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1. Evaluating and Improving Small Subunit rRNA PCR Primer Coverage for Bacteria, Archaea, and Eukaryotes Using Metagenomes from Global Ocean Surveys

   https://doi.org/10.1128/mSystems.00565-21  

   McNichol, Jesse ; Berube, Paul M. ; Biller, Steven J. ; Fuhrman, Jed A. ( June 2021 , mSystems)

   Gilbert, Jack A. (Ed.)

   ABSTRACT Small subunit rRNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly used primers with >300 million rRNA gene sequences retrieved from globally distributed marine metagenomes. The best-performing primers compared to 16S rRNA of bacteria and archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering cyanobacterial and chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA specific; 82%)—demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median R 2 = 0.98, n  = 272), indicating amplicons can produce equally accurate community composition data compared with shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly completely universal primer set that could accurately quantify biogeochemically important taxa in ecosystems ranging from the deep sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data. IMPORTANCE PCR amplification and sequencing of marker genes is a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time but will work optimally only if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally distributed short-read oceanic metagenomes. Our results demonstrate that primer sets vary widely in performance, and that at least for marine systems, rRNA amplicon data from some primers lack significant biases compared to metagenomes. We also show that it is theoretically possible to create a nearly universal primer set for diverse saline environments by defining a specific mixture of a few dozen oligonucleotides, and present a software pipeline that can guide rational design of primers for any environment with available meta’omic data. 

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2. Finding needles in a haystack—Extensive diversity in the eustigmatophyceae revealed by community metabarcode analysis targeting the rbc L gene using lineage‐directed primers

   https://doi.org/10.1111/jpy.13196  

   Fawley, Marvin W. ; Fawley, Karen P. ; Cahoon, Aubrey Bruce ; Lane, ed., C. ( August 2021 , Journal of Phycology)

   Sequences from the Stramenopile class Eustigmatophyceae are rarely reported in metabarcoding studies, and when they have been reported, there are very few haplotypes. We hypothesized that the paucity of eustigmatophyte species detected in these studies may be a result of the metabarcoding techniques used, which have primarily employed universal ribosomal RNA gene regions. In this study, we examined environmental DNA samples from 22 sites in southwestern Virginia, some of which had previously been studied using ribosomal RNA analysis. We used metabarcoding techniques targeting the plastidrbcL gene with new primers designed to produce a 370 bp amplicon from all lineages of the Eustigmatophyceae in a reference collection. The amplicons were then analyzed with DADA2 to produce amplicon sequence variants (ASVs). Our results revealed 184rbcL haplotypes that can be tentatively assigned to the Eustigmatophyceae from these sites, representing much higher diversity than has been detected by ribosomal DNA‐based studies. The techniques employed can be used for future studies of population structure, ecology, distribution, and diversity of this class. With these techniques, it should be possible to make realistic estimates of the species‐level diversity of the Eustigmatophyceae on local, regional, and perhaps global scales.

    

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3. Feedback Regulation between Aquatic Microorganisms and the Bloom-Forming Cyanobacterium Microcystis aeruginosa

   https://doi.org/10.1128/AEM.01362-19  

   Zhang, Meng ; Lu, Tao ; Paerl, Hans W. ; Chen, Yiling ; Zhang, Zhenyan ; Zhou, Zhigao ; Qian, Haifeng ; Drake, Harold L. ( November 2019 , Applied and Environmental Microbiology)

