Microbial Diversity Across an Oxygen Gradient Using Large-scale Phylogenetic-based Analysis of Marine Metagenomes

Insufficient reference sequence data for annotation of unknown environmental sequences and metagenomes has driven efforts to find alternative annotation methods that mitigate biases from missing information. The use of phylogenetic-placement algorithms shows promise as a robust sequence annotation technique that deals with missing reference information by allowing for annotation of sequences at internal nodes of a phylogenetic tree. However, using these methods for community level surveys of the thousands of genes found in metagenomes requires powerful computational systems and sophisticated software workflows. The main goal of this thesis is to outline a phylogenetic analysis pipeline built to process environmental metagenomic samples using the pplacer software suite, and a pilot study performed with this software pipeline to investigate community-level patterns in gene diversity for a marine oxygen minimum zone (OMZ) off the coast of Chile, South America. Reference sequence data was used to create a custom database and custom reference packages for 9,204 functional housekeeping genes, along with small sub-unit ribosomal genes (SSU) by Domain. A comparative analysis of metagenomic samples from the OMZ using our pipeline shows that while functional and SSU genes show similar spatial patterns of diversity across the oxygen gradient, higher overall diversity was identified via the functional genes. Ecologically relevant functional genes showed higher levels of diversity than either the total from all functional genes or SSU ribosomal genes, underlining the importance of diversity in ecosystem functions

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

This is the author's accepted manuscriptFinal version available from NAS via the DOI in this recordPhytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer.A.M. and T.A.R. are funded by the Royal Society, through Newton and University Research fellowships, respectively. This work is supported in part by research grants from The Gordon and Betty Moore Foundation (GBMF5514), Leverhulme Trust (PLP-2014-147), and the University of Exeter. The University of Exeter OmniLog facility is supported by a Wellcome Trust Institutional Strategic Support Award WT105618MA. Phylogenetic reconstructions were computed on the Data Intensive Academic Grid (National Science Foundation, MRI-R2 Project DBI-0959894)

Phylogenetic-Based Characterization of Microbial Eukaryote Community Structure and Diversity of an Estuary in the Salish Sea

Coastal estuaries are highly dynamic environments. Due to a multitude of environmental variables, microbial communities in these systems are generally complex and difficult to predict. A majority of studies in estuaries have focused on relating environmental factors to changes in community response through indirect measurements such as biomass or chlorophyll. Though these studies are important for our understanding of these systems, they treat community members as a “black box” by focusing on the environmental input and biological output of the system on a broad scale. However, community composition and diversity dictates how cohabitants respond to both environmental stimuli and each other, which in turn impacts their community-level response. We utilized high throughput sequencing of the small subunit ribosomal RNA gene to phylogenetically characterize the diversity of microbial eukaryotes in a coastal estuary located in the Northeastern Salish Sea, and explore spatial and temporal patterns in community structure. Sampling was carried out daily to biweekly for four years during the summer seasons of 2013 to 2016. While diatoms and dinoflagellates were found to be the most abundant amplicon sequences in our samples, there was a diverse assemblage of less represented amplicon sequences that showed spatial and temporal patterns. We also found that our assessment of stability in this system varied based on timescale. Our results highlight the importance of repeated sampling to characterize microbial eukaryotes in dynamic environments, and the importance of including small and/or rare taxa in future ecological studies of these systems. We also discuss current challenges and limitations of this methodological approach to evaluate microbial eukaryotes

The biodiversity of phytate cycling in soils

Phytic acid, myo-inositol hexakisphosphate, InsP6, is the major storage form of phosphate in seeds and grains that constitute a major part of the diets of monogastric animals such as swine and poultry. Monogastrics lack enough enzymes in the right part of the gut to digest dietary phytate. Consequently, phytases, a group of enzymes capable of releasing inorganic phosphate from phytate, are added to commercial poultry and swine diets. These adjunct phytases are a major sector of the global enzyme market with an estimated value of $5 billion in 2015. There is continued commercial interest in the discovery and development of more effective and cost-effective enzymes. The soil environment is microbially diverse and therefore offers significant potential for the isolation of novel phytases. In this thesis, I have developed new methods for the culture-dependent isolation of phytases from different soil environments by first analysing phytase activity of the soil microbiome using HPLC. The isolation of a multiple inositol polyphosphate phosphatase, MINPP, from Acinetobacter sp. represents one of the first phytases of its kind to be isolated from the soil environment. This study provides a robust characterisation of the protein, identifying an outstanding long-term stability at room temperature and activity from 37.6-101.3% over 755 days. The expression of the phytase was examined using β-galactosidase and qPCR assays which showed that while expression was enhanced in the presence of impure phytate by β-galactosidase, it was significantly repressed in the qPCR experiment. Additionally, a long-term phytase isolation experiment was performed using well-characterised Rothamsted soils. Of the sixty-six isolates that were re-streaked from mixed plates, seventeen showed a diverse array of phytase degradation profiles, highlighting the diversity of phytase activity in the soil environment. I have also undertaken metagenomic analysis to examine the diversity of phytases in environmental and enteric environments. This highlighted the dominance of MINPP genes in enteric environments above all other known classes of phytases. In soil and aquatic metagenomes, the relative abundances were significantly less than in enteric environments and here the Multiple Inositol Polyphosphate Phosphatase (MINPP) gene was not the overwhelming majority, instead the Beta-Propeller Phytase (BPPhy), Histidine Acid Phytase (HAPhy) and Protein Tyrosine Phosphatase-like Phytase (PTPhy) were equal to or even higher in abundances. Additionally, in this analysis I also examined the prospect of horizontal gene transfer in the MINPP dataset using the program T-rex. Here I identified multiple HGT events occurring between both enteric and environmental bacteria, with one transfer occurring between environments

