Molecular biology of B vitamin metabolism genes and their regulation in Phaeodactylum tricornutum

Thiamine (Vitamin B1) in its diphosphate form is an essential cofactor for virtually all organisms. Thiamine biosynthesis is an expensive metabolic process involving suicidal and low-turnover enzymes, and many organisms have lost the ability to synthesise thiamine and depend on an exogenous source. Those prototrophs that synthesise thiamine, including species of bacteria, fungi, green algae and plants, have been shown to tightly regulate their thiamine-related genes in response to exogenous thiamine. In many cases this is via riboswitches, sequences in mRNA that fold into a tertiary structure (aptamer) able to specifically recognise a ligand and mediate a change of genetic expression in response. In diatoms, marine algae responsible for ~20 % of net primary productivity, THIC, which encodes one of the first enzymes in the thiamine biosynthetic pathway, has been predicted to have a thiamine pyrophosphate (TPP) riboswitch in the 3’UTR. Additionally, THIC has been previously observed to be downregulated by cobalamin (vitamin B12) supplementation in diatoms reflecting the interconnectedness and co-regulation of different B vitamin metabolisms. The aim of this thesis is to investigate different aspects of the regulation of thiamine and cobalamin-related genes and the transport and metabolism of thiamine and cobalamin in diatoms. Homology-based bioinformatic tools confirmed that all available diatom genomes contain homologues of THIC encoding the first enzyme in the pyrimidine branch of the thiamine biosynthesis pathway in bacteria, and/or NMT1, which catalyses the equivalent step in fungi. Additionally, it was found that many of these genes, and those coding for SSSP, a putative thiamine transporter, had predicted TPP aptamers in their 3’UTRs. A conserved polyadenylation site was found overlapping the diatom TPP aptamer sequences, which might be involved in a hypothetical mechanism of action. However, experiments using RT-qPCR and 3’-RACE showed that the THIC and SSSP genes in Phaeodactylum tricornutum did not respond to thiamine supplementation at the transcriptional or post-transcriptional level, even though thiamine is taken up by the cells. Furthermore, unlike in the green alga Chlamydomonas reinhardtii, which has experimentally characterised TPP riboswitches, the diatoms P. tricornutum and Thalassiosira pseudonana were insensitive to pyrithiamine, a thiamine antimetabolite that primarily inhibits growth by binding TPP riboswitches and downregulating thiamine biosynthesis genes. Reporter constructs confirmed that the PtTHIC regulatory sequences (promoter, 5’UTR, 3’UTR) could not regulate heterologous genes in response to thiamine supplementation. Nor did the PtTHIC aptamer respond in C. reinhardtii chimeric constructs. Finally, site-directed mutagenesis of the PtTHIC aptamer did not alter endogenous thiamine levels in P. tricornutum, in contrast to equivalent mutations in other organisms with confirmed riboswitches. Together, these results demonstrate that the predicted TPP aptamer in the PtTHIC gene does not act as a riboswitch. RT-qPCR experiments suggested that PtTHIC is downregulated by cobalamin supplementation similarly to cobalamin-independent methionine synthase (PtMETE). A motif-prediction algorithm revealed a conserved 14 bp motif in the promoters of THIC, METE and other cobalamin-downregulated genes in diatoms that could indicate the co-regulation of cobalamin and thiamine metabolism. Reporter construct experiments confirmed that the PtTHIC promoter could downregulate a reporter in response to cobalamin and that the PtMETE promoter with a mutation in the motif did not drive the expression of a reporter, demonstrating that the conserved motif is necessary for gene expression. Finally, a CRISPR/Cas9 method was developed in collaboration with other members of the group to generate knock-out mutants of several genes in P. tricornutum. The THIC knock-out did not require thiamine for growth in f/2 minimal media, suggesting P. tricornutum can obtain thiamine or its pyrimidine moiety from an alternative source or pathway. A knock-out of SSSP demonstrated that this gene is required for thiamine uptake. The METE knock-out was auxotrophic for cobalamin and the Cobalamin Acquisition Protein 1 (CBA1) knock-out confirmed it is necessary for cobalamin uptake. Taken together, the results in this thesis provide new insights into thiamine and cobalamin metabolism, transport and genetic regulation in diatoms. Unexpected results such as the unaltered growth of the THIC knock-out and the lack of function of the P. tricornutum predicted TPP riboswitches stand in contrast with existing knowledge of thiamine metabolism in other organisms. This raises important questions to understand the role of thiamine in the physiology, ecology and evolution of an algal group with global ecological relevance

Exploring Protein Interactions Through the Development of in silico Methodologies and Genetic Analysis

Significant advances in sequencing technologies over the past twenty years have drastically changed scientific approaches to solving biological problems. A shift towards big data driven research and computational predictions has been made possible by vast sequence and structural databases, populated by sequences determined by modern sequencing methods. This thesis focuses on the use of these resources to predict protein function in the form of ligand-binding sites, in the determination of positions in proteins that are pivotal in virus pathogenesis, and in the elucidation of amino acids in myosin proteins which are adapted to the mass of mammalian species. Firstly, a tool developed to predict protein-ligand interactions is introduced that utilises up-to-date data resources, tools, and machine learning algorithms to identify, and assign confidence to, candidate ligand-binding positions in proteins. This work builds on 3DLigandSite in the first major update since its release. A new webserver is presented in this work for users to query their protein sequences for probable ligand-binding residues. Sequence analysis of conventional myosin sequences identified positions associated with increased body mass and no association with clade in the second area explored in this thesis. In beta-cardiac myosin, nine positions were found to adjust contraction velocity when mutated, highlighting the predictive power from alignments combined with a measurable phenotype. This further shows the time- and cost-saving benefits of combined computational and experimental methods. Finally, differentially conserved positions were identified between SARS-CoV and SARS-CoV-2, determining probable variants for the source of phenotypic differences between these viruses. A webserver for other researchers to perform these analyses was developed, enabling users to query coronavirus sequences for these variants. Our analysis included all sequences available from GISAID at the time of writing. Some of the DCPs identified using these methods are shown to be found in the ACE2 binding site of the spike protein, and in close vicinity to serine protease binding sites, which are essential for virus entry into the host