   ABSTRACT The frequency and intensity of cyanobacterial blooms are increasing worldwide. Interactions between toxic cyanobacteria and aquatic microorganisms need to be critically evaluated to understand microbial drivers and modulators of the blooms. In this study, we applied 16S/18S rRNA gene sequencing and metabolomics analyses to measure the microbial community composition and metabolic responses of the cyanobacterium Microcystis aeruginosa in a coculture system receiving dissolved inorganic nitrogen and phosphorus (DIP) close to representative concentrations in Lake Taihu, China. M. aeruginosa secreted alkaline phosphatase using a DIP source produced by moribund and decaying microorganisms when the P source was insufficient. During this process, M. aeruginosa accumulated several intermediates in energy metabolism pathways to provide energy for sustained high growth rates and increased intracellular sugars to enhance its competitive capacity and ability to defend itself against microbial attack. It also produced a variety of toxic substances, including microcystins, to inhibit metabolite formation via energy metabolism pathways of aquatic microorganisms, leading to a negative effect on bacterial and eukaryotic microbial richness and diversity. Overall, compared with the monoculture system, the growth of M. aeruginosa was accelerated in coculture, while the growth of some cooccurring microorganisms was inhibited, with the diversity and richness of eukaryotic microorganisms being more negatively impacted than those of prokaryotic microorganisms. These findings provide valuable information for clarifying how M. aeruginosa can potentially modulate its associations with other microorganisms, with ramifications for its dominance in aquatic ecosystems. IMPORTANCE We measured the microbial community composition and metabolic responses of Microcystis aeruginosa in a microcosm coculture system receiving dissolved inorganic nitrogen and phosphorus (DIP) close to the average concentrations in Lake Taihu. In the coculture system, DIP is depleted and the growth and production of aquatic microorganisms can be stressed by a lack of DIP availability. M. aeruginosa could accelerate its growth via interactions with specific cooccurring microorganisms and the accumulation of several intermediates in energy metabolism-related pathways. Furthermore, M. aeruginosa can decrease the carbohydrate metabolism of cooccurring aquatic microorganisms and thus disrupt microbial activities in the coculture. This also had a negative effect on bacterial and eukaryotic microbial richness and diversity. Microcystin was capable of decreasing the biomass of total phytoplankton in aquatic microcosms. Overall, compared to the monoculture, the growth of total aquatic microorganisms is inhibited, with the diversity and richness of eukaryotic microorganisms being more negatively impacted than those of prokaryotic microorganisms. The only exception is M. aeruginosa in the coculture system, whose growth was accelerated. 

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4. Defining the Sphagnum Core Microbiome across the North American Continent Reveals a Central Role for Diazotrophic Methanotrophs in the Nitrogen and Carbon Cycles of Boreal Peatland Ecosystems

   https://doi.org/10.1128/mbio.03714-21  

   Kolton, Max ; Weston, David J. ; Mayali, Xavier ; Weber, Peter K. ; McFarlane, Karis J. ; Pett-Ridge, Jennifer ; Somoza, Mark M. ; Lietard, Jory ; Glass, Jennifer B. ; Lilleskov, Erik A. ; et al ( February 2022 , mBio)

   Martiny, Jennifer B. (Ed.)

