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 - 5 of 5

Assessing Taxonomic Issues with the Genera Anabaena, Aphanizomenon and Nostoc Using Morphology, 16S rRNA and efp genes
(University of Waterloo, 2009-01-08T18:53:31Z) Beltrami, Orietta
Cyanobacteria are an ancient lineage of gram-negative photosynthetic prokaryotes that play an important role in the nitrogen cycle in terrestrial and aquatic systems. Widespread cyanobacterial blooms have prompted numerous studies on the classification of this group, however defining species is problematic due to lack of clarity as to which characters best define the various taxonomic levels. The genera Anabaena, Aphanizomenon and Nostoc form one of the most controversial groups and are typically paraphyletic within phylogenetic trees and share similar morphological characters. This study’s purpose was to determine the taxonomic and phylogenetic relationships among isolates from these three genera using 16S rRNA and bacterial elongation factor P (efp) gene sequences as well as morphological analyses. These data confirmed the non-monophyly of Anabaena and Aphanizomenon and demonstrated that many of the isolates were intermixed among various clades in both gene phylogenies. In addition, the genus Nostoc was clearly not monophyletic and this finding differed from previous studies. The genetic divergence of the genus Nostoc was confirmed based on 16S rRNA gene sequence similarities (≥85.1%), and the isolates of Anabaena were genetically differentiated, contrary to previous studies (16S rRNA gene sequence similarities ≥89.4%). The morphological diversity was larger than the molecular diversity, since the statistical analysis ANOSIM showed that the isolates were morphologically well differentiated; however, the 16S rRNA gene sequence similarities showed some isolates as being related at the species level. Planktonic and benthic strains were not distinguished phylogenetically, although some well-supported clusters were noted. Cellular measurements (length and width of vegetative cells, end cells, heterocysts and akinetes) were noted to be the morphological characters that best supported the differentiation among isolates, more than qualitative characterization. Among the metric parameters, the length of akinetes resulted in better differentiation among isolates. The efp gene sequence analyses did not appear to be useful for the taxonomic differentiation at lower taxonomic levels, but gave well-supported clusters for Aphanizomenon that was supported by the morphological analyses. Both gene regions gave similar trees with the exception of the Aphanizomenon isolates which clustered together in phylogenetic trees based on the efp gene. This differed from the 16S rRNA gene in which this genus was paraphyletic with Anabaena species that were similar in morphology to Aphanizomenon. Hence, the application of multiple taxonomic criteria is required for the successful delineation of cyanobacterial species.
Assessment of toxic cyanobacterial abundance at Hamilton Harbour from analysis of sediment and water
(University of Waterloo, 2014-05-09) Jonlija, Miroslava
The western embayment of Lake Ontario, Hamilton Harbour, is one of the most polluted sites in the Laurentian Great Lakes and in recent years has seen a reoccurrence of cyanobacterial blooms. This study uses a multidisciplinary approach to examine the presences of toxic Cyanobacteria in the harbour in order to gain insight into these recurrent blooms. Microscopic analyses of phytoplankton samples collected during the 2009 summer-fall sampling season from two locations within the harbour showed the spatial and seasonal diversity of the contemporary cyanobacterial community. Microcystis colonies relative abundances in relation to total algal numbers were estimated. The lowest and highest relative abundances of Microcystis in the phytoplankton population were 0.6% and 9.7%, respectively, and showed seasonal variability between stations. Fourteen cyanobacterial genera comprising six families and three orders were identified and for which the most abundant filamentous genera during the summer-fall sampling season were Planktothrix, Aphanizomenon and Limnothrix. Potential microcystin producers Microcystis, Planktothrix, Aphanizomenon and Dolichospermum were also present and during the sampling period Microcystis was recorded at both stations on all dates, however, its relative abundance was below 10 % throughout the study period. The composition and abundance of filamentous cyanobacteria were observed to be positively statistically correlated to water quality environmental parameters dissolved nitrates (NO3/NO2), dissolved inorganic carbon (DIC), and conductivity. Redundancy analysis (RDA) found that 53.35% total variance of Aphanizomenon was correlated to low water column NO3/NO2 and conductivity, and higher water column DIC. 58.13% of the relative abundance of Planktothrix was correlated to high concentrations of dissolved nitrates, while 51.69% of total variance of Limnothrix was correlated to higher DIC and lower water column dissolved nitrate concentrations. Information about past cyanobacterial communities was obtained from the sediment core analysis, using paleolimnological and modern molecular methods. The age of the 100.5 cm long sediment core retrieved from the deepest part of Hamilton Harbour was established to be 140 years (1869-2009), using the Constant Rate of Supply (CRS) 210Pb age model. This age was not sufficient to provide information of harbour’s environmental conditions, presence of the blooms, and triggers for their occurrence before European settlement in the area. Results of the HPLC analysis of fossil pigments indicated that the dominant members of the algal community have not changed over the 140 years and that