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 - 4 of 4

Bacterial biogeography of the rare Charitable Research Reserve
(University of Waterloo, 2015-09-30) Seuradge, Brent
Soil microbial communities play a dominant role in global biogeochemical cycles, with profound effects on agriculture, ecosystem stability, human health, and global climate. As a result, assessing their biogeographic patterns can help to further reveal mechanisms shaping their diversity and function in the environment. Furthermore, due to extensive spatial heterogeneity and environmental gradients, there is potential for overlooking key biogeographical patterns, critical metabolic processes, and novel bacterial taxa existing within deeper soil horizons that can be highly dependent on changes in land-usage. Additionally, an active area of research in soil microbial biogeography is assessing the extent to which current environmental or past historical factors constrain microbial community assemblages. The objectives of this study were to examine and characterize depth-dependent bacterial community characteristics across multiple land-use types to explore subsurface biogeographical patterns. I collected soil samples across seven distinct land-use types to depths of 45 cm, including old-growth and mature forests, decommissioned, and active agricultural fields from the rare Charitable Research Reserve (Cambridge, Ontario). Bacterial communities were characterized by sequencing of bacterial 16S rRNA gene amplicons coupled with multivariate statistical analyses from 376 soil samples. In addition, to explore functional and metabolic characteristics of collected soils, the PICRUSt algorithm was used to predict metagenomes of uncharacterized taxa. Soil bacterial communities across all sites were strongly influenced by depth. Upper soils (0–15 cm) and open field sites maintained higher bacterial alpha-diversity than deeper soils and forested sites. The magnitude of soil depth effects appeared to differ across environment types highlighting that land-use type also plays a significant role in shaping communities; bacterial communities across the field sites (i.e., grasslands and agricultural sites) were shown to be more strongly affected than forested sites. Soil pH, which exhibited a large gradient across samples, appeared to be largely responsible for differential shifts in communities with depth across land-use types especially considering that C, NH4+, NO3‾, moisture, and texture showed generally consistent trends with depth across all sites. This observation was further corroborated by NPMANOVA and CCA, which highlighted that pH was among the top explanatory variable explaining >15% of the variation in the dataset. This finding further emphasizes that pH is a strong predictor of bacterial community composition, not only across surface soils, but also within the soil subsurface. Overall, the impact of pH on soil bacterial community composition exceeded that of depth. The effect of land-use type on subsurface bacterial communities was found to be largely attributed to differences in dominant plant communities. Field sites were characterized by tall grasses whereas forest sites were characterized by woody tree species. Considering that plant inputs (i.e., root exudates, litter) are translocated through soils over time and affect the physicochemical environment, these findings further enforce that plants play important roles in structuring soil bacterial communities across environment types. In addition, contrary to evidence from the aboveground plant communities and site histories, there was no direct evidence of bacterial community succession throughout soils across the field sites sampled in this investigation. Instead, edaphic factors including soil texture, particularly sand, silt, clay, and moisture, appeared to govern changes in overall community composition across the field sites, highlighting the importance of the immediate physicochemical environment in shaping soil bacterial communities. Soils across all sites and depths were dominated by members of the Proteobacteria (33.2%), Actinobacteria (27.8%), Acidobacteria (14.9%), Chloroflexi (6.6%), Gemmatimonadetes (4.7%), Bacteroidetes (3.0%), Nitrospirae (2.1%), Firmicutes (2.3%), Verrucomicrobia (1.7%), and Latescibacteria (formerly WS3; 1.2%). In addition to observing trends in specific phyla with depth (e.g., Proteobacteria and Bacteroidetes), data also highlighted consistent depth-specific changes in OTU relative abundances. Although the majority of significant correlations were negative (indicating a decrease in abundance with increasing depth), Spearman’s correlation analysis found evidence for consistent positively correlated OTUs with depth. Notably, all positively depth-correlated OTUs were affiliated with uncultivated bacteria, further highlighting that subsurface environments are poorly studied. Correlation analyses were also conducted for pH. Nitrospirae and Chloroflexi members were among the top strongly and positively correlated taxa with pH, consistent with previous studies. Acidomicrobiia and Solibacteres classes, members of the Acidobacteria phylum, were found to be strongly and negatively correlated with pH, which is also consistent with previous research. These results further demonstrate the importance of pH in shaping soil bacterial communities considering that many taxa are adapted to narrow and specific growth and pH ranges. The PICRUSt results reflected observations noted in the taxonomy-based analysis. “Transporter” associated genes appeared to show differential abundances across land-use type. Forest sites, in particular site CA, a mature forest environment, had the lowest abundance of “transporter” associated genes. This result may further highlight pH effects on soil bacterial communities, considering that site CA had samples with the lowest pH and, consequently, the lowest species diversity. Overall, this research has set up baseline observations of bacterial community dynamics at the rare Charitable Research Reserve expanding on the few studies that have included soil depth as an environmental gradient and paving the way for future investigations. In addition, this study exemplifies important global environmental gradients including depth, land-usage, and soil biogeochemistry operating at smaller geographical scales across consistent underlying geology. Furthermore, this work has added insight concerning the interplay of the immediate physicochemical environment and past historical legacies in shaping soil microbial communities. Future research with the dataset generated will further explore bacterial taxa that vary in relation to pH and depth, in addition to phylogenetically novel taxa existing at low relative abundance, providing additional insight into the unexplored biodiversity of soil microbial communities.