   ABSTRACT Peat mosses of the genus Sphagnum are ecosystem engineers that frequently predominate over photosynthetic production in boreal peatlands. Sphagnum spp. host diverse microbial communities capable of nitrogen fixation (diazotrophy) and methane oxidation (methanotrophy), thereby potentially supporting plant growth under severely nutrient-limited conditions. Moreover, diazotrophic methanotrophs represent a possible “missing link” between the carbon and nitrogen cycles, but the functional contributions of the Sphagnum -associated microbiome remain in question. A combination of metagenomics, metatranscriptomics, and dual-isotope incorporation assays was applied to investigate Sphagnum microbiome community composition across the North American continent and provide empirical evidence for diazotrophic methanotrophy in Sphagnum -dominated ecosystems. Remarkably consistent prokaryotic communities were detected in over 250 Sphagnum SSU rRNA libraries from peatlands across the United States (5 states, 17 bog/fen sites, 18 Sphagnum species), with 12 genera of the core microbiome comprising 60% of the relative microbial abundance. Additionally, nitrogenase ( nifH ) and SSU rRNA gene amplicon analysis revealed that nitrogen-fixing populations made up nearly 15% of the prokaryotic communities, predominated by Nostocales cyanobacteria and Rhizobiales methanotrophs. While cyanobacteria comprised the vast majority (>95%) of diazotrophs detected in amplicon and metagenome analyses, obligate methanotrophs of the genus Methyloferula (order Rhizobiales ) accounted for one-quarter of transcribed nifH genes. Furthermore, in dual-isotope tracer experiments, members of the Rhizobiales showed substantial incorporation of 13 CH 4 and 15 N 2 isotopes into their rRNA. Our study characterizes the core Sphagnum microbiome across large spatial scales and indicates that diazotrophic methanotrophs, here defined as obligate methanotrophs of the rare biosphere ( Methyloferula spp. of the Rhizobiales ) that also carry out diazotrophy, play a keystone role in coupling of the carbon and nitrogen cycles in nutrient-poor peatlands. IMPORTANCE Nitrogen availability frequently limits photosynthetic production in Sphagnum moss-dominated high-latitude peatlands, which are crucial carbon-sequestering ecosystems at risk to climate change effects. It has been previously suggested that microbial methane-fueled fixation of atmospheric nitrogen (N 2 ) may occur in these ecosystems, but this process and the organisms involved are largely uncharacterized. A combination of omics (DNA and RNA characterization) and dual-isotope incorporation approaches illuminated the functional diversity of Sphagnum -associated microbiomes and defined 12 bacterial genera in its core microbiome at the continental scale. Moreover, obligate diazotrophic methanotrophs showed high nitrogen fixation gene expression levels and incorporated a substantial amount of atmospheric nitrogen and methane-driven carbon into their biomass. Thus, these results point to a central role for members of the rare biosphere in Sphagnum microbiomes as keystone species that couple nitrogen fixation to methane oxidation in nutrient-poor peatlands. 

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5. The respiratory microbiota of three cohabiting beluga whales (Delphinapterus leucas) under human care

   https://doi.org/10.3389/fmars.2023.1168623  

   Smith, Susan A. ; Ropati, Destiny V. ; Santoferrara, Luciana F. ; Romano, Tracy A. ; McManus, George B. ( December 2023 , Frontiers in Marine Science)

   We sampled the respiratory mucus from voluntary blowhole exhalations (“blow”) of three healthy beluga whales (Delphinapterus leucas) under professional human care. Blow samples were collected from three resident belugas, one adult male (M1) and two adult females (F1, F2), with voluntary behaviors via non-invasive methods over three days in July 2021 (four days for M1). Samples were weighed and examined microscopically for the enumeration of eukaryotic and prokaryotic microbes, and then were used to evaluate carbon substrate use and taxonomic diversity of prokaryotic communities in the host respiratory sytem. Microscopical observations and 18S rRNA gene sequencing indicated the presence of eukaryotic microbiota, the ciliate generaPlanilaminaandKyaroikeusin all three individuals. Exposure of samples to different metabolic carbon substrates indicated significant differences in the number of carbon sources usable by the prokaryotic communities of different whales (range: 11-25 sources), as well as a signficantly decreased diversity of carbon sources used by the community in the habitat water (5 sources). Sequencing of the hypervariable V4 region of the 16S rRNA gene revealed 19 amplicon sequence variants (ASVs) that were present in all whale samples. The oldest femaleD. leucas(F2) had the lowest overall diversity, and was significantly different from M1 and F1 in taxon composition, including an anomalously low ratio of Baccillota: Bacteroidota (0.01) compared to the other whales. In comparisons of microbial community composition, M1 had a significantly higher diversity than F1 and F2. These results suggest that attention should be given to regular microbiome sampling, and indicate a need for the pairing of microbiome and clinical data for animals in aquaria. Overall, these data contribute to the growing database on the core respiratory microbiota in cohabiting cetaceans under professional human care, indicate the utility of non-invasive sampling, and help characterize a baseline for healthyD. leucas.

    

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