Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.

Three novel virophage genomes discovered from Yellowstone Lake metagenomes.

UNLABELLED: Virophages are a unique group of circular double-stranded DNA viruses that are considered parasites of giant DNA viruses, which in turn are known to infect eukaryotic hosts. In this study, the genomes of three novel Yellowstone Lake virophages (YSLVs)--YSLV5, YSLV6, and YSLV7--were identified from Yellowstone Lake through metagenomic analyses. The relative abundance of these three novel virophages and previously identified Yellowstone Lake virophages YSLV1 to -4 were determined in different locations of the lake, revealing that most of the sampled locations in the lake, including both mesophilic and thermophilic habitats, had multiple virophage genotypes. This likely reflects the diverse habitats or diversity of the eukaryotic hosts and their associated giant viruses that serve as putative hosts for these virophages. YSLV5 has a 29,767-bp genome with 32 predicted open reading frames (ORFs), YSLV6 has a 24,837-bp genome with 29 predicted ORFs, and YSLV7 has a 23,193-