Phylogenetic relationships of the Wolbachia of nematodes and arthropods

Wolbachia are well known as bacterial symbionts of arthropods, where they are reproductive parasites, but have also been described from nematode hosts, where the symbiotic interaction has features of mutualism. The majority of arthropod Wolbachia belong to clades A and B, while nematode Wolbachia mostly belong to clades C and D, but these relationships have been based on analysis of a small number of genes. To investigate the evolution and relationships of Wolbachia symbionts we have sequenced over 70 kb of the genome of wOvo, a Wolbachia from the human-parasitic nematode Onchocerca volvulus, and compared the genes identified to orthologues in other sequenced Wolbachia genomes. In comparisons of conserved local synteny, we find that wBm, from the nematode Brugia malayi, and wMel, from Drosophila melanogaster, are more similar to each other than either is to wOvo. Phylogenetic analysis of the protein-coding and ribosomal RNA genes on the sequenced fragments supports reciprocal monophyly of nematode and arthropod Wolbachia. The nematode Wolbachia did not arise from within the A clade of arthropod Wolbachia, and the root of the Wolbachia clade lies between the nematode and arthropod symbionts. Using the wOvo sequence, we identified a lateral transfer event whereby segments of the Wolbachia genome were inserted into the Onchocerca nuclear genome. This event predated the separation of the human parasite O. volvulus from its cattle-parasitic sister species, O. ochengi. The long association between filarial nematodes and Wolbachia symbionts may permit more frequent genetic exchange between their genomes

Investigating Genetic (IN)Compatibility Between Temperate Phages and CRISPR-CAS Systems in Staphylococcus Aureus

Prokaryotic organisms employ various mechanisms for defending against parasitism by viruses and other mobile genetic elements. One form of defense comprises the adaptive immune systems derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci and CRISPR-associated (cas) genes. CRISPR-Cas immune systems enable the acquisition of heritable resistance to specific mobile genetic elements on the basis of nucleic acid sequence recognition, but do not necessarily discriminate between target elements which are burdensome and those which are beneficial. My thesis is concerned with the consequences of CRISPR-Cas immunity directed at a particular breed of bacterial DNA viruses, known as temperate phages, which cause both harmful (lytic) and benign (lysogenic) infections under different conditions. Initial studies investigating prokaryotic CRISPR-Cas immunity seemed to indicate that functional, DNA-targeting systems cannot stably co-exist with their target elements in vivo. For example, in studies where immunity was directed at temperate phages, DNA-targeting CRISPR-Cas systems were found to prevent both lysogenic and lytic infections except when targeting was altogether abrogated via mutation or inhibition of the CRISPR-Cas system. The first part of my thesis work includes in vivo experiments which challenged the generality of this view, with regard to the different types of DNA-targeting CRISPR-Cas systems. Namely, I demonstrated that a staphylococcal branch of the ‘type III’ CRISPR-Cas systems is capable of tolerating lysogenic infections by specific temperate phages which are otherwise targeted during lytic infections. I further established that the capacity for conditional temperate phage tolerance results from a transcription-dependent targeting modality which was not anticipated for this particular DNA-targeting type III system. In contrast, I observed only the expected genetic escape outcomes when temperate phages were targeted by a ‘type II’ CRISPR-Cas system with a transcription-independent (Cas9-based) DNA targeting modality. These findings laid the groundwork for subsequent studies of CRISPR-Cas immunity to phages in Staphylococcus aureus hosts, and guided my colleagues towards in vitro characterization of the type III system’s transcription-dependent targeting mechanism. CRISPR-Cas systems have been identified in about 50% of sequenced bacterial genomes, and the factors which influence this distribution are still not fully understood. My description of conditional tolerance by a staphylococcal, type III CRISPR-Cas system illustrated that, in principle, these particular systems could stably co-exist with their temperate phage target elements in lysogenic hosts while maintaining their ability to protect against lytic infections. During the second part of my thesis work, I set out to define additional phenotypic consequences for the lysogenized lineages of S. aureus which maintain conditional tolerance, in an effort to better understand how this phenomenon might influence the distribution and stability of type III systems among natural isolates. Notably, I found that the maintenance of certain temperate-phage-targeting systems can incur fitness costs in lysogenic populations. I showed, furthermore, that these costs are potentially greater if more than one temperate phage is targeted in populations of double lysogens, but that they can be alleviated by mutations which do not abrogate phage targeting during lytic infections. Collectively, these findings imply that long-term maintenance of type III systems in natural populations of lysogens might require additional evolutionary fine-tuning, particularly among lineages which are prone to multiple infection

The Janthinobacterium sp. HH01 genome encodes a homologue of the V. cholerae CqsA and L. pneumophila LqsA autoinducer synthases

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes

The Hos2p Histone De-acetylase Promotes the Successful Completion of Cytokinesis in Schizosaccharomyces pombe.

