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1. Metatranscriptomics yields new genomic resources and sensitive detection of infections for diverse blood parasites

   https://doi.org/10.1111/1755-0998.13091  

   Galen, Spencer C. ; Borner, Janus ; Williamson, Jessie L. ; Witt, Christopher C. ; Perkins, Susan L. ( December 2019 , Molecular Ecology Resources)

   Metatranscriptomics is a powerful method for studying the composition and function of complex microbial communities. The application of metatranscriptomics to multispecies parasite infections is of particular interest, as research on parasite evolution and diversification has been hampered by technical challenges to genome‐scale DNA sequencing. In particular, blood parasites of vertebrates are abundant and diverse although they often occur at low infection intensities and exist as multispecies infections, rendering the isolation of genomic sequence data challenging. Here, we use birds and their diverse haemosporidian parasites to illustrate the potential for metatranscriptome sequencing to generate large quantities of genome‐wide sequence data from multiple blood parasite species simultaneously. We used RNA‐sequencing of 24 blood samples from songbirds in North America to show that metatranscriptomes can yield large proportions of haemosporidian protein‐coding gene repertoires even when infections are of low intensity (<0.1% red blood cells infected) and consist of multiple parasite taxa. By bioinformatically separating host and parasite transcripts and assigning them to the haemosporidian genus of origin, we found that transcriptomes detected ~23% more total parasite infections across all samples than were identified using microscopy and DNA barcoding. For single‐species infections, we obtained data for >1,300 loci from samples with as lowmore »as 0.03% parasitaemia, with the number of loci increasing with infection intensity. In total, we provide data for 1,502 single‐copy orthologous loci from a phylogenetically diverse set of 33 haemosporidian mitochondrial lineages. The metatranscriptomic approach described here has the potential to accelerate ecological and evolutionary research on haemosporidians and other diverse parasites.

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2. METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks

   https://doi.org/10.1186/s40168-021-01213-8  

   Zhou, Zhichao ; Tran, Patricia Q. ; Breister, Adam M. ; Liu, Yang ; Kieft, Kristopher ; Cowley, Elise S. ; Karaoz, Ulas ; Anantharaman, Karthik ( February 2022 , Microbiome)

   Advances in microbiome science are being driven in large part due to our ability to study and infer microbial ecology from genomes reconstructed from mixed microbial communities using metagenomics and single-cell genomics. Such omics-based techniques allow us to read genomic blueprints of microorganisms, decipher their functional capacities and activities, and reconstruct their roles in biogeochemical processes. Currently available tools for analyses of genomic data can annotate and depict metabolic functions to some extent; however, no standardized approaches are currently available for the comprehensive characterization of metabolic predictions, metabolite exchanges, microbial interactions, and microbial contributions to biogeochemical cycling.

   We present METABOLIC (METabolic And BiogeOchemistry anaLyses In miCrobes), a scalable software to advance microbial ecology and biogeochemistry studies using genomes at the resolution of individual organisms and/or microbial communities. The genome-scale workflow includes annotation of microbial genomes, motif validation of biochemically validated conserved protein residues, metabolic pathway analyses, and calculation of contributions to individual biogeochemical transformations and cycles. The community-scale workflow supplements genome-scale analyses with determination of genome abundance in the microbiome, potential microbial metabolic handoffs and metabolite exchange, reconstruction of functional networks, and determination of microbial contributions to biogeochemical cycles. METABOLIC can take input genomes from isolates, metagenome-assembled genomes, ormore »single-cell genomes. Results are presented in the form of tables for metabolism and a variety of visualizations including biogeochemical cycling potential, representation of sequential metabolic transformations, community-scale microbial functional networks using a newly defined metric “MW-score” (metabolic weight score), and metabolic Sankey diagrams. METABOLIC takes ~ 3 h with 40 CPU threads to process ~ 100 genomes and corresponding metagenomic reads within which the most compute-demanding part of hmmsearch takes ~ 45 min, while it takes ~ 5 h to complete hmmsearch for ~ 3600 genomes. Tests of accuracy, robustness, and consistency suggest METABOLIC provides better performance compared to other software and online servers. To highlight the utility and versatility of METABOLIC, we demonstrate its capabilities on diverse metagenomic datasets from the marine subsurface, terrestrial subsurface, meadow soil, deep sea, freshwater lakes, wastewater, and the human gut.

   METABOLIC enables the consistent and reproducible study of microbial community ecology and biogeochemistry using a foundation of genome-informed microbial metabolism, and will advance the integration of uncultivated organisms into metabolic and biogeochemical models. METABOLIC is written in Perl and R and is freely available under GPLv3 athttps://github.com/AnantharamanLab/METABOLIC.

