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1. Pangenomic comparison of globally distributed Poribacteria associated with sponge hosts and marine particles

   https://doi.org/10.1038/s41396-018-0292-9  

   Podell, Sheila; Blanton, Jessica M.; Neu, Alexander; Agarwal, Vinayak; Biggs, Jason S.; Moore, Bradley S.; Allen, Eric E. (October 2018, The ISME Journal)

   Abstract Candidatus Poribacteria is a little-known bacterial phylum, previously characterized by partial genomes from a single sponge host, but never isolated in culture. We have reconstructed multiple genome sequences from four different sponge genera and compared them to recently reported, uncharacterized Poribacteria genomes from the open ocean, discovering shared and unique functional characteristics. Two distinct, habitat-linked taxonomic lineages were identified, designated Entoporibacteria (sponge-associated) and Pelagiporibacteria (free-living). These lineages differed in flagellar motility and chemotaxis genes unique to Pelagiporibacteria, and highly expanded families of restriction endonucleases, DNA methylases, transposases, CRISPR repeats, and toxin–antitoxin gene pairs in Entoporibacteria. Both lineages shared pathways for facultative anaerobic metabolism, denitrification, fermentation, organosulfur compound utilization, type IV pili, cellulosomes, and bacterial proteosomes. Unexpectedly, many features characteristic of eukaryotic host association were also shared, including genes encoding the synthesis of eukaryotic-like cell adhesion molecules, extracellular matrix digestive enzymes, phosphoinositol-linked membrane glycolipids, and exopolysaccharide capsules. Complete Poribacteria 16S rRNA gene sequences were found to contain multiple mismatches to “universal” 16S rRNA gene primer sets, substantiating concerns about potential amplification failures in previous studies. A newly designed primer set corrects these mismatches, enabling more accurate assessment of Poribacteria abundance in diverse marine habitats where it may have previously been overlooked. 

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2. 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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3. Expanded Phylogenetic Diversity and Metabolic Flexibility of Mercury-Methylating Microorganisms

   https://doi.org/10.1128/mSystems.00299-20  

   McDaniel, Elizabeth A.; Peterson, Benjamin D.; Stevens, Sarah L.; Tran, Patricia Q.; Anantharaman, Karthik; McMahon, Katherine D. (August 2020, mSystems)

   Kent, Angela D. (Ed.)

   ABSTRACT Methylmercury is a potent bioaccumulating neurotoxin that is produced by specific microorganisms that methylate inorganic mercury. Methylmercury production in diverse anaerobic bacteria and archaea was recently linked to the hgcAB genes. However, the full phylogenetic and metabolic diversity of mercury-methylating microorganisms has not been fully unraveled due to the limited number of cultured experimentally verified methylators and the limitations of primer-based molecular methods. Here, we describe the phylogenetic diversity and metabolic flexibility of putative mercury-methylating microorganisms by hgcAB identification in publicly available isolate genomes and metagenome-assembled genomes (MAGs) as well as novel freshwater MAGs. We demonstrate that putative mercury methylators are much more phylogenetically diverse than previously known and that hgcAB distribution among genomes is most likely due to several independent horizontal gene transfer events. The microorganisms we identified possess diverse metabolic capabilities spanning carbon fixation, sulfate reduction, nitrogen fixation, and metal resistance pathways. We identified 111 putative mercury methylators in a set of previously published permafrost metatranscriptomes and demonstrated that different methylating taxa may contribute to hgcA expression at different depths. Overall, we provide a framework for illuminating the microbial basis of mercury methylation using genome-resolved metagenomics and metatranscriptomics to identify putative methylators based upon hgcAB presence and describe their putative functions in the environment. IMPORTANCE Accurately assessing the production of bioaccumulative neurotoxic methylmercury by characterizing the phylogenetic diversity, metabolic functions, and activity of methylators in the environment is crucial for understanding constraints on the mercury cycle. Much of our understanding of methylmercury production is based on cultured anaerobic microorganisms within the Deltaproteobacteria , Firmicutes , and Euryarchaeota. Advances in next-generation sequencing technologies have enabled large-scale cultivation-independent surveys of diverse and poorly characterized microorganisms from numerous ecosystems. We used genome-resolved metagenomics and metatranscriptomics to highlight the vast phylogenetic and metabolic diversity of putative mercury methylators and their depth-discrete activities in thawing permafrost. This work underscores the importance of using genome-resolved metagenomics to survey specific putative methylating populations of a given mercury-impacted ecosystem. 

