ICTC12 Abstract Book

Abstract book for the 12th International Conference on Toxic Cyanobacteria

Next-generation sequencing (NGS) for assessment of microbial water quality: current progress, challenges, and future opportunities

Water quality is an emergent property of a complex system comprised of interacting microbial populations and introduced microbial and chemical contaminants. Studies leveraging next-generation sequencing (NGS) technologies are providing new insights into the ecology of microbially mediated processes that influence fresh water quality such as algal blooms, contaminant biodegradation, and pathogen dissemination. In addition, sequencing methods targeting small subunit (SSU) rRNA hypervariable regions have allowed identification of signature microbial species that serve as bioindicators for sewage contamination in these environments. Beyond amplicon sequencing, metagenomic and metatranscriptomic analyses of microbial communities in fresh water environments reveal the genetic capabilities and interplay of waterborne microorganisms, shedding light on the mechanisms for production and biodegradation of toxins and other contaminants. This review discusses the challenges and benefits of applying NGS-based methods to water quality research and assessment. We will consider the suitability and biases inherent in the application of NGS as a screening tool for assessment of biological risks and discuss the potential and limitations for direct quantitative interpretation of NGS data. Secondly, we will examine case studies from recent literature where NGS based methods have been applied to topics in water quality assessment, including development of bioindicators for sewage pollution and microbial source tracking, characterizing the distribution of toxin and antibiotic resistance genes in water samples, and investigating mechanisms of biodegradation of harmful pollutants that threaten water quality. Finally, we provide a short review of emerging NGS platforms and their potential applications to the next generation of water quality assessment tools.Singapore-MIT Alliance for Research and Technology. Center for Environmental Sensing and Modelin

Recovery and characterization of viral diversity from aquatic short- and long-read metagenomes

