Fluorescently labeled prey surrogates in combination with fluorescence-activated cell sorting successfully discriminate actively feeding mixotrophs in a lake water sample

Mixotrophic protists are capable of acting both as primary producers and primary consumers at the base of the aquatic food web, thus constituting key organisms in ecosystems where they are abundant. However, their identity, abundance, ecological dynamics, and biogeochemical impact in aquatic ecosystems remain understudied in comparison to classically demarcated autotrophs or heterotrophs. In this study, we make use of fluorescently labeled prey and fluorescence-activated cell sorting to taxonomically identify actively-feeding individual mixotrophic flagellates from lake water. Replicated experiments were carried out to assess the performance of three different fluorescently labeled prey types and a fluorescent dye targeting food vacuoles. In the experiments, water from an oligotrophic lake was exposed independently to each type of reporter and cells were individually sorted based on fluorescent signals for predation and chlorophyll a. A total of 927 individual single cells were successfully recovered, with all fluorescent reporters exhibiting high sensitivity for putative mixotrophic taxa: overall, 87% of the occurrences could be assigned to dictyochophytes, 9% to chrysophytes, and 3% to dinoflagellates. As a result, we were able to detect cryptic diversity within pedinellid algae and report a Prorocentrum-like freshwater occurrence. We argue that this procedure can be a valuable tool to uncover relevant and unexpected active mixotrophic species in a wider range of aquatic environments, and could easily be coupled to other techniques to describe the finer details of the trophic status of aquatic microbial communities

Shotgun metagenomes from productive lakes in an urban region of Sweden

Urban lakes provide multiple benefits to society while influencing life quality. Moreover, lakes and their microbiomes are sentinels of anthropogenic impact and can be used for natural resource management and planning. Here, we release original metagenomic data from several well-characterized and anthropogenically impacted eutrophic lakes in the vicinity of Stockholm (Sweden). Our goal was to collect representative microbial community samples and use shotgun sequencing to provide a broad view on microbial diversity of productive urban lakes. Our dataset has an emphasis on Lake Malaren as a major drinking water reservoir under anthropogenic impact. This dataset includes short-read sequence data and metagenome assemblies from each of 17 samples collected from eutrophic lakes near the greater Stockholm area. We used genome-resolved metagenomics and obtained 2378 metagenome assembled genomes that de-replicated into 514 species representative genomes. This dataset adds new datapoints to previously sequenced lakes and it includes the first sequenced set of metagenomes from Lake Malaren. Our dataset serves as a baseline for future monitoring of drinking water reservoirs and urban lakes

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions

Choice of methodology and surrogate prey are decisive for the quality of protistan bacterivory rate estimates

Microeukaryote predation on bacteria is a fundamental phenomenon to understand energy and nutrient dynamics at the base of the aquatic food web. To date, the most prevalent way to estimate grazing rates is by using epifluorescence microscopy to enumerate ingestion events of fluorescently labelled tracers (FLTs) after short-term incubation experiments. However, this approach can be sensitive to the type of FLT, requires skillful preparation of the samples and is limited to small sample sizes. We tested the susceptibility of rate estimates to the choice of prey and made a side-by-side comparison between microscopy and flow cytometry when recording ingestion by a bacterivorous flagellate. Short-term uptake experiments were established using 5 types of FLTs differing in quality (living, dead or inert) and size (large or small), with Ochromonas triangulata as a model flagellate. The experiments showed that (1) each of the different prey types yielded different clearing rates, ranging from 0.5 to 3.6 nl cell-1 h-1, with the largest differences (3-fold or higher) between small prey (lower rates) and large prey (higher rates); (2) the cytometry estimate differed significantly from the microscopy estimate in 3 out of 4 experimental configurations; and (3) the precision of the cytometric analysis was greater, with >3-fold higher uncertainty associated with microscopy counting. Our results validate that flow cytometry provides a more precise bacterivory estimate, and that the choice of FLT influences the grazing rate estimate to a high extent regardless of the analytical method used

A genomic catalog of Earth’s microbiomes

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes

Redox gradient shapes the abundance and diversity of mercury-methylating microorganisms along the water column of the Black Sea

