Biology

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This is the collection for the University of Waterloo's Department of Biology.

Research outputs are organized by type (eg. Master Thesis, Article, Conference Paper).

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Now showing 1 - 7 of 7

Development and analysis of molecular methods for functional metagenomics of the human gut microbiome
(University of Waterloo, 2016-03-21) Lam, Kathy; Charles, Trevor
Interest in the human microbiome has risen quickly in recent years as the microbes that live in and on our body have been implicated in a growing number of human health and disease states. This interest has been supported by advances in DNA sequencing technology that have allowed us to obtain vast amounts of sequence data, and yet we have difficulty assigning function to many of the gene sequences obtained. As research on the role of these microorganisms continues, there will be an increased need for high-throughput methods that can provide knowledge of microbial gene function. Functional metagenomics is one such method, and it relies on first cloning environmental DNA to generate metagenomic libraries that are maintained in Escherichia coli and second, screening the cloned DNA for particular functions of interest. This powerful function-first method allows for the isolation of genes whose role may not have been predicted using DNA sequence homology. This thesis describes the analysis of techniques used in functional metagenomics research, as well as the development of new strategies to aid in functional screening of metagenomic libraries, particularly those constructed from gut-derived DNA. The work is divided into four data chapters that each explore a distinct aspect of the functional metagenomics approach. The first data chapter describes the evaluation of a pooled strategy for sequencing cosmid clones that were previously isolated in functional screens of metagenomic libraries. Ninety-two large-insert clones were pooled for Illumina-sequencing and the assembled sequence data were evaluated against reference sequence data that were obtained from individual barcoded Illumina sequencing of the same clones. The results indicated that a pooled strategy works well provided that sufficient sequencing depth is obtained and that pooled clones do not share sequence similarity to the extent that would be problematic for assembly of short reads that derive from those clones. The second data chapter is an exploration of possible causes for the known cloning bias of metagenomic libraries, by comparing environmental DNA before cloning to the DNA cloned in the final metagenomic library in E. coli. For a human gut metagenomic library, DNA was sampled and Illumina-sequenced at three different steps during the construction of the library. Analyses of the sequence data showed that there was indeed major bias in the final library, but that the bias was not due to fragmentation of the DNA during the cloning process as has been previously suggested; rather, the data were consistent with alternative hypotheses that suggest bias occurs after the DNA is introduced into E. coli, and analyses provide support for the hypothesis that spurious transcription of foreign DNA in E. coli may be contributing to the bias of libraries. Bias was also examined for a soil metagenomic library using 16S rRNA gene sequencing and though broad phylum-level biases were not as severe as observed for the human gut library, analyses revealed a bias in the relative abundance of individual OTUs. The third data chapter describes efforts to develop Bacteroides thetaiotaomicron (B. theta) VPI-5482 as a surrogate host for screening metagenomic libraries constructed from human gut-derived DNA. In this strategy, metagenomic libraries that have been constructed in E. coli can be transferred to B. theta using triparental conjugation. A member of the Bacteroidetes was chosen to specifically address the likely barrier to gene expression in E. coli of DNA that originates from this phylum. To allow the library to be replicated in B. theta, a B. theta-compatible library cloning vector was constructed, and this vector was used to generate genomic and metagenomic clone libraries. A metagenomic library was successfully screened in B. theta, leading to functional complementation of a B. theta mutant strain unable to grow on chondroitin sulfate as sole carbon source. However, further examination of the complemented clones indicated that the library clone DNA had integrated into the B. theta mutant genome. To address this problem, an alternative method for screening was devised, and although this method demonstrates that screening in B. theta remains feasible, more work is required to optimize the conjugation efficiency and the level of throughput. The fourth and last data chapter is an exploration of the use of transcriptional terminator elements in library cloning vectors, inspired by the results of previous chapters. Two unidirectional transcriptional terminators were added to a copy number-inducible fosmid vector, flanking the cloning site, with the intention of reducing insert-born transcription into the vector backbone. The terminators were tested using a reporter gene to confirm their functionality in this context, and derivative vectors were generated for future testing of whether or in what contexts terminators may help alleviate cloning bias in metagenomic libraries. The work described in this thesis contributes to method advancement for functional metagenomics through the analysis of a cost-effective strategy for sequencing library clones, the examination of potential causes of sequence bias in metagenomic libraries, the development of a surrogate host for more productive functional screening, and the consideration of vector elements that may improve metagenomic library stability in E. coli.