cyanobacteria were regular members of the phytoplankton community. The composition of the major chlorophyll pigments indicated high presence of Chlorophyta and Bacillariophyta in the harbour at all times. The main algal groups identified on the basis of marker pigments presence, besides the Chlorophyta and Bacillariophyta, were the Dinophyta and the Cryptophyta. The presence of a scytonemin derivative, compound B, indicated that cyanobacterial blooms were occurring in past, before the first officially recorded blooms in the 1960s. Cyanobacterial pigments presence indicated that Cyanobacteria have been a regular but not dominant feature of Hamilton Harbour phytoplankton in the past. To our knowledge, this study is the first one examining fossil pigments from Hamilton Harbour. Results of the PCR-DGGE molecular analysis of 16S rRNA-V3 gene fragments from sedimentary DNA revealed the presence of thirteen cyanobacterial genotypes. The temporal change in the cyanobacterial community composition was indicated by the increasing number of species over time, from the oldest to the most recent sediment layers. The deepest sediment strata showed the lowest number (two bands) and intensity of bands. The most recent sediment layer had the greatest numbers (11) and intensity of bands. This increased diversity indicated changing environmental conditions in the harbour, primarily nutrient pollution and worsening water quality. Results of the PCR-DGGE molecular analysis of mcyE-AMT gene fragments showed that Microcystis aeruginosa and Planktothrix rubescens were two microcystin producers present in Hamilton Harbour over the last 80 years. The persistent presence and resilience of these two genera indicated a more serious and longer-term issue of toxic blooms than previously recognized. Historical records show that noticeable anthropogenic impact on Lake Ontario environment has been measurable since the 1780s, the first dramatic impact on the Lake Ontario watershed was evident from the mid1880s, the earliest evidence of eutrophication in the lake occurred between 1820 and 1850, while human induced environmental changes in Hamilton Harbour date back ca. 350 years. In the 1960s, cyanobacterial blooms were first officially recognized in the harbour and the lower Great Lakes. The present research is the first report of the mcyE module and AMT domain of microcystin genes being amplified from sediment of North American lakes, and showed that toxic Cyanobacterial have been regular members of Hamilton Harbour phytoplankton community for almost a century. This research considerably deepened the knowledge of the past toxic cyanobacterial blooms in Hamilton Harbour and their possible causes. It also showed that in the absence of historical records, both the PCR-DGGE method and the mcyE-AMT gene may be used for reconstruction of the past toxic blooms not only in the Laurentian Great Lakes, but also in other aquatic regions of the world impacted by toxic cyanobacterial blooms. Also, it demonstrated the utility of the combined molecular and paleolimnological analyses, which might become a useful tool in the determination of the bloom causes factors and in the mitigation of the future production of toxic blooms.
Bacterial Communities in the Glenmore Reservoir with an emphasis on the Cyanobacteria
(University of Waterloo, 2021-08-30) Aljoudi, Ghadeer
Lakes and reservoirs play an essential role as a source for freshwater that can be potable after proper treatment, or for irrigation to meet the water needs of the population, industry, and agriculture. However, freshwaters bodies face significant challenges (including pollutant loads and climate change) that may lead to water quality degradation; among these are taste and odour events and harmful bacterial blooms. In Calgary, Alberta, Canada, runoff from agriculture and residential development in urbanized watersheds also contributes to water quality deterioration in the Elbow River, which flows into the Glenmore Reservoir and serves as one of the primary sources of drinking water for the city. While harmful algal blooms (i.e., Cyanobacteria) have not occurred in the Glenmore Reservoir historically, increased pressures (e.g., stormwater discharges, potential erosion and runoff after wildfire) on source water quality in the city’s wildfire-prone source watersheds have the potential to increase nutrient (especially bioavailable phosphorus) loading to the reservoir; this can promote algal blooms (City of Calgary, 2018). Therefore, to enable identification of source water quality shifts and treatment challenges that may impact the provision of adequate amounts of safe drinking water, it is important to monitor both water quality and bacterial and cyanobacterial communities within the reservoir. This research focused on identifying and detecting the bacterial and cyanobacterial communities that present in oligotrophic Glenmore Reservoir in Calgary, AB. The 16S rRNA gene next-generation sequencing (NGS) was applied to investigate bacterial and cyanobacterial composition in the reservoir and detect any problematic taxa that may lead to concerns regarding the quality of the drinking water. Major findings revealed that Actinobacteria, which are known to produce geosmin and 2-MIB (taste and odour compounds), were the second most dominant phylum and may be metabolically active residents due to their adaptation to nutrient-poor environments and thus potential sources of T&O (taste and odour). In addition, sequences from the cyanobacterial community matched two potential toxin-producing genera: Synechococcus sp. and Planktothrix agardhii. This preliminary analysis can provide baseline information against which subsequent, more detailed investigations of reservoir bacterial and cyanobacterial communities may be compared.