MOLECULAR CHARACTERIZATION OF FACTORS CONSTRAINING THE SUCCESS AND TOXICITY OF \u3ci\u3eMICROCYSTIS\u3c/i\u3e BLOOMS

Harmful cyanobacterial blooms (cyanoHABs) have detrimental effects on freshwater lakes and reservoirs around the world. CyanoHABs severely reduce water quality, altering the food web and disrupting fishing and tourism industries. In addition, many bloom-forming cyanobacteria have the capacity to produce potent toxins, making the negative impacts of cyanoHABs of ecological and economic importance and a serious public health risk. Microcystis spp., which are the typically dominating cyanobacteria in blooms, often produce thick scums, taste and odor compounds and the hepatotoxins, microcystin, implicated in water advisories, human and animal poisonings and drinking water shutdowns globally. Primarily driven by anthropogenic nutrient loading and climate change, Microcystis blooms are on the rise, not only increasing in frequency but also intensity. Nitrogen (N) and phosphorus (P) are considered the most important nutrients in driving bloom formation and persistence but historically management practices have solely focused on P abatements. Large inputs of N into the environment have now been tied to the increased prevalence of toxic Microcystis. To better understand the role of N and other environmental factors impacting the success and toxicity of Microcystis, a number of molecular techniques were employed to characterize bloom community dynamics and the physiology of Microcystis. Results from environmental studies generated new hypotheses about the role of heterotrophic bacteria in N-cycling and microcystin degradation, while observations from laboratory studies provided novel insight into the metabolism of N by Microcystis and subsequent microcystin production was proposed. Together, the findings presented here can be extrapolated to the natural environment and provide greater insight into the mechanisms that contribute to cyanoHAB expansion and toxicity

Metagenomic analysis of the biodiversity and seasonal variation in the meromictic Antarctic lake, Ace Lake

Ace lake is a stratified lake in the Vestfold Hills, Antarctica. The presence of a thick ice-cover for ~11 months of the year and a strong salinity gradient are responsible for its permanent stratification. Taxonomy analyses showed depth-based segregation of its microbial community, including viruses. Functional potential analyses of the lake taxa highlighted their roles in nutrient cycling. In this thesis, the seasonal changes in Ace Lake microbial community were studied using a time-series of metagenomes utilizing the Cavlab metagenome analysis pipeline. Statistical analyses of taxa abundance and environmental factors revealed the effects of the polar light cycle, with 24 hours of daylight in summer and no sunlight in winter, on the phototrophs identified in the lake, indicating the importance of light-based primary production in summer to prevail through the dark winter. Analysis of viral data generated from the metagenomes showed the presence of viruses, including a ‘huge phage’, throughout the lake, with a diverse population existing in the oxic zone. Analysis of virus-host associations of phototrophic bacteria revealed that the availability of light, rather than viral predation, was probably responsible for seasonal variations in host abundances. Genomic variation in Synechococcus and Chlorobium populations, analysed using metagenome-assembled genomes (MAGs) from Ace Lake, revealed phylotypes that highlighted their adaptation to the lake environment. Synechococcus phylotypes were linked to complex interaction with viruses, whereas some Chlorobium phylotypes were inferred to interact with Synechococcus. Some Chlorobium phylotypes were also inferred to have improved photosynthetic capacity, which might contribute to the very high abundance of this species in Ace Lake. Comparative genomic analysis of Chlorobium was performed using MAGs from Ace Lake, Ellis Fjord, and Taynaya Bay and the genome of a non-Antarctic Chlorobium phaeovibrioides. A single Chlorobium species, distinct from the non-Antarctic species, was prevalent in the oxycline of all three stratified systems, highlighting its endemicity to the Vestfold Hills. Potential Chlorobium viruses, representing generalist viruses, were identified in aquatic systems from the Vestfold Hills and the Rauer Islands, indicating a widespread geographic distribution. Seasonal variation in the Chlorobium population appeared to be caused by reliance on sunlight rather than the impact of viral predation, and was inferred to benefit the host by restricting the ability of specialist viruses to establish effective lifecycles. The findings in this thesis highlight the seasonal influence on Ace Lake biodiversity, the adaptations and potential interactions of the two key species Synechococcus and Chlorobium, and the endemicity of Ace Lake Chlorobium to the Vestfold Hills

