Metagenomic analyses of modern stromatolitic mats from Hamelin Pool, Shark Bay, Western Australia

The living stromatolites of Shark Bay on the western coast of Australia are marine systems that present unique opportunities to address fundamental questions in diverse fields ranging from microbiology, geology, evolution, functional genomics, and biotechnology. They are one of the most dominant and persistent parts of Earth’s fossil records and thus highly significant from evolutionary and ecological perspectives. Also, these stromatolites are an ideal biological system for studying survival strategies of resident microorganisms, as well as the potential for novel bioactive discovery. Previous studies utilising culture and non-culture methods confirmed the high diversity of microbial life in this environment. To further our understanding of these geobiological structures, detailed analyses of associated microbial communities and their functional characteristics are crucial. The emergence of next-generation sequencing technologies provides unprecedented access to detailed genetic information on the systems in Shark Bay, and allows us to address fundamental ecological, adaptive, and evolutionary questions. To further delineate microbial community composition and elucidate for the first time the genetic potential of the resident communities, in this study, we have sequenced two distinct stromatolitic mat metagenomes by 454 shotgun pyrosequencing and 16S rDNA tagging. The libraries were annotated using the MetaGenomic Rapid Annotation using Subsystem Technology (MG-RAST) and Integrated Microbial Genome with Microbiome Samples-Expert Review (IMG/MER). In addition to phylogenetic analyses, we focused on key subsystems and identified the diversity and abundance of genes involved in critical processes such as a stromatolite formation (e.g. EPS turnover, nutrient cycling), adaptive responses (e.g. osmo, antibiotics and heavy metal), cell communication (e.g. quorum sensing), and bioactive production (eg. non-ribosomal peptide synthesis). Clustering analyses of the different stromatolites samples, based primarily on subsystems, have revealed processes that are shared in a given sample and potentially distinguish the Shark Bay stromatolites from other ecosystems. The functional genetic complexity of microorganisms associated with the Shark Bay stromatolites has been shown for the first time and vastly improves our understanding of these ancient ecosystems, while providing a platform to further explore the potential of these systems at the expression level

Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Murray, S. A., Ruvindy, R., Kohli, G. S., Anderson, D. M., & Brosnahan, M. L. Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1. Scientific Reports, 9(1), (2019): 14532, doi:10.1038/s41598-019-51074-3.Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.We thank the Australian Research Council for Funding (FT120100704). We thank Chowdhury Sarowar for the toxicity measurements, at the Sydney Institute of Marine Science. Support to MB and DA was provided by MIT Sea Grant (NA14OAR4170077) and the Woods Hole Center for Oceans and Human Health (National Science Foundation award OCE-1840381 and National Institute of Environmental Health Sciences award 1-P01-ES028938–01). We are grateful for assistance from David Kulis, Claire Mullen, and Isaac Rosenthal for assistance in the collection and processing of Salt Pond samples

The application of qPCR assays for the early detection of toxic Alexandrium in eastern australian waters

University of Technology Sydney. Faculty of Science.Harmful algal blooms that produce Paralytic Shellfish Toxins (PSTs) are prevalent and affect shellfish harvesting areas worldwide. PSTs have caused shellfish harvesting closures and product recalls, resulting in economic losses, as well as brand damage and damage to the wider economy including the tourism industry. In Tasmania, it is known that four PST producing species co-occur, comprising , . , . and . In particular, of these, three species are morphologically almost identical, the species of the former species complex (. , . , and . ), which cannot be differentiated using light microscopy. Therefore, phytoplankton monitoring using light microscopy and total PST in shellfish using High Performance Liquid Chromatography (HPLC) may not be sufficient to allow for an early warning with enough time to take appropriate shellfish harvesting management decisions. In this thesis, quantitative Polymerase Chain Reaction (qPCR) assays are investigated as an in-field early warning system, as well as a tool for long-term risk assessment of PST-associated harmful algal blooms. A commercial on-farm pipeline based on the collection and filtration of water samples using a custom designed gravity filter, a cell lysis, and a qPCR assay based on 4 was also developed and validated. QPCR assays based on ribosomal DNA (rDNA) ‘barcoding’ regions and an assay based on a gene associated with PST biosynthesis (4) were found to be generally specific, sensitive and efficient. The efficacy of an rDNA-based assay for cyst quantification was demonstrated, showing potential for its use as a long-term risk assessment tool for a new harvest area. However, qPCR assays based on rDNA gene regions were found to overestimate cell abundances. An analysis of rDNA copy number variation among strains of species of showed a variation of up to 3-5 orders of magnitude within a species, and was correlated significantly with genome size, which also varied within a species. An analysis of the variation in genomic copies of 4 genes showed variation as well, however this was of a lesser degree, of up to one order of magnitude. A positive correlation was found between 4 copies per cell and the total PST produced per cell, showing that the dosage effect may contribute to the regulation of PST biosynthesis

Correction: McLennan et al. Assessing the Use of Molecular Barcoding and qPCR for Investigating the Ecology of Prorocentrum minimum (Dinophyceae), a Harmful Algal Species. Microorganisms 2021, 9, 510

The authors wish to make the following corrections to this paper [...

Viral Communities of Shark Bay Modern Stromatolites

Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients

Data_Sheet_1_Viral Communities of Shark Bay Modern Stromatolites.xlsx

<p>Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients.</p

An On-Farm Workflow for Predictive Management of Paralytic Shellfish Toxin-Producing Harmful Algal Blooms for the Aquaculture Industry

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire’s buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7–106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L–1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture

The evolution of living beings started with prokaryotes and in interaction with prokaryotes

In natural world, no organism exists in absolute isolation, and thus every organism must interact with the environment and other organisms. Next-generation sequencing technologies are increasingly revealing that most of the cells in the environment resist cultivation in the laboratory and several prokaryotic divisions have no known cultivated representatives. Based on this, we hypothesize that species that live together in the same ecosystem are more or less dependent upon each other and are very large in diversity and number, outnumbering those that can be isolated in single-strain laboratory culture. In natural environments, bacteria and archaea interact with other organisms (viruses, protists, fungi, animals, plants, and human) in complex ecological networks, resulting in positive, negative, or no effect on one or another of the interacting partners. These interactions are sources of ecological forces such as competitive exclusion, niche partitioning, ecological adaptation, or horizontal gene transfers, which shape the biological evolution. In this chapter, we review the biological interactions involving prokaryotes in natural ecosystems, including plant, animal, and human microbiota, and give an overview of the insights into the evolution of living beings. We conclude that studies of biological interactions, including multipartite interactions, are sources of novel knowledge related to the biodiversity of living things, the functioning of ecosystems, the evolution of the cellular world, and the ecosystem services to the living beings