A cell cycle checkpoint at the G2/M boundary of fission yeast cells ensures that the G2/M transition of daughter cells only occurs after successful cytokinesis in the mother cell. The Lst complex is necessary for proper functioning of this checkpoint and is orthologous to a human histone de-aceylase complex (HDAC3/NCOR2-SMRT), with a known role in cytokinesis. However, the fission yeast orthologue of the human HDAC3 histone de-acetylase, Hos2p, was never previously characterized with respect to the Lst complex. Through a combination of phenotypic analyses on hos2 mutants, live-cell imaging of Hos2p localization, live-cell imaging of cytokinesis dynamics as they relate to Hos2p’s cytokinetic regulatory function, and co-immunoprecipitation experiments, I showed that Hos2p is indeed a member of the Lst complex, and ensures faithful cytokinesis through its de-acetylase activity. Additionally, I used Western Blotting to show that the Lst complex is a stress-responsive complex that up-regulates expression of its Lstlp sub-unit in response to a sub-set of environmental stressors known to turn on the fission yeast core environmental stress response (CESR). Finally, I show that Hos2p inhibits growth and produces various abnormal phenotypes in a dose-dependent manner, although the biological significance of these observations is unclear

Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD's overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism

Identification of Protein-Protein Interactions of Amyotrophic Lateral Sclerosis Associated Protein TDP-43

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by progressive degeneration of upper and lower motor neurons in the brain and spinal cord. Multiple mutations are found in some of the proteins associated with ALS, including superoxide dismutase (SOD1), fused in sarcoma (FUS) and trans-activation response DNA-binding protein (TDP-43). TDP-43 is a DNA and RNA binding protein, well conserved, and ubiquitously expressed in all tissues. TDP-43 resides in the nucleus and sometimes shuttles between nucleus and cytoplasm. Mutations in TDP-43 leads to mislocalization of TDP-43 to the cytosol where it was ubiqutinated and hyperphosphsorylated, ultimately leading to neuronal cell death. The aim of this project is to identify and compare binding partners of both wild type (WT) and mutant TDP-43 using yeast two hybrid screening (Y2H). We identified PICK1 (Protein interacting with protein C-kinase) that binds to both wild type and disease causing mutant (M337V) TDP-43. Interestingly, PICK1 also interacts with other TDP-43 mutants (D169G, Q331K, G298S, and A315T), although the affinity of interaction is weaker

Increasing Production of Therapeutic mAbs in CHO Cells through Genetic Engineering

Between 2014 and 2018, the global market for therapeutic monoclonal antibodies (mAbs) rose from $60 billion to$115.2 billion with a projected value of $300 billion by 2025. These molecules are used to effectively treat some of the most challenging illnesses from auto-immune diseases to cancer. While mAbs are highly valuable with potent applications, their production at scale remains an outstanding challenge. These molecules are largely produced in Chinese Hamster Ovary (CHO) cells that require highly specific conditions to produce a useful product. Genetic engineering presents one solution to overcome productivity limits. With the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) systems, engineering the CHO genome has never been easier. CRISPR/Cas9 allows for site-specific editing and gene integration. Within the CHO genome, a variety of sites have been identified that warrant further investigation for editing. Among these sites is the gene C12orf35. The deletion of C12orf35 has been shown to lead to increased productivity in CHO cells. Additionally, C12orf35 has been identified as a site with a high transcription rate, implying that genes at this site are likely to be expressed more frequently. The gene coding for mammalian target of rapamycin (mTOR) has been demonstrated to alter CHO cell phenotype characteristics such as cell size, viable cell density, and antibody productivity when expressed transiently. This study aims to evaluate the potential synergism of deleting the gene C12orf35 by editing the gene coding for mTOR between a cut site made in C12orf35. Splicing the gene coding for mTOR at this site has the potential combined benefit of disrupting C12orf35 while simultaneously stably expressing the mTOR gene at a highly transcribed site in the CHO genome

Investigating HOX Protein Requirement for Tarsus Determination in Drosophila melanogaster

Generally, all bilaterans examined have similar conservation of HOX protein structure, function, expression, and requirement. However, at the level of being the same, it is unknown whether the HOX protein, Antennapedia, is required for tarsus determination in Drosophila melanogaster as in Tribolium casteneum, or whether the requirement of HOX proteins in determination of body parts diverges in insects. I proposed to use a heat shock-inducible nanobody (UAS- NSlmb-vhhGFP4 driven by hsp-GAL4) activated during the third larval stage in all cells to degrade thoracically expressed HOX proteins (Sex combs reduced, Antennapedia, and Ultrabithorax) tagged with green fluorescence protein (GFP) derivatives; GFP, YFP, CFP, or 17 amino acid epitope of GFP. Due to difficulties in establishing CRISPR mediated homologous recombination, only the initial steps have been completed, but the system is now established to determine whether HOX requirement for tarsus determination is conserved in insects