Investigating Cyanobacterial Communities Across Canada and Characterizing the Seasonality of Cyanobacterial Toxin and Nitrogen Metabolism Genes in the Turkey Lakes Watershed Using Metagenomics
(University of Waterloo, 2023-08-08) Abed, Hadeel
The long evolutionary history of gram-negative cyanobacteria has enabled them to tolerate extreme conditions, succeed in numerous habitats, and exploit special nutrients, all while resisting harmful anthropogenic and climate change exacerbated influences. Increases in harmful cyanobacterial blooms (cHABs) pose considerable challenges to the provision of safe drinking water globally. Cyanobacterial blooms are most often recorded in eutrophic lakes where they have sufficient nutrients for proliferation, however, this has led to the misunderstanding that they do not pose risks in oligotrophic lakes because of the lower nutrient availability needed to form harmful blooms. Yet, the absence of visual surface blooms does not correlate to the absence of cyanobacteria as they can be abundant at different depths within the water column. Cyanobacteria may release toxic metabolites into water sources and if left untreated, these toxins threaten drinking water security, including those from oligotrophic lakes, hence, the high demand for research in drinking water treatability sectors and sustainable forested watershed management. The distribution of cyanobacterial communities across Canada was investigated in this study (Chapter 2); samples were collected by various forWater HQP from five major forested ecozones: Atlantic and Pacific Maritime, Boreal Shield and Plains, and the Montane Cordillera. Unique cyanobacterial ASVs obtained from V4-16S rRNA region gene sequencing of all samples were phylogenetically analysed using Maximum Likelihood tree models to observe cyanobacterial taxonomy and biogeography within the various research platforms. Understanding the composition of cyanobacterial communities is a valuable tool in the proactive monitoring of threats to water systems because by observing microbial communities, it can help identify major toxin producing species and illustrate the overall quality of the source water. This research demonstrated that cyanobacteria across Canada are highly diverse and spread across the country, cyanobacteria are unaffected by geographical restrictions as the same taxa can be observed in any of the five ecozones, and there is a significant gap in sequenced picocyanobacteria (0.2-2 μm) in genomic libraries. Whole genome shotgun metagenomic sequencing uses untargeted sequencing to characterize the functional gene diversity and biochemical potential of entire environmental DNA microbial communities. Its application is a valuable tool in observing the potential for toxin synthesis and taste/odour compounds that may negatively impact the state of drinking/recreational water systems. As the only toxin regulated in Canada (1.5 μg/L drinking water, 10 μg/L recreational waters), microcystin damages the liver and kidneys by acting as hepatocytes and disrupting intracellular protein regulation. Lipid A is the toxic component of lipopolysaccharides (endotoxins produced by gram-negative bacteria) which initiate dangerous immune responses and is the culprit for most gram-negative diseases. Alternatively, nitrogen- fixation by cyanobacteria converts atmospheric nitrogen into usable intracellular N2 supplies, allowing for optimal nitrogen availability that is crucial for proliferation and gene expression. The potential for organisms to produce such toxins or be capable of fixing nitrogen is dependent on the presence of biosynthetic gene clusters encoding the necessary genes. Several gene markers were targeted that represent the presence of potential biosynthesis of toxins and N2-metabolic abilities. This research (Chapter 3) characterized seasonal distribution of two toxin gene families: microcystin (mcy) and lipoxygenase (lpx), and three nitrogen metabolism genes families: cyanase (cynS), nitrogenase (nif), and a global nitrogen regulator (ntcA). Genes within the biosynthetic mcy gene cluster signify the production of microcystin, and lpx genes encode for lipid A endotoxins; the cynS gene is involved in fixing cyanate (an organic nitrogen source), the nif gene cluster encodes the primary nitrogen fixation enzyme family, nitrogenase, and ntcA is a global nitrogen regulator crucial for all nitrogen assimilation and acquisition within cyanobacteria. The target toxin genes (mcy, lpx) were chosen for their potential to harm individuals infected with contaminated water, and the target nitrogen-metabolism genes (cynS, nif, ntcA) were chosen due to their capability of providing nitrogen to cyanobacteria which allows them to proliferate, thrive, and express genes even when nitrogen levels in the water are not optimal. The target gene abundances were observed over six sampling points, including under an ice-covered period, using metagenomic analysis, to identify if there is a link among seasonal gene observations.