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3. Bioavailable iron titrations reveal oceanic Synechococcus ecotypes optimized for different iron availabilities

   https://doi.org/10.1038/s43705-022-00132-5  

   Gilbert, Naomi E. ; LeCleir, Gary R. ; Strzepek, Robert F. ; Ellwood, Michael J. ; Twining, Benjamin S. ; Roux, S. ; Pennacchio, C. ; Boyd, Philip W. ; Wilhelm, Steven W. ( July 2022 , ISME Communications)

   The trace metal iron (Fe) controls the diversity and activity of phytoplankton across the surface oceans, a paradigm established through decades of in situ and mesocosm experimental studies. Despite widespread Fe-limitation within high-nutrient, low chlorophyll (HNLC) waters, significant contributions of the cyanobacteriumSynechococcusto the phytoplankton stock can be found. Correlations among differing strains ofSynechococcusacross different Fe-regimes have suggested the existence of Fe-adapted ecotypes. However, experimental evidence of high- versuslow-Fe adapted strains ofSynechococcusis lacking, and so we investigated the transcriptional responses of microbial communities inhabiting the HNLC, sub-Antarctic region of the Southern Ocean during the Spring of 2018. Analysis of metatranscriptomes generated from on-deck incubation experiments reflecting a gradient of Fe-availabilities reveal transcriptomic signatures indicative of co-occurringSynechococcusecotypes adapted to differing Fe-regimes. Functional analyses comparing low-Fe and high-Fe conditions point to various Fe-acquisition mechanisms that may allow persistence of low-Fe adaptedSynechococcusunder Fe-limitation. Comparison of in situ surface conditions to the Fe-titrations indicate ecological relevance of these mechanisms as well as persistence of both putative ecotypes within this region. This Fe-titration approach, combined with transcriptomics, highlights the short-term responses of the in situ phytoplankton community to Fe-availability that are often overlooked by examining genomic content or bulk physiological responses alone. These findings expandmore »our knowledge about how phytoplankton in HNLC Southern Ocean waters adapt and respond to changing Fe supply.

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4. Trait-Based Comparison of Coral and Sponge Microbiomes

   https://doi.org/10.1038/s41598-020-59320-9  

   Fiore, Cara L. ; Jarett, Jessica K. ; Steinert, Georg ; Lesser, Michael P. ( February 2020 , Scientific Reports)

   Corals and sponges harbor diverse microbial communities that are integral to the functioning of the host. While the taxonomic diversity of their microbiomes has been well-established for corals and sponges, their functional roles are less well-understood. It is unclear if the similarities of symbiosis in an invertebrate host would result in functionally similar microbiomes, or if differences in host phylogeny and environmentally driven microhabitats within each host would shape functionally distinct communities. Here we addressed this question, using metatranscriptomic and 16S rRNA gene profiling techniques to compare the microbiomes of two host organisms from different phyla. Our results indicate functional similarity in carbon, nitrogen, and sulfur assimilation, and aerobic nitrogen cycling. Additionally, there were few statistical differences in pathway coverage or abundance between the two hosts. For example, we observed higher coverage of phosphonate and siderophore metabolic pathways in the star coral,Montastraea cavernosa, while there was higher coverage of chloroalkane metabolism in the giant barrel sponge,Xestospongia muta. Higher abundance of genes associated with carbon fixation pathways was also observed inM. cavernosa, while inX. mutathere was higher abundance of fatty acid metabolic pathways. Metagenomic predictions based on 16S rRNA gene profiling analysis were similar, and there was high correlation betweenmore »the metatranscriptome and metagenome predictions for both hosts. Our results highlight several metabolic pathways that exhibit functional similarity in these coral and sponge microbiomes despite the taxonomic differences between the two microbiomes, as well as potential specialization of some microbially based metabolism within each host.

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5. MetaboDirect: an analytical pipeline for the processing of FT-ICR MS-based metabolomic data

   https://doi.org/10.1186/s40168-023-01476-3  

   Ayala-Ortiz, Christian ; Graf-Grachet, Nathalia ; Freire-Zapata, Viviana ; Fudyma, Jane ; Hildebrand, Gina ; AminiTabrizi, Roya ; Howard-Varona, Cristina ; Corilo, Yuri E. ; Hess, Nancy ; Duhaime, Melissa B. ; et al ( February 2023 , Microbiome)

   Microbiomes are now recognized as the main drivers of ecosystem function ranging from the oceans and soils to humans and bioreactors. However, a grand challenge in microbiome science is to characterize and quantify the chemical currencies of organic matter (i.e., metabolites) that microbes respond to and alter. Critical to this has been the development of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which has drastically increased molecular characterization of complex organic matter samples, but challenges users with hundreds of millions of data points where readily available, user-friendly, and customizable software tools are lacking.

   Here, we build on years of analytical experience with diverse sample types to develop MetaboDirect, an open-source, command-line-based pipeline for the analysis (e.g., chemodiversity analysis, multivariate statistics), visualization (e.g., Van Krevelen diagrams, elemental and molecular class composition plots), and presentation of direct injection high-resolution FT-ICR MS data sets after molecular formula assignment has been performed. When compared to other available FT-ICR MS software, MetaboDirect is superior in that it requires a single line of code to launch a fully automated framework for the generation and visualization of a wide range of plots, with minimal coding experience required. Among the tools evaluated, MetaboDirect is alsomore »uniquely able to automatically generate biochemical transformation networks (ab initio) based on mass differences (mass difference network-based approach) that provide an experimental assessment of metabolite connections within a given sample or a complex metabolic system, thereby providing important information about the nature of the samples and the set of microbial reactions or pathways that gave rise to them. Finally, for more experienced users, MetaboDirect allows users to customize plots, outputs, and analyses.

   Application of MetaboDirect to FT-ICR MS-based metabolomic data sets from a marine phage-bacterial infection experiment and aSphagnumleachate microbiome incubation experiment showcase the exploration capabilities of the pipeline that will enable the research community to evaluate and interpret their data in greater depth and in less time. It will further advance our knowledge of how microbial communities influence and are influenced by the chemical makeup of the surrounding system. The source code and User’s guide of MetaboDirect are freely available through (https://github.com/Coayala/MetaboDirect) and (https://metabodirect.readthedocs.io/en/latest/), respectively.

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