Viruses are the most abundant biological entities in marine ecosystems and play an essential role in global biogeochemical cycles. They have important ecological functions as drivers of bacterial populations through lytic infections and contribute to bacterial genetic diversification. Unfortunately, their study is severely limited by the difficulty to culture and isolate them in lab conditions. Culture-independent techniques such as metagenomics can complement culture-based approaches to capture more phage diversity. However, the vast majority of viral sequences recovered through these methods are uncharacterized and therefore do not provide any information about their interactions with the bacterial community, a phenomenon that has been named “viral dark matter”. In this thesis, several bioinformatic techniques are applied to both short- and long-read metagenomic datasets to recover biological information from marine viral sequences contained therein. A pipeline for recovering viral sequences based on a reference genome was developed and applied to the study of myophages infecting the alphaproteobacterial SAR11 clade, one of the most abundant bacterioplankton groups in surface marine and freshwater ecosystems. We were able to recover 22 new genomes which include the first genomes of myophages infecting LD12, the SAR11 freshwater clade. These sequences are underrepresented in datasets derived from the viral fraction, suggesting a bias of either technical or biological nature. Surprisingly, this family of phages code for an operon which resembles the secretion system type VIII operon in Escherichia coli. The function of this phage operon is still unknown. Next, a long-read dataset from the Mediterranean Sea was explored for viral contigs to contrast phage recovery between long- and short-read datasets. The analysis revealed that while long-read assemblies resulted in viral sequences of better quality, there was a sizable amount of intra-clade viral diversity that was not included in the assemblies. This viral diversity only found in long reads is even greater than previously thought. This untapped diversity could aid biotechnological efforts as evidenced by the discovery of new endolysins. Finally, a tool (Random Forest Assignment of Hosts, or RaFAH) for assigning hosts to phage sequences obtained from metagenomic datasets was created. The tool is based on a machine learning tool trained with phage protein clusters generated de novo. Benchmarking shows that RaFAH is on par with other state-of-the-art classifiers and is able to classify phage contigs at the level of Kingdom, which makes it the first classifier to accurately detect Archaea viruses from metagenomic samples. A feature importance analysis reveals that the protein clusters with the most predictive power are those involved in host recognition.Los bacteriófagos (”fagos”) son los organismos más abundantes en los ecosistemas marinos y tienen un papel esencial en los ciclos biogeoquímicos globales. Asimismo, influencian la evolución de las poblaciones bacterianas que infectan y contribuyen a la diversificación del acervo genético bacteriano. Desgraciadamente, su estudio se ve limitado por la dificultad de cultivar y aislar estos organismos en el laboratorio. El uso de técnicas que no requieren cultivo, como la metagenómica, pueden complementar el cultivo en laboratorio para recuperar una mayor diversidad de fagos. Sin embargo, la inmensa mayoría de secuencias virales recuperadas mediante metagenómica no pueden ser caracterizadas, por lo que no proporcionan ninguna información sobre sus interacciones con la comunidad bacteriana, un fenómeno que se ha nombrado “materia oscura viral”. En esta tesis se han utilizado múltiples procesos bioinformáticos en colecciones de metagenomas de lectura corta y larga para caracterizar las secuencias virales que contienen. Se ha desarrollado un procedimiento para recuperar secuencias virales a partir de un genoma de referencia y se ha aplicado al estudio de miofagos que infectan al clado SAR11 de las Alfaproteobacteria, uno de los grupos de bacterioplankton más abundantes en agua dulce y agua salada de superficie. Se consiguió recuperar 22 nuevos genomas que incluyen el primer genoma que infecta LD12, el subclado de SAR11 de agua dulce. Estos genomas están poco representados en colecciones obtenidas de la fracción viral, lo que sugiere que las afecta un sesgo técnico o biológico. Sorprendentemente, esta familia de fagos contiene un operón similar al sistema de secreción tipo VIII de Escherichia coli. La función de este operón es aún desconocida. Asimismo, se contrastó la recuperación de secuencias víricas entre colecciones de lectura corta y larga utilizando colecciones obtenidas en el mar Mediterráneo. Los resultados muestran que aunque los ensamblajes derivados de las lecturas largas producen secuencias virales de mejor calidad, en el proceso se pierde una gran cantidad de diversidad intraclado. Esta diversidad es mucho mayor de la recuperada con lecturas cortas, y podría explotarse para aplicaciones biotecnológicas, como el descubrimiento de nuevas endolisinas. Finalmente, se desarrolló un programa (Random Forest Assignment of Hosts, o RaFAH) para asignar hospedadores a secuencias virales obtenidas de colecciones metagenómicas. El programa se basa en el uso de algoritmos de machine learning entrenados con grupos de proteínas creados de novo. RaFAH muestra un rendimiento similar a otros clasificadores de secuencias y es capaz de clasificar secuencias víricas al nivel taxonómico de Reino, siendo así el primer clasificador capaz de detectar fagos que infectan arqueas con precisión. El análisis de importancia de rasgo revela que los grupos de proteínas con mayor poder predictivo son aquellos involucrados en el reconocimiento del hospedador

Bacterial populations and human pathogens in irrigation water sources

Poor microbial water quality can cause foodborne disease from contaminated fresh produce. In Australia, regulations and certification standards lack a consistent and specific approach to defining safe water sources, making it challenging for the fresh produce industry to monitor water for human health risks. The US fresh produce industry has developed the ‘AgWater App’ to predict microbial contamination in irrigation water sources. Freshcare, Australia’s most common fresh produce safety standard, requires preharvest water to meet E. coli <100 cfu per 100mL. This thesis uses the ‘AgWater App’ approach and Freshcare criteria to determine significant correlations between physicochemical water quality and E. coli, highlighting the impact of sediment-water exchanges and environmental factors. However, site-specific influences make it challenging to design a tool that can be applied widely. The thesis also investigates the suitability of E. coli as an indicator of microbial water quality for fresh produce irrigation. Metagenomic 16S rRNA data for unculturable bacteria were used to identify food safety-related taxa present in irrigation water sources, including Bacteroides, Cyanobacteria, and Proteobacteria. Inferred functional pathways implicated in infectious diseases were also identified. Spatio-temporal trends in the data showed seasonal variation and sediment-water exchanges that impact the bacterial community dynamics. This thesis contributes to the discourse of assigning risk to irrigation water sources, providing insights into future applications of metagenomics in produce safety. The research underscores the need for evidence-based approaches to defining safe water sources and the importance of considering sediment-water exchanges and environmental factors in monitoring for human health risks. It also highlights the limitations of using E. coli as the sole indicator of food safety concerns in irrigation water sources