Spatiotemporal Shifts in Cyanobacterial Communities in a Northern Temperate Watershed – Applications of Next-Generation Sequencing and Implications for Monitoring and Climate Change Adaptation
(University of Waterloo, 2021-08-05) Cameron, Ellen S
Cyanobacteria, a group of photosynthetic bacteria, threaten water quality and drinking water resources globally through the production of potent toxins and the formation of dense surface blooms. These bloom events are increasing in intensity, frequency, and duration due to warming climates and anthropogenic land use and require monitoring programs for water quality management. However, cyanobacteria vary both spatially and temporally and if sampling efforts do not reflect this variation, potentially toxic organisms may be undetected or underestimated. This thesis explores the spatiotemporal trends of cyanobacterial communities in a series of interconnected, oligotrophic lakes in a northern temperate watershed (Turkey Lakes Watershed; North Part, ON) using next-generation sequencing (NGS). Next-generation sequencing of marker genes allows for rapid characterization of environmental communities and has become increasingly accessible, allowing for interdisciplinary applications. Optimal approaches in data handling and analysis are debated due to key challenges arising due to the data structure. Amplicon sequencing samples will vary in library sizes—the total number of reads—but this variation is not biologically meaningful and library sizes must be normalized to account for these differences. Rarefying, the process of subsampling to a normalized size, is frequently used to account for this variation but has been highly criticized due to the omission of valid data. To address the concerns of data omission, repeated iterations of rarefying were evaluated as a normalization technique in diversity analyses (Chapter 2). Repeatedly rarefying was demonstrated to characterize variation introduced through subsampling for applications in diversity analyses. This technique was implemented in the subsequent analysis of cyanobacterial communities in this thesis. Cyanobacterial communities are dynamic exhibiting heterogeneity in their spatial and temporal distribution in lakes. This spatiotemporal variation is driven by environmental conditions and physical characteristics (e.g., cell size, cell density) of taxa and can subsequently create challenges in monitoring. The spatiotemporal variation of cyanobacterial communities was characterized on both a diurnal scale (Chapter 3) and seasonal scale (Chapter 4) through amplicon sequencing of the V4 region of the 16S rRNA gene. Although the lakes in this study did not have visible bloom biomass, cyanobacterial sequences comprised up to 56% of the bacterial community and were frequently dominated by sequences classified as picocyanobacterial genera, which range from 0.2 – 2.0 µm in diameter. This dominance exemplifies the inability to rely on visual detection as a monitoring technique. In both studies, trends in the spatiotemporal variation varied between the lake sites due to differences in morphometry, thermal stratification and surrounding landscape processes demonstrating the impact of system specific characteristics on cyanobacterial dynamics. In combination with warming climates in temperate zones, cyanobacterial growth habits may change and appear as significant components of the bacterial community as early as May in oligotrophic lakes contrasting the previous perception of peak occurrence in the late summer requiring monitoring protocols to re-evaluate appropriate sampling time frames in temperate systems. The research conducted in this thesis identifies key areas for developing ecologically relevant sampling guidelines for cyanobacterial monitoring in lakes. Monitoring protocols are frequently developed from characteristics of common bloom forming taxa resulting in reliance on visual observation of biomass at the surface of the water and focusing sampling efforts to the summer months when blooms typically occur. This research demonstrated the flaws in these assumptions and provides a discussion on appropriate recommendations. Specifically, cyanobacterial community dynamics were demonstrated to be impacted by system specific characteristics and sampling protocols must be tailored to reflect the (i) physicochemical characteristics of the system, and (ii) ecological community structure. The research presented herein demonstrates the need for re-evaluation of current guidelines due to shifts in cyanobacterial growth habits in response to warming climates, and the reported dominance of picocyanobacteria which may impose toxicity risks despite the absence of visible biomass.
Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics
(University of Waterloo, 2014-02-25) Verastegui Pena, Yris Milusqui
Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial “indicator species” for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.

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Browsing Biology by Subject "16S rRNA gene"