>Magister Scientiae - MSc / The high rate of rediscovery of known compounds has led to a decline in the discovery of novel natural products. The high biodiversity of organisms growing in extreme conditions such as oceans has led to the increased interest by researchers for their use as a source of novel natural products. Marine bacteria are known for their extensive biosynthetic capacity to produce diverse natural products, which are suitable for various biotechnology applications such as in agriculture, for treatment of fungal plant pathogens, and as antibiotics, for treatment of bacterial infections. This study aimed at discovering novel secondary metabolites from marine bacteria previously associated with novel marine invertebrate species endemic to the South African coast. The methodologies used in this study included a bioassay guided fractionation coupled to genome sequencing and mining. For the bioassay guided fractionation approach, the study first focused on screening marine bacteria for antimicrobial activity when cultured on 4 different media, against fungal strains previously shown to be virulent olive trunk pathogens. In parallel, the bacterial isolates with the most inhibitory activity against the fungal pathogens were also screened for antimicrobial activity against 4 indicator strains including Gram-negative Escherichia coli 1699 (E. coli), Pseudomonas putida, and Gram-positive Staphylococcus epidermidis ATCC14990, and Bacillus cereus ATCC10702. One of the marine bacterial isolates, PE6-126, showed diverse antimicrobial activity including antibacterial and antifungal activity against the tested strains. The genome sequencing data revealed that this isolate was B. cereus based on the average nucleotide identity (ANI) (>99%) to reference strains. antiSMASH analysis of the genome revealed nine predicted secondary metabolite clusters including bacteriocins (2), non-ribosomal peptide synthetase (NRPS) (2), siderophore (1), sactipeptide (1), betalactone (1), linear azol(in)e-containing peptides (LAP) - bacteriocin (1) and a terpene (1). Some of these pathways had low to no sequence similarity to known pathways, indicating the potential of these pathways to produce novel compounds. One of the pathways showed very high sequence similarity to the thuricin CD pathway in Bacillus thuringiensis. Considering that thuricin CD has been reported to have antimicrobial activity against B. cereus (ATCC1072), it was hypothesised that it could also be produced by PE6-126. However, the antimicrobial extract from PE6-126 was tested for sensitivity to proteinase K and heat treatment, which thuricin CD is known to be sensitive to. The results revealed that the antimicrobial activity was not lost after treatment, implying that a different metabolite could be responsible for the anti-B. cereusactivity. In addition, PE6-126 initially displayed antimicrobial activity against a multi-drug resistant E. coli 1699, suggesting some of the antimicrobial compound/(s) produced by this strain could potentially be novel. The bioassay-guided fractionation approach coupled to Liquid Chromatography Mass Spectrometry (LC-MS) did not lead to identification of the antimicrobial compound/(s), therefore it remains a question whether the secondary metabolite pathways predicted by antiSMASH lead to the production of the active compound/(s).The results from this study showed that even well studied species have the potential to synthesize as yet undescribed compounds, based on the novelty of some of the pathways. This study highlights the importance of employing a genome-guided approach in drug discovery, as there may be many novel compounds to discover from biosynthetic pathways that have not yet been characterised. Further research is needed to identify the antimicrobial compound/(s) produced by PE6-126.

Philosophiae Doctor - PhD / 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.

Philosophiae Doctor - PhD / 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.

As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. / Dissertation / Doctor of Philosophy (PhD) / Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target.