Microbial indicators for environmental stress and ecosystem health assessments

Bettina Glasl established the first microbial baseline for the Great Barrier Reef (GBR) and quantified the diagnostic potential of multiple reef microbiomes. She found that the seawater microbiome was the most suitable microbiome for a microbial indicator program. Her research provides a framework for the integration of microbial observatories in reef monitoring programs

Next-generation sequencing, morphology, and culture-based methods reveal diverse algal assemblages throughout the Florida springs

Algae are a group of highly diverse photosynthetic organisms found in variety of habitats. As the primary energy base in ecosystems, knowledge of the diversity and presence of certain algal lineages is paramount to our understanding of the trophic state of aquatic habitats. In recent years, the state of Florida has seen an increase of both marine and freshwater algal blooms. Similarly, filamentous algae have begun outcompeting vascular macrophytes throughout many of Florida’s springs as nutrient enrichment from anthropogenic sources increases. Traditionally, the Florida algal spring communities have been assessed using classic morphological methods, which may underrepresent the true biodiversity present. Therefore, the goal of this study was to conduct a more complete diversity assessment implementing next-generation sequencing techniques (NGS) with morphological analyses and culturing methods. While morphological methods identified a wide variety of algal taxa, belonging to 4 phyla (Bacillariophyta, Charophyta, Chlorophyta, and Cyanobacteria), next-generation sequencing techniques provided greater detail of the diatom community. This is particularly important as many diatom taxa are used as indicators of water quality. We noted discrepancies between these two methods, highlighting how NGS techniques may complement the use of morphological analyses when analyzing algal diversity in this system. Culturing methods also revealed the presence of two taxa new to science (Nodosilinea fontisand Brasilonema variegatus), indicating these springs may represent a potential source of novel cyanobacteria. Taken together, this study showcases Florida springs are rich in algal diversity and a combination of methods is required for more complete biodiversity assessments. Future studies implementing such methods will aid in the preservation and conservation of these ecosystems

From Genes to Ecosystems: Resource Availability and DNA Methylation Drive the Diversity and Abundance of Restriction Modification Systems in Prokaryotes

Together, prokaryotic hosts and their viruses numerically dominate the planet and are engaged in an eternal struggle of hosts evading viral predation and viruses overcoming defensive mechanisms employed by their hosts. Prokaryotic hosts have been found to carry several viral defense systems in recent years with Restriction Modification systems (RMs) were the first discovered in the 1950s. While we have biochemically elucidated many of these systems in the last 70 years, we still struggle to understand what drives their gain and loss in prokaryotic genomes. In this work, we take a computational approach to understand the underlying evolutionary drivers of RMs by assessing ‘big data’ signals of RMs in prokaryotic genomes and incorporating molecular data in trait-based mathematical models. Focusing on the Cyanobacteria, we found a large discrepancy in the frequency of RMs per genome in different environmental contexts, where Cyanobacteria that live in oligotrophic nutrient conditions have few to no RMs and those in nutrient-rich conditions consistently have many RMs. While our models agree with the observation that increased nutrient inputs make the selective pressure of RMs more intense, they were unable to reconcile the high numbers of RMs per genome with their potent defensive properties- a situation of apparent overkill. By incorporating viral methylation, an unavoidable effect of RMs, we were able to explain how organisms could carry over 15 RMs. With this discovery, we then tried and reassess the distribution of methyltransferases, an essential component of RMs that can also have alternate physiological rolls in the cell. We expand on conventional wisdom, that methyltransferases that are widely phylogenetically conserved are associated with global cellular regulation. However, we also find that organisms with high numbers of RMs also have a surprising amount of conservation in the methyltransferases that they carry. This data suggests caution should be used in associating phylogenic signals with functional rolls in methyltransferases as different functional rolls seem to overlap in their phylogenetic signal. Indeed, we suggest trait-based modeling may be the best tool in elucidating why organisms with a high selective pressure to maintain RMs appear to have conserved methyltransferase