Discovering new viral lineages and estimating their abundance in wastewater
(University of Waterloo, 2022-09-27) Ellmen, Isaac; Charles, Trevor; Nissimov, Jozef
Wastewater surveillance of SARS-CoV-2 has emerged as a critical tool for tracking the spread of COVID-19. In addition to estimating the relative case numbers using qPCR, SARS-CoV-2 genomic RNA can be extracted from wastewater and sequenced. The sequenced genomes provide information about which lineages, in particular which variants of concern (VOCs) are present in a community. Wastewater RNA sequencing data has two distinct challenges: First, the genomes are highly fragmented and the alignments often have poor genome coverage. Second, the samples are comprised of a mixture of genomes so mutations cannot be directly attributed to a single lineage. In this thesis, I explore methods to overcome these two challenges to extract useful information from the samples. First, I look at the problem of determining the relative abundance of VOCs. Most existing techniques only consider mutations which are unique to a particular VOC which massively reduces the amount of usable data. I introduce a new technique which extends mean and median frequencies over shared mutations in order to make use of the huge pool of shared mutations. Next, I investigate strategies for designing single-amplicon sequencing methods. I look at selecting single amplicons which are well-conserved and rich in information. I also design a single amplicon which is capable of amplifying multiple coronaviruses. I conclude the SARS-CoV-2 work by providing a technique which can identify novel lineages and sublineages from wastewater sequencing runs. Finally, I show that the techniques for analyzing SARS-CoV-2 in wastewater can also be applied to an important plant pathogen, the Tomato Brown Rugose Fruit Virus.
Experimental characterization of a catalytically active flagellin variant in Clostridium haemolyticum
(University of Waterloo, 2017-04-18) Bandukwala, Hina; Doxey, Andrew; Charles, Trevor
The bacterial flagellum is made up of approximately 20,000 subunits of the monomeric protein, flagellin, and plays a role in cell motility and pathogenesis. The extreme sequence diversity within the hypervariable region of flagellin genes observed across phyla suggests hidden functional diversity. This thesis outlines the discovery of the first family of flagellin variants with proteolytic activity. A multi-faceted approach revealed a conserved HExxH motif within the hypervariable region (HVR) of these flagellin variants. The motif is characteristic of the Gluzincin family of thermolysin-like peptidases and was found to be conserved in 74 bacterial species spanning over 32 genera. Experimental validation began with the recombinant expression and purification of the HVR of the flagellin FliA(H) from the species Clostridium haemolyticum, an animal pathogen. An approach using mass spectrometry and proteomics revealed that the substrate specificity of this flagellin protease is similar to that of zinc-dependant matrix metallopeptidases (MMPs). Furthermore, peptide sequencing of harvested C.haemolyticum flagellar filaments revealed that the proteolytic flagellin was the second most dominant flagellin component and was also shown to have MMP-like protease activity. Considering the expanded functional repertoire of this organelle in the recent years, this flagellin-associated protease may play a role in chemotaxis, biofilm formation, adhesion and pathogenesis.
Functional characterization and bioinformatic analysis of glutamine synthetases of Ochrobactrum anthropi
(University of Waterloo, 2020-12-22) Alzahrani, Saud; Charles, Trevor
Glutamine synthetase (GS) is an essential enzyme required for the conversion of ammonium (NH4+) into glutamine. It is commonly classified into GSI, GSII, and GSIII based on molecular size, number of subunits, underlying regulation and enzyme structure. GSI is further subdivided into GSI-α and GSI-β. The latter form contains a conserved motif (NLYDLP) for the adenylation of a tyrosine residue near the active site and insertion of a specific 25-amino acid residue domain. Five ORFs were described to have GS activity in the genome of Ochrobactrum anthropi ATCC 49188. However, there are no studies regarding their functional significance and bioinformatic analysis. Therefore, with thorough bioinformatic analysis, I identified and classified these five putative distantly related GSs. Moreover, I modified the previously reported conserved motif (NLYDLP) for adenylation of tyrosine at the N-terminus of GSI-β to N/D-LYDLP. Using this modified motif as criterion as well as insertion of specific 25 amino acids, I identified the chromosome I GS (Oant_2087) of O. anthropi as GSI-β. Since those features were absent in the GS from pONAT01(Oant_4491) and the two GS of chromosome II (Oant_3936 and Oant_3881), they were identified as GSI-α. Chromosome II GS (Oant_4157) is GSII type. Further, my results from the bioinformatic analysis strongly indicate that GS on pONAT01 was acquired through horizontal gene transfer from either Ensifer adhaerens plasmid or Ensifer adhaerens chromosome 1. Interestingly, this transferred enzyme was found to be functional in O. anthropi as knocking it out from pONAT01 of O. anthropi resulted in 50% reduction in enzyme activity. All these findings will provide an insight the underlying mechanism of regulation for the five GSs present in O. anthropi and could serve as the basis for further investigation into the molecular functions of these five GSs, and the plasmid based one in particular.
Glyphosate as a Source of Phosphorus for Bacteria: Selection of an Enrichment Culture for Glyphosate Degradation, and Analysis of the C-P Lyase Pathway