Method development for the analysis of soil bacterial communities
(University of Waterloo, 2015-01-23) Bartram, Andrea
Due to the tremendous diversity and abundance of microbes in environmental and host-associated environments, adequate characterization of these samples remains a challenge for microbiologists. In order to increase the depth of sampling for diverse bacterial communities, this thesis research developed a novel method for sequencing and assembly of millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; ~200 nucleotides), using Illumina-based next-generation sequencing. To confirm reproducibility and identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNA from known cultured bacterial isolates (>1 million post-assembly sequences) and from an Arctic tundra soil sample (>6 million post-assembly sequences). These Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e.g. 454 pyrosequencing); the assembly of paired–end offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane and may be readily modified to target other variable regions or genes. Soil pH is an important determinant of microbial community composition and diversity, yet few studies have characterized the specific effects of pH on individual bacterial taxa within bacterial communities, both abundant and rare. Composite soil samples were collected over two years from an experimentally maintained pH gradient ranging from 4.5 to 7.5 from the Craibstone Experimental Farm (Craibstone, Scotland). Extracted nucleic acids were characterized by bacterial and group-specific denaturing gradient gel electrophoresis (DGGE) and were sequenced using the Illumina sequencing method describe above. Both methods demonstrated comparable and reproducible shifts within higher taxonomic bacterial groups (e.g. Acidobacteria, Alphaproteobacteria, Verrucomicrobia, and Gamma-proteobacteria) across the pH gradient. In addition, non-negative matrix factorization (NMF) was used for the first time on 16S rRNA gene data to identify positively interacting (i.e. co-occurring) operational taxonomic unit (OTU) clusters (i.e. “components”), with abundances that correlated strongly with pH, and sample year to a lesser extent. The OTUs identified by NMF were visualized within principle coordinate analyses of UniFrac distances and subjected to taxonomic network analysis (SSUnique), which plotted OTU abundance and similarity against established taxonomies. Most pH-dependent OTUs identified here would not have been identified by previous methodologies for microbial community profiling and were unrelated to known lineages. Methods to limit and reduce carbon emissions are becoming increasingly important for circumventing future impacts of climate change. Biochar is a recalcitrant aromatic-carbon compound formed during pyrolysis in an anoxic environment. The use of lignocellulosic waste material as an input for biochar generation acts as a carbon sink when applied as a soil amendment. Biochar added to soil has been shown to have beneficial effects on crop yield, soil pH, nutrient retention, and fertilizer requirement. However, impacts of biochar applications on soil microbial communities are not well characterized. In order to assess the impact of biochar application on soil microbial communities, two studies were conducted: a multi-year Canadian field trial and a controlled microcosm study. Together, these studies enabled the assessment of the microbial response to biochar, both with and without the influence of above-ground vegetation, respectively. Field trial samples were collected in 2010, with rhizosphere and bulk soil taken from agricultural plots planted with corn, switchgrass, and soybean, amended with either 0 or 20 t ha-1 of dry biochar. The field experiment was also performed on two contrasting soil types: a sandy soil and a loam soil. The microcosm study was conducted over a period of twenty weeks, with biochar added at rates equivalent to 0, 20, 40, and 60 t ha-1 to a loam soil in an anoxic incubation system (1 L Mason jar). Nucleic acids were extracted from these soil samples and used as template for bacterial 16S rRNA gene fingerprinting (denaturing gradient gel electrophoresis; DGGE) and amplicon sequencing (101,448,506 assembled sequences generated by Illumina). The resulting fingerprints and PCoA plots based on UniFrac distances indicated that the largest factor governing the microbial community in the field study was soil type, followed by plant type. On the other hand, the corresponding PCoA plots for the microcosm study showed strong separation between biochar-amended samples and controls, in addition to separation corresponding to incubation time. DGGE fingerprints for the microcosm study showed a predominant biochar-associated band. The corresponding sequence in the Illumina libraries classified as an uncultured Gammaproteobacteria clone and increased in abundance in biochar-amended samples and was absent from the no-biochar controls. These results indicate that microbial communities detected in the field were controlled primarily by soil type and vegetation cover rather than biochar application, but strong biochar-dependent shifts were observed in the microcosm study.