In the global context of seawater deoxygenation triggered by climate change and anthropogenic activities, changes in redox gradients impacting biogeochemical transformations of pollutants, such as mercury, become more likely. Being the largest anoxic basin worldwide, with high concentrations of the potent neurotoxic methylmercury (MeHg), the Black Sea is an ideal natural laboratory to provide new insights about the link between dissolved oxygen concentration and hgcAB gene-carrying (hgc+) microorganisms involved in the formation of MeHg. We combined geochemical and microbial approaches to assess the effect of vertical redox gradients on abundance, diversity, and metabolic potential of hgc+ microorganisms in the Black Sea water column. The abundance of hgcA genes [congruently estimated by quantitative PCR (qPCR) and metagenomics] correlated with MeHg concentration, both maximal in the upper part of the anoxic water. Besides the predominant Desulfobacterales, hgc+ microorganisms belonged to a unique assemblage of diverse—previously underappreciated—anaerobic fermenters from Anaerolineales, Phycisphaerae (characteristic of the anoxic and sulfidic zone), Kiritimatiellales, and Bacteroidales (characteristic of the suboxic zone). The metabolic versatility of Desulfobacterota differed from strict sulfate reduction in the anoxic water to reduction of various electron acceptors in the suboxic water. Linking microbial activity and contaminant concentration in environmental studies is rare due to the complexity of biological pathways. In this study, we disentangle the role of oxygen in shaping the distribution of Hg-methylating microorganisms consistently with MeHg concentration, and we highlight their taxonomic and metabolic niche partitioning across redox gradients, improving the prediction of the response of marine communities to the expansion of oxygen-deficient zones. IMPORTANCE Methylmercury (MeHg) is a neurotoxin detected at high concentrations in certain marine ecosystems, posing a threat to human health. MeHg production is mainly mediated by hgcAB gene-carrying (hgc+) microorganisms. Oxygen is one of the main factors controlling Hg methylation; however, its effect on the diversity and ecology of hgc+ microorganisms remains unknown. Under the current context of seawater deoxygenation, mercury cycling is expected to be disturbed. Here, we show the strong effect of oxygen gradients on the distribution of potential Hg methylators. In addition, we show for the first time the significant contribution of a unique assemblage of potential fermenters from Anaerolineales, Phycisphaerae, and Kiritimatiellales to Hg methylation, stratified in different redox niches along the Black Sea gradient. Our results considerably expand the known taxonomic diversity and ecological niches prone to the formation of MeHg and contribute to better apprehend the consequences of oxygen depletion in seawater

Goldberger-Treiman relation and Wu-type experiment in the decuplet sector

The leading-order chiral Lagrangian for the baryon octet and decuplet states coupled to Goldstone bosons and external sources contains six low-energy constants. Five of them are fairly well known from phenomenology, but the sixth one is practically unknown. This coupling constant provides the strength of the (p-wave) coupling of Goldstone bosons to decuplet states. Its size and even sign are under debate. Quark model and QCD for a large number of colors provide predictions, but some recent phenomenological analyses suggest even an opposite sign for the Delta-pion coupling. The Goldberger-Treiman relation connects this coupling constant to the axial charge of the Delta baryon. This suggests a Wu-type experiment to determine the unknown low-energy constant. While this is not feasible in the Delta sector because of the large hadronic width of the Delta, there is a flavor symmetry related process that is accessible: the weak semileptonic decay of the Omega baryon to a spin 3/2 cascade baryon. A broad research program is suggested that can pin down at least the rough size and the sign of the last unknown low-energy constant of the leading-order Lagrangian. It encompasses experimental measurements, in particular the forward-backward asymmetry of the semileptonic decay, together with a determination of the quark-mass dependences using lattice QCD for the narrow decuplet states and chiral perturbation theory to extrapolate to the Delta sector. Besides discussing the strategy of the research program, the present work provides a feasibility check based on a simple leading-order calculation.Comment: 7 page

mOTUpan: a robust Bayesian approach to leverage metagenome-assembled genomes for core-genome estimation

Recent advances in sequencing and bioinformatics have expanded the tree of life by providing genomes for uncultured environmentally relevant clades, either through metagenome-assembled genomes or through single-cell genomes. While this expanded diversity can provide novel insights into microbial population structure, most tools available for core-genome estimation are sensitive to genome completeness. Consequently, a major portion of the huge phylogenetic diversity uncovered by environmental genomic approaches remains excluded from such analyses. We present mOTUpan, a novel iterative Bayesian method for computing the core genome for sets of genomes of highly diverse completeness range. The likelihood for each gene cluster to belong to core or accessory genome is estimated by computing the probability of its presence/absence pattern in the target genome set. The core-genome prediction is computationally efficient and can be scaled up to thousands of genomes. It has shown comparable estimates to state-of-the-art tools Roary and PPanGGOLiN for high-quality genomes and is capable of using genomes at lower completeness thresholds. mOTUpan wraps a bootstrapping procedure to estimate the quality of a specific core-genome prediction, as the accuracy of each run will depend on the specific completeness distribution and the number of genomes in the dataset under scrutiny. mOTUpan is implemented in the mOTUlizer software package, and available at github.com/moritzbuck/mOTUlizer, under GPL 3.0 license

Exploring environmental intra-species diversity through non-redundant pangenome assemblies

At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different environmental niches and can have a profound influence on microbial community dynamics. As a consequence, any individual genome or population will contain merely a fraction of the total genetic diversity of any operationally defined "species", whose ecological potential can thus be only fully understood by studying all of their genomes and the genes therein. This concept, known as the pangenome, is valuable for studying microbial ecology and evolution, as it partitions genomes into core (present in all the genomes from a species, and responsible for housekeeping and species-level niche adaptation among others) and accessory regions (present only in some, and responsible for intra-species differentiation). Here we present SuperPang, an algorithm producing pangenome assemblies from a set of input genomes of varying quality, including metagenome-assembled genomes (MAGs). SuperPang runs in linear time and its results are complete, non-redundant, preserve gene ordering and contain both coding and non-coding regions. Our approach provides a modular view of the pangenome, identifying operons and genomic islands, and allowing to track their prevalence in different populations. We illustrate this by analysing intra-species diversity in Polynucleobacter, a bacterial genus ubiquitous in freshwater ecosystems, characterized by their streamlined genomes and their ecological versatility. We show how SuperPang facilitates the simultaneous analysis of allelic and gene content variation under different environmental pressures, allowing us to study the drivers of microbial diversification at unprecedented resolution