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4. Anaerobic fungi in the tortoise alimentary tract illuminate early stages of host-fungal symbiosis and Neocallimastigomycota evolution

   https://doi.org/10.1038/s41467-024-47047-4  

   Pratt, Carrie J.; Meili, Casey H.; Jones, Adrienne L.; Jackson, Darian K.; England, Emma E.; Wang, Yan; Hartson, Steve; Rogers, Janet; Elshahed, Mostafa S.; Youssef, Noha H. (March 2024, Nature Communications)

   Abstract Anaerobic gut fungi (AGF,Neocallimastigomycota) reside in the alimentary tract of herbivores. While their presence in mammals is well documented, evidence for their occurrence in non-mammalian hosts is currently sparse. Culture-independent surveys of AGF in tortoises identified a unique community, with three novel deep-branching genera representing >90% of sequences in most samples. Representatives of all genera were successfully isolated under strict anaerobic conditions. Transcriptomics-enabled phylogenomic and molecular dating analyses indicated an ancient, deep-branching position in the AGF tree for these genera, with an evolutionary divergence time estimate of 104-112 million years ago (Mya). Such estimates push the establishment of animal-Neocallimastigomycotasymbiosis from the late to the early Cretaceous. Further, tortoise-associated isolates (T-AGF) exhibited limited capacity for plant polysaccharides metabolism and lacked genes encoding several carbohydrate-active enzyme (CAZyme) families. Finally, we demonstrate that the observed curtailed degradation capacities and reduced CAZyme repertoire is driven by the paucity of horizontal gene transfer (HGT) in T-AGF genomes, compared to their mammalian counterparts. This reduced capacity was reflected in an altered cellulosomal production capacity in T-AGF. Our findings provide insights into the phylogenetic diversity, ecological distribution, evolutionary history, evolution of fungal-host nutritional symbiosis, and dynamics of genes acquisition inNeocallimastigomycota. 

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5. RelocaTE2: a high resolution transposable element insertion site mapping tool for population resequencing

   https://doi.org/10.7717/peerj.2942  

   Chen, Jinfeng; Wrightsman, Travis R.; Wessler, Susan R.; Stajich, Jason E. (January 2017, PeerJ)

   Background Transposable element (TE) polymorphisms are important components of population genetic variation. The functional impacts of TEs in gene regulation and generating genetic diversity have been observed in multiple species, but the frequency and magnitude of TE variation is under appreciated. Inexpensive and deep sequencing technology has made it affordable to apply population genetic methods to whole genomes with methods that identify single nucleotide and insertion/deletion polymorphisms. However, identifying TE polymorphisms, particularly transposition events or non-reference insertion sites can be challenging due to the repetitive nature of these sequences, which hamper both the sensitivity and specificity of analysis tools. Methods We have developed the tool RelocaTE2 for identification of TE insertion sites at high sensitivity and specificity. RelocaTE2 searches for known TE sequences in whole genome sequencing reads from second generation sequencing platforms such as Illumina. These sequence reads are used as seeds to pinpoint chromosome locations where TEs have transposed. RelocaTE2 detects target site duplication (TSD) of TE insertions allowing it to report TE polymorphism loci with single base pair precision. Results and Discussion The performance of RelocaTE2 is evaluated using both simulated and real sequence data. RelocaTE2 demonstrate high level of sensitivity and specificity, particularly when the sequence coverage is not shallow. In comparison to other tools tested, RelocaTE2 achieves the best balance between sensitivity and specificity. In particular, RelocaTE2 performs best in prediction of TSDs for TE insertions. Even in highly repetitive regions, such as those tested on rice chromosome 4, RelocaTE2 is able to report up to 95% of simulated TE insertions with less than 0.1% false positive rate using 10-fold genome coverage resequencing data. RelocaTE2 provides a robust solution to identify TE insertion sites and can be incorporated into analysis workflows in support of describing the complete genotype from light coverage genome sequencing. 

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