(University of Waterloo, 2024-12-17) Van Dyk, Adrian; Charles, Trevor
Glyphosate is one of the world’s most widely used herbicides, the active ingredient in Roundup as well as many generic herbicide brands. Though it is one of the safer herbicides, its ubiquity necessitates further study of the pathways that bacteria can use to degrade it. This study investigates the ability of bacteria to break the strong carbon-phosphorus bond of glyphosate. The carbon-phosphorus bond has the highest activation energy of the bonds within glyphosate, and breaking this bond is therefore a rate-limiting step of complete glyphosate mineralization. By optimizing a defined media and supplementing with glyphosate as the only source of phosphorus, the ability of bacterial species to grow on glyphosate as the sole source of phosphorus can be tested and compared to their growth when easily accessible ortho-phosphate is provided. This defined media was used to create an enrichment culture to study which species are selected for by glyphosate as the sole source of phosphorus, and compare them to cultures selected on easily accessible ortho-phosphate. Glyphosate as sole source of phosphorus selected for a different suite of species, especially those in the Rhizobiaceae family, than ortho-phosphate selected for. The Rhizobiaceae genus Agrobacterium was found to contain particularly good degraders of glyphosate, and a deletion and replacement experiment revealed that the C-P lyase pathway is required for glyphosate degradation in an Agrobacterium pusense isolate. Genome and growth comparisons between A. pusense, A. tumefaciens, and A. fabrum isolates revealed them all to be able to degrade glyphosate, and that they share very similar C-P lyase operons, despite having significant heterogeneity in their genomes. Additionally, a commercial enzyme linked immunosorbent assay was used to quantify glyphosate degradation. This work adds support to the theory that C-P lyase is required for glyphosate degradation in Agrobacterium, and sets the stage for additional research into glyphosate degradation pathways by bacteria.
Metabolic engineering strategies for biomanufacturing of chemicals using Yarrowia lipolytica and Escherichia coli
(University of Waterloo, 2024-04-24) Kefale, Teshager Bitew; Charles, Trevor
This dissertation advances metabolic engineering by optimizing the genetic and metabolic capabilities of Yarrowia lipolytica and Escherichia coli to enhance their applications in biotechnology. It focuses on improving Y. lipolytica's mannitol and amino acids production by varying fermentation temperatures and employing techniques like shake flask fermentation, HPLC, and NMR. Notably, mannitol production was enhanced through targeted modifications of FBP1 gene at elevated temperatures. RNAseq analyses highlighted shifts in metabolic pathways under thermal stress, markedly in lipid, sugar and amino acids metabolism. Additionally, a dual-gRNA CRISPR-Cas9 system was integrated within the pCRISPRYL2 plasmid, noticeably improving genetic editing precision by overcoming the constraints of the non-homologous end joining (NHEJ) pathway. Furthermore, the study pioneered a Cell-Free Metabolic Engineering (CFME) strategy to synthesize 5-Aminolevulinic Acid (5-ALA) utilizing optimized enzymatic reactions and operational conditions, presenting a scalable and eco-friendly alternative to conventional whole-cell systems. In parallel, engineered E. coli demonstrated robust heme production capabilities in both whole-cell and cell-free systems. Heme derivatives, including valuable pigments like biliverdin, Phycocyanobilin (PCB) and Phycoerythrobilin (PEB) were also produced at a 1L bioreactor scale utilizing E. coli engineered with unexplored enzymes. Overall, this work not only expands the scope of metabolic engineering but also sets a foundational work for future innovations in biomanufacturing.
The Proximon: Representation, Evaluation, and Applications of Metagenomic Functional Interactions
(University of Waterloo, 2017-09-19) Vey, Gregory; Charles, Trevor
The effective use of metagenomic functional interactions represents a key prospect for a variety of applications in the field of functional metagenomics. By definition, metagenomic operons represent such interactions but many operon predictions protocols rely on information about orthology and/or gene function that is frequently unavailable for metagenomic genes. In this thesis, I introduce the proposition of the proximon as a unit of functional interaction that is intended for use in metagenomic scenarios where supplemental information is sparse. The proximon is defined as a series of co-directional genes where minimal intergenic distance exists between any two consecutive member genes within the same proximon. In particular, the proximon is presented here as a biological abstraction aimed at facilitating bioinformatics and computational goals. In this thesis, proximons are constructed as information theoretic entities and employed in a variety of contexts related to functional metagenomics. I begin by implementing a computational representation for proximon data and demonstrate its utility through the deployment of a public database. Next, I perform a formal validation where proximons are contrasted against known operons by using the Escherichia coli K-12 model organism as a gold standard to measure the extent to which proximons emulate actual operons. This is followed by a demonstration of how proximon data can be applied to infer potential functional networks and depict potential functional modules. I conclude by enumerating the limitations of the research performed here and I present objectives and goals for future work.

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