<p>Nonribosomal peptides (NRPs) are a major class of natural products known for their biological activities and are employed therapeutically as immunosupressants, anticancer agents, and antibiotics. Nonribosomal peptides are microbial products, biosynthesized by large assembly line-like enzymes, known as nonribosomal peptide synthetases (NRPSs) that can be found in large gene clusters within the genome. With the advent of genome sequencing, the gene clusters for known NRPs are easily identified within producing organisms, but more strikingly, this sequencing reveals that microbes often contain many gene clusters with no known products suggesting traditional methods of isolation are overlooking the majority of NRPs.</p> <p>Extensive studies of NRPS functions have revealed assembly line logic for the biosynthesis of NRPs and using this knowledge, the NRP products of NRPS gene clusters can be predicted. In this research, products from both a simple dimodular NRPS from <em>Staphylococcus aureus </em>and a complex 11 module NRPS from <em>Delftia acidovorans </em>were predicted and used to successfully identify and isolate two novel NRPs, aureusimine and delftibactin.<em> </em>Theses compounds fell outside traditional NRP activities, one being a virulence regulator and the other a gold-complexing metallophore. Subsequent biosynthetic studies of the aureusimine gene cluster within the heterologous host, <em>Escherichia coli</em>, provide insight into NRPS flexibility for the creation of NRP natural variants and highlighted the utility of <em>E. coli </em>for the heterologous production of NRPs.</p> <p>Realizing single NRP predictions are not always accurate, a strategy was devised to use a genomically predicted NRP fragment barcode databases with the LC-MS/MS dereplication algorithm, iSNAP, to chemoinformatically identify and physically locate genetically predicted NRPs within crude extracts. This final contribution eliminates the need for bioactivity guided approaches to discovery and provides a strategy to systematically discover all predicted NRPs from cryptic gene clusters. This thesis delivers strategies and technologies for the directed discovery of NRPs from microbial sources.</p> / Doctor of Philosophy (PhD)

Plants and animals are characterized by intimate, enduring, often indispensable, and always complex associations with microbes. Therefore, it should come as no surprise that when the genome of a eukaryote is sequenced, a medley of bacterial sequences are produced as well. These sequences can be highly informative about the interactions between the eukaryote and its bacterial cohorts; unfortunately, they often comprise a vanishingly small constituent within a heterogeneous mixture of microbial and host sequences. Genomic analyses typically avoid the bacterial sequences in order to obtain a genome sequence for the host. Metagenomic analysis typically avoid the host sequences in order to analyze community composition and functional diversity of the bacterial component. This dissertation describes the development of a novel approach at the intersection of genomics and metagenomics, aimed at the extraction and characterization of bacterial sequences from heterogeneous sequence data using phylogenomic and bioinformatic tools. To achieve this objective, three interoperable workflows were constructed as modular computational pipelines, with built-in checkpoints for periodic interpretation and refinement. The MetaMiner workflow uses 16S small subunit rDNA analysis to enable the systematic discovery and classification of bacteria associated with a host genome sequencing project. Using this information, the ReadMiner workflow comprehensively extracts, assembles, and characterizes sequences that belong to a target microbe. Finally, AssemblySifter examines the genes and scaffolds of the eukaryotic genome for sequences associated with the target microbe. The combined information from these three workflows is used to systemically characterize a bacterial target of interest, including robust estimation of its phylogeny, assessment of its signature profile, and determination of its relationship to the associated eukaryote. This dissertation presents the development of the described methodology and its application to three eukaryotic genome projects. In the first study, the genomic sequences of a single, known endosymbiont was extracted from the genome sequencing data of its host. In the second study, a highly divergent endosymbiont was characterized from the assembled genome of its host. In the third study, genome sequences from a novel bacterium were extracted from both the raw sequencing data and assembled genome of a eukaryote that contained significant amounts of sequence from multiple competing bacteria. Taken together, these results demonstrate the usefulness of the described approach in singularly disparate situations, and strongly argue for a sophisticated, multifaceted, supervised approach to the characterization of host-associated microbes and their interactions. / Ph. D.