Profiling skin-associated archaea and bacteria with 16S rRNA and cpn60 genes
(University of Waterloo, 2021-08-13) Umbach, Alexander
As the largest organ of the mammalian body, skin is associated with commensal microorganisms that impact host health. Characterizing host-microbe associations is critical to our understanding of skin health, function, and disease, and the potential co-evolutionary relationships that have occurred throughout mammalian and prokaryotic evolution. The research within this thesis focused on profiling the bacteria and archaea that inhabit mammalian skin using two phylogenetic marker genes: cpn60 and 16S rRNA. The cpn60 gene was applied to mammalian skin swab samples to provide increased taxonomic resolution for microbial populations on mammalian skin. Datasets previously generated using the 16S rRNA gene were included to assess archaeal populations associated with the skin and skin-associated surfaces. Previous research into the mammalian skin microbiome using the 16S rRNA gene identified evidence for phylosymbiosis within the Perissodactyla and Artiodactyla, as well as highlighted core taxa common to all sampled mammalian skin. The increased taxonomic resolution provided by the cpn60 gene has the potential to reveal additional co-evolutionary patterns and can more thoroughly probe specific populations of the mammalian skin microbiome. Chapter 2 of this thesis describes a newly generated cpn60 gene dataset sourced from the mammalian skin microbiome of Carnivora, Perissodactyla, Artiodactyla, and Primate hosts. Significant patterns of phylosymbiosis for Artiodactyla and Perissodactyla were confirmed when using weighted (p = 4.43x10-2) and unweighted (p = 4.36x10-2) UniFrac metrics, which are observations not made previously with a comparable 16S rRNA gene dataset. Using the cpn60 gene, specific Staphylococcaceae communities were successfully delimited from their genus classifications, with improved species-level resolution for Macrococcus, Staphylococcus, and Salinicoccus, compared to the 16S rRNA gene dataset. Additionally, Jeotgalicoccus halophilus was detected broadly within mammalian skin microbiomes, representing a first report of widescale association of this species with mammalian skin. These results demonstrate associations between mammalian hosts and skin-associated taxa warrants further investigation. Future amplicon-based skin microbiome studies focusing on host-microbe interactions would benefit from continued use of the cpn60 gene given the increased taxonomic resolution that it provides. Limited skin-related archaea research has not yet allowed for a consensus on the prevalence of skin-associated archaea. Recent studies suggest that archaea are consistently detected and relatively abundant on human skin, with skin “archaeomes” dominated by putative ammonia oxidizers of the Nitrososphaeria class (Thermoproteota phylum - formerly Thaumarchaeota). Chapter 3 evaluated new and existing 16S rRNA gene sequence data sourced from mammalian skin and skin-associated surfaces, generated with two commonly used universal prokaryotic primers sets, to assess archaeal prevalence, relative abundance, and taxonomic distributions. Archaeal 16S rRNA gene sequences were detected in only 17.5% of 1,688 sample high-throughput sequence data, with most of the archaea-positive samples associated with non-human mammalian skin. Only 5.9% of human-associated skin sample datasets contained sequences affiliated with archaeal 16S rRNA genes. When detected, the relative abundance of sequences affiliated with archaeal ASVs was less than 1% for most mammalian skin samples and did not exceed 2% for any samples. Although several computer keyboard microbial profiles were dominated by Nitrososphaeria sequences, all other skin microbiome datasets tested were primarily composed of sequences affiliated with Methanobacteriota and Halobacteriota phyla. Our findings revise downwards recent estimates of human skin archaeal distributions and relative abundances, especially those affiliated with the Nitrososphaeria, reflecting a limited and infrequent archaeal presence within the mammalian skin microbiome. This work provides insight in the microbial communities of mammalian skin-associated bacteria and archaea, their relationships with mammalian hosts. Increased sequencing depth and mammalian host representation could reveal additional co-evolutionary patterns, and the species-level resolution provided by the cpn60 gene could be used to target additional microbial populations of interest. Likewise, additional research into the mammalian skin archaeome would benefit from further comparisons of universal and archaea-specific primers and help elucidate potential differences in abundance and distribution caused by regionality.

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Browsing Biology by Subject "16S rRNA"