Host-microbe symbiosis and coevolution in coral reef invertebrates

Paul O'Brien used the topic of coevolution to study the microbiome of coral reef invertebrates. He found that a) the evolutionary history of the host is reflected in the microbiome, b) a subset of microbial species display strong patterns of cophylogeny, and c) the genomes of those microbes show evidence of adaptation to the host. Through the light of coevolution, this thesis has deepened our understanding of the structure, function and importance of the microbiome of coral reef invertebrates

Identification and activity of bacteria consuming key intermediates of carbon and sulfur cycling in coastal sands

Coastal and shelf sediments are hot spots for carbon remineralization and also for carbon fixation. Here, a large fraction of organic carbon is mineralized under anoxic conditions by microorganisms via fermentation or respiration of fermentation products such as molecular hydrogen (H2) and acetate. Reduced inorganic metabolites released during these anaerobic processes and inorganic carbon are used by light-independent chemolithoautotrophs for socalled secondary production. However, still little is known about the in situ relevant organisms and how they contribute to key processes like chemoautotrophy as well as H2 and acetate turnover. To understand how inorganic carbon at sediment surfaces is turned over we surveyed the diversity of candidate bacterial chemolithoautotrophs in 13 tidal and sublittoral sediments and identified ubiquitous core groups of Gammaproteobacteria mainly affiliating with sulfuroxidizing bacteria. In a novel methodological approach we quantified dark carbon fixation by scintillography of specific microbial populations extracted and flow-sorted from sediments that were short term incubated with 14C-bicarbonate. Here, we show that uncultured Gammaproteobacteria dominate dark carbon fixation in coastal sediments and three distinct gammaproteobacterial clades made up more than half of dark carbon fixation in a tidal sediment. Meta- and single cell genomics along with metatranscriptomics provided evidence for a largely sulfur-based carbon fixation. These chemolithoautotrophic gammaproteobacterial clades also accounted for a substantial fraction of the microbial community in 1,000 to 2,000 year old subsurface sediments, suggesting that burial of chemolithoautotrophic bacteria could possibly be a yet-unrecognized mechanism of carbon sequestration. Microbial scavenging of H2 is an essential process in anoxic carbon mineralization, because only low H2 levels make H2-forming fermentation thermodynamically feasible. In a sediment metagenome we identified a high diversity of genes encoding the NiFe uptake hydrogenases of numerous yet-uncultured, potentially H2-oxidizing bacteria. Metatranscriptomics together with incubation experiments suggested uncultured Desulfobacteraceae, in particular the sulfate-reducing Sva0081-clade, as important H2 oxidizers in anoxic sediments. On the contrary, Gammaproteobacteria and Flavobacteria encoding O2-tolerant hydrogenases are possibly involved in H2 oxidation in oxic sediments. In a third study, we quantified the relative contribution of single bacterial populations to total acetate assimilation. Here, we showed that acetate was assimilated by physiologically and phylogenetically distinct bacterial groups such as Gammaproteobacteria, sulfate-reducing Desulfobacteraceae and Desulfobulbaceae as well as likely lithoheterotrophic sulfur-oxidizing Roseobacter-clade bacteria. We identified uncultured Gammaproteobacteria as a major contributor to acetate assimilation under oxic and anoxic conditions accounting for 31-62% of the total acetate assimilation. In summary, this thesis contributes to our understanding how distinct bacterial populations turn over key metabolites of organic carbon degradation in marine sediments. The quantification of uptake of 14C-labeleld model compounds by defined populations is a major step forward in the identification of key organisms in element cycling in marine sediments

Taxonomic and environmental annotation of bacterial 16S rRNA gene sequences via Shannon entropy and database metadata terms

Microbial ecology seeks to describe the diversity and distribution of microorganisms in various habitats within the context of environmental variables. High throughput sequencing has greatly boosted the number and scope of projects aiming to study and analyse these organisms, with ever-increasing amounts of data being generated. Amplicon based taxonomic analysis, which determines the presence of microbial taxa in different environments on the basis of marker gene annotations, often uses percentage identity as the main metric to determine sequence similarity against databases. This data is then used to study the distribution of biodiversity as well as the response of microbial communities to stressors. However, the 16S rRNA gene displays varying degrees of sequence conservation along its length and is therefore prone to provide different results depending on the part of 16S rRNA gene used for sequencing and analysis. Furthermore, sequence alignment is primarily performed using the popular BLAST sequence alignment tool, which incurs a great computational performance penalty although newer, more efficient tools are being developed. A new approach that is fast and more accurate is critically needed to process the avalanche of data. Additionally, repositories of environmental metadata can provide contextual information to sequence annotations, potentially enhancing analysis if they can be incorporated into bioinformatics pipelines. The overarching aim of this work was to enhance the taxonomic annotation of bacterial sequences by developing a weighted scheme that utilizes inherent evolutionary conservation in the bacterial 16S rRNA gene sequences and by adding contextual, environmental information pertaining to these sequences in a systematic fashion