>Magister Scientiae - MSc / The high rate of rediscovery of known compounds has led to a decline in the discovery of novel natural products. The high biodiversity of organisms growing in extreme conditions such as oceans has led to the increased interest by researchers for their use as a source of novel natural products. Marine bacteria are known for their extensive biosynthetic capacity to produce diverse natural products, which are suitable for various biotechnology applications such as in agriculture, for treatment of fungal plant pathogens, and as antibiotics, for treatment of bacterial infections. This study aimed at discovering novel secondary metabolites from marine bacteria previously associated with novel marine invertebrate species endemic to the South African coast. The methodologies used in this study included a bioassay guided fractionation coupled to genome sequencing and mining. For the bioassay guided fractionation approach, the study first focused on screening marine bacteria for antimicrobial activity when cultured on 4 different media, against fungal strains previously shown to be virulent olive trunk pathogens. In parallel, the bacterial isolates with the most inhibitory activity against the fungal pathogens were also screened for antimicrobial activity against 4 indicator strains including Gram-negative Escherichia coli 1699 (E. coli), Pseudomonas putida, and Gram-positive Staphylococcus epidermidis ATCC14990, and Bacillus cereus ATCC10702. One of the marine bacterial isolates, PE6-126, showed diverse antimicrobial activity including antibacterial and antifungal activity against the tested strains. The genome sequencing data revealed that this isolate was B. cereus based on the average nucleotide identity (ANI) (>99%) to reference strains. antiSMASH analysis of the genome revealed nine predicted secondary metabolite clusters including bacteriocins (2), non-ribosomal peptide synthetase (NRPS) (2), siderophore (1), sactipeptide (1), betalactone (1), linear azol(in)e-containing peptides (LAP) - bacteriocin (1) and a terpene (1). Some of these pathways had low to no sequence similarity to known pathways, indicating the potential of these pathways to produce novel compounds. One of the pathways showed very high sequence similarity to the thuricin CD pathway in Bacillus thuringiensis. Considering that thuricin CD has been reported to have antimicrobial activity against B. cereus (ATCC1072), it was hypothesised that it could also be produced by PE6-126. However, the antimicrobial extract from PE6-126 was tested for sensitivity to proteinase K and heat treatment, which thuricin CD is known to be sensitive to. The results revealed that the antimicrobial activity was not lost after treatment, implying that a different metabolite could be responsible for the anti-B. cereus activity. In addition, PE6-126 initially displayed antimicrobial activity against a multi-drug resistant E. coli 1699, suggesting some of the antimicrobial compound/(s) produced by this strain could potentially be novel. The bioassay-guided fractionation approach coupled to Liquid Chromatography Mass Spectrometry (LC-MS) did not lead to identification of the antimicrobial compound/(s), therefore it remains a question whether the secondary metabolite pathways predicted by antiSMASH lead to the production of the active compound/(s). The results from this study showed that even well studied species have the potential to synthesize as yet undescribed compounds, based on the novelty of some of the pathways. This study highlights the importance of employing a genome-guided approach in drug discovery, as there may be many novel compounds to discover from biosynthetic pathways that have not yet been characterised. Further research is needed to identify the antimicrobial compound/(s) produced by PE6-126.

<p>Nonribosomal peptides, polyketides, and fatty acids comprise a distinct subset of microbial secondary metabolites produced by similar biosynthetic methods and exhibit broad structural diversity with a high propensity for biological activity. Dedicated studies of these specific microbial small molecules have identified numerous potent actions towards human cells with many clinical translations. Interestingly, most therapeutically used nonribosomal peptides and polyketides were discovered from soil bacteria, meanwhile, bacteria that have co-evolved within a human context, the human microbiota, have barely been explored for secondary metabolites. The central goal of this thesis is to explore the secondary metabolome of human microbiota for nonribosomal peptides and polyketides, which are hypothesized to possess biological activities significant within the human host context. Candidate organisms were chosen for their established connections to human health and evidence suggestive of secondary metabolite production. Specifically, questions about gene to molecule prediction capability, metabolite production, structural diversity, and biological activity were explored from studies of the dental caries linked Streptococcus mutans UA159, from the probiotic Lactobacillus plantarum WCFS1, and the Crohn’s disease associated Faecalibacterium prausnitzii.</p> / Master of Science (MSc)