>Magister Scientiae - MSc / Pharmaceutical research and development strategies rely on the constant discovery of novel natural products as potential drugs. Recent studies have shown that the microorganisms associated with sponges are the true producers of some previously isolated compounds. This study created a large collection of bacterial symbionts associated with the South African marine sponge, Hamacantha esperioides. The bioactivity assays performed, showed that 44 isolates produced compounds with antimicrobial or anti-inflammatory activity. The successful identification of novel species that produce potential natural products highlights the importance of cultivation-dependent methods. To further screen for natural products, a cultivation-independent approach was used. A sequenced-based method, based on the biosynthetic genes of polytheonamide, was developed to screen for proteusins in sponge metagenomic DNA and the genomes of bacterial symbionts. The degenerate primers could amplify the targeted genes from DNA known to contain homologues. Evaluation of the primers' specificity showed non-specific amplification of genes, some containing similar conserved domains as the target genes. This study demonstrated that the use and development of cultivation-dependent and -independent screens are important for the discovery of novel natural products from the symbiotic bacteria of South African sponges. / National Research Foundation (NRF)

Thesis advisor: Marc Snapper / Thesis advisor: Steven Bruner / Peptide natural products have diverse, elaborate scaffolds and are important leads in the development of new drugs. A complete understanding of the natural biosynthetic pathways of these compounds can improve chemical syntheses and boost bioengineering efforts. There are two classes of peptide natural products: ribosomal and nonribosomal peptides. Ribosomally produced and posttranslationally modified peptides (RiPPs) are produced by the ribosome using the 20 canonical amino acids and undergo extensive tailoring to yield the active natural products. Nonribosomal peptides (NRPs) are assembled through an enzyme dependent system and can incorporate over 500 different amino and acyl building blocks to impart complexity. These peptides can also undergo additional tailoring to further modify the core peptide. The microviridins are a class of RiPPs that are modified by two ATP dependent ligases to create a total of three macrocyclic bonds. We have solved the three dimensional protein structures of each of these ligases to establish the mechanism of substrate recognition and cyclization. Vancomycin is a NRP that contains five nonproteinogenic aromatic amino acids that are necessary for biological activity. One of these amino acids is derived from a polyketide pathway and undergoes a four-electron oxidation by a cofactor independent dioxygenase, DpgC. We have solved the structure of this enzyme and have established a radical mechanism. We have investigated this mechanism using synthetic probes and mutagenesis. We have examined O₂ binding using molecular dynamics and mutagenesis. NRPs are synthesized by the multidomain, modular nonribosomal peptide synthetases (NRPSs) in an enzyme templated, ATP-dependent manner. We have synthesized domain specific probes to study the structures and mechanisms of these pathways. Our continued work will provide the insight necessary to manipulate these pathways to provide biologically active compounds. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

>Magister Scientiae - MSc / The continued emergence of bacterial resistance to the antibiotics currently employed to treat several diseases has added to the urgency to discover and develop novel antibiotics. It is well established that natural products have been the source of the most effective antibiotics that are currently being used to treat infectious diseases and they remain a major source for drug production. Natural products derived from marine microorganisms have received much attention in recent years due to their applications in human health. One of the biggest bottlenecks in the drug discovery pipeline is the rediscovery of known compounds. Hence, dereplication strategies such as genome sequencing, genome mining and LCMS/MS among others, are essential for unlocking novel chemistry as it directs compound discovery away from previously described compounds. In this study, the genome of a marine microorganism, Thalassomonas viridans XOM25T was mined and its antimicrobial activity was assessed against a range of microorganisms. Genome sequencing data revealed that T. viridans is a novel bacterium with an average nucleotide identity of 81% to its closest relative T. actiniarum. Furthermore, genome mining data revealed that 20% of the genome was committed to secondary metabolisms and that the pathways were highly novel at a sequence level. To our knowledge, this species has not previously been exploited for its antimicrobial activity. Hence, the aim of this study was to screen for bioactivity and identify the biosynthetic gene/s responsible for the observed bioactivity in T. viridans using a bioassay-and-genome- guided isolation approach to assess the bioactive agent. The bioassay-guided fractionation approach coupled to LCMS/MS led to the identification of a novel antimicrobial protein, TVP1. Bioinformatic analyses showed that TVP1 is a novel antimicrobial protein that is found in the tail region of a prophage in the T. viridans genome. Phage-derived proteins have previously been shown to induce larval settlement in some marine invertebrates. Since the mechanism of action of TVP1 remains unknown, it remains a speculation whether it may offer a similar function. More research is required to determine the biotechnological application and the role of TVP1 in its host and natural environment.

