to a greater extent than it did nodule dry weight and conversely the addition of N~ + led to a greater decrease in nodule dry weight than it did AM colonization. In spite of this decline, AM benefited the host by improving host N nutrition and relieving N-feedback inhibition of the export amino acid asparagine on BNF. These AM induced benefits did come at a cost to the host though, the dual inoculated plants had higher below ground respiratory costs and subsequently higher photosynthetic rates to compensate for the increased demand for C. The higher photosynthetic rates associated with dual inoculation were as a result of symbiont induced sink stimulation and not due to the improved nutrition of the host, as shown by the photosynthetic and nutrient response ratios. However, the respiratory costs associated with the uptake of soil nutrients were lower in AM colonized roots, thus showing an increased efficiency in nutrient gain by AM colonized roots. This improvement in host N nutrition as a result of AM colonization, coupled with the lower respiratory costs of AM nutrition led to the conclusion that under certain growing conditions nodules can become redundant and possibly parasitic.
Investigating the antimicrobial potential of Thalassomonas actiniarum
(University of the Western Cape, 2020) Pheiffer, Fazlin; Trindade, Marla
bioassay guided isolation approach was then used to isolate the high molecular weight antibacterial compound (50kDa-100kDa) from T. actiniarum fermentations. With common protein isolation, purification and detection methods failing to provide insight into the nature of the antibacterial compound, we hypothesized that the active agent is not proteinaceous in nature and may be a high molecular weight exopolysaccharide. Extraction and antibacterial screening of the exopolysaccharide fraction from T. actiniarum showed antibacterial activity as well as lytic activity when subjected to a zymography assay using Pseudomonas putida whole cells as a substrate. Additionally, the biosynthetic pathways for the production of poly-β-1, 6-N-acetyl-glucosamine (PNAG), an exopolysaccharide involved in biofilm formation and chondroitin sulfate, a known and industrially important glycosaminoglycan with antibacterial and anti-inflammatory activity was identified and the mechanism may be novel. Genome mining identified a variety of novel secondary metabolite gene clusters which could potentially encode other novel bioactivities. Therefore a bioassay guided isolation, focused on the small (<3kDa) molecules, was pursued. Secondary metabolites were extracted, fractionated and screened for biofilm inhibition, antibacterial and anticancer activity and activity was observed in all assays. Active fractions were dereplicated by UHPLC-QToF-MS and compounds of interest were isolated using mass guided preparative HPLC. The purity of the isolated compounds was assessed using UHPLC-QToF-MS and NMR and the structure of the target compounds elucidated. Structures that could be determined were the bile acids cholic acid and 3-oxo cholic acid and although not responsible for the observed activities, this is the first report of bile acid production for this genus. This is the first study investigating the bioactive potential of the strain and the first demonstrating that T. actiniarum is a promising source of potentially novel pharmaceutically relevant natural products depicted through both culture-dependent and culture-independent approaches.
Investigating the antimicrobial potential of Thalassomonas actiniarum
(University of Western Cape, 2020) Pheiffer, Fazlin; Trindade, Marla; van Zyl, Leonardo
The World Health Organisation predicts that by the year 2050, 10 million people could die annually as a result of infections caused by multidrug resistant bacteria. Individuals with compromised immune systems, caused by underlying disease such as HIV, MTB and COVID-19, are at a greater risk. Antibacterial resistance is a global concern that demands the discovery of novel drugs. Natural products, used since ancient times to treat diseases, are the most successful source of new drug candidates with bioactivities including antibiotic, antifungal, anticancer, antiviral, immunosuppressive, anti-inflammatory and biofilm inhibition. Marine bioprospecting has contributed significantly to the discovery of novel bioactive NPs with unique structures and biological activities, superior to that of compounds from terrestrial origin. Marine invertebrate symbionts are particularly promising sources of marine NPs as the competition between microorganisms associated with invertebrates for space and nutrients is the driving force behind the production of antibiotics, which also constitute pharmaceutically relevant natural products.

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