Future climate scenarios in the Baltic Sea project increasing sea surface temperature, as well as increasing precipitation and river runoff resulting in decreased salinity. These changes can severely impact the dynamics and function of brackish water communities, specifically phytoplankton. Phytoplankton are a significant source of organic matter to other trophic levels, and some species can be toxic. Their response to future climate conditions is of great relevance for the health of humans and aquatic ecosystems. The aim of this thesis was to assess the potential for climate-induced changes, such as decreasing salinity, to affect phytoplankton dynamics, physiology and chemical profiles in the Baltic Sea.      Phytoplankton successional patterns in the Baltic Proper consist of a spring bloom where diatoms and dinoflagellates co-occur and a summer bloom dominated by filamentous/colonial cyanobacteria. The consensus is that future warmer conditions will promote filamentous/colonial cyanobacteria blooms. This thesis shows that phytoplankton biomass in the spring bloom was lower in years with milder winters compared with cold winters. This suggests that in terms of annual carbon export to higher trophic levels, loss of biomass from the spring bloom is unlikely to be compensated by summer cyanobacteria. High frequency sampling of phytoplankton performed in this thesis revealed a strong relationship between the dynamics of pico- and filamentous cyanobacteria. Large genetic diversity was found in cyanobacterial populations with high niche differentiation among the same species. At community level, high temperature and low salinity were the main factors shaping the summer cyanobacterial composition. These conditions may promote the predominance of opportunistic filamentous cyanobacteria, e.g. Nodularia spumigena. This species produces various bioactive compounds, including non-ribosomal peptides such as the hepatotoxin nodularin. In this work, N. spumigena subpopulations evolved different physiological strategies, including chemical profiles, to cope with salinity stress. This high phenotypic plasticity ensures survival in future climate conditions. Under salinity stress, some subpopulations displayed shorter filaments as a trade-off. This indicates that the future freshening of the Baltic Sea may promote grazing on filamentous cyanobacteria and modify carbon flows in the ecosystem. In this thesis, Baltic N. spumigena chemotypes and genotypes grouped into two main clusters without influence of geographical origin. Thus, chemical profiling can be used to explore conspecific diversity in closely genetically related N. spumigena subpopulations.      Overall, this thesis has significantly expanded the knowledge on phytoplankton community and population responses to short- and long-term environmental changes, relevant to project the impacts of future climate conditions in the Baltic Sea.

La colistine, antibiotique appartenant à la famille des polymyxines, est un polypeptide cyclique, cationique, ciblant les membranes bactériennes. Elle est produite par Paenibacillus polymyxa via des complexes multi-enzymatiques appelés Non-Ribosomal Peptides Synthétases (NRPS). Dans le cas de la mucoviscidose, et malgré des effets secondaires importants, la colistine est utilisée comme ultime recours pour lutter contre les bactéries Gram-négatives multirésistantes responsables d’infections pulmonaires dont Pseudomonas aeruginosa. Jusqu’ici les systèmes génétiques à l’origine de la production de la colistine étaient peu connus. Au cours de cette étude, nous avons caractérisé par LC-MS haute résolution des molécules antimicrobiennes, dont des colistines, produites par un nouveau Paenibacillus. Afin d’identifier et de cloner le cluster de gène responsable de la production de ces antibiotiques, une banque d’ADN génomique a été construite et criblée par homologie de séquence avec des systèmes de production déjà connus. Ce criblage a permis de sélectionner quatre clones d’intérêt. L’étude in silico de leurs séquences a permis d’identifier les différents modules d’un nouveau cluster NRPS qui serait à l’origine de la synthèse de variants de la colistine. À terme, cette découverte pourrait permettre de mieux contrôler la production de la colistine et d’obtenir des composés plus actifs et/ou présentant des effets secondaires amoindris. En parallèle à ce premier travail, nous avons également recherché la présence de nouvelles NRPS chez une centaine de micro-organismes issus d’une station d’étude environnementale du laboratoire (vasière intertidale). Ce travail a permis de découvrir des nouvelles séquences et d’isoler un nouveau micro-organisme producteur d’antibiotique(s). / Colistin is a cationic cyclic polypeptide antibiotic belonging to the polymyxin family and targeting bacterial membranes. It is produced by Paenibacillus polymyxa through a Nonribosomal Peptide Synthetase (NRPS) mechanism. In the context of cystic fibrosis (CF), colistin is used for the treatment of lung infections caused by multiresistant Gram-negative bacteria including Pseudomonas aeruginosa. Unfortunately, this molecule is also known for its strong side effects. So far, genetic systems controlling the production of polymyxins were little known. In this study we characterized by High-resolution LC-MS the antimicrobial molecules, including colistins, of a new Paenibacillus. A genomic library of this strain was constructed and screened to identify genes involved in the production of these antibiotics. A degenerated PCR screening was performed and allowed to select four clones in the genomic library. In silico study allowed to identify a new NRPS gene cluster responsible for the biosynthesis of colistin variants. In the future, this work might allow the harnessing of the production of colistin derived structures, more active and/or showing fewer side effects. In parallel, a second investigation was performed in order to find new NRPS genes in a collection of one hundred intertidal mudflat bacterial isolates. This work has allowed the identification of new sequences and the characterization of a new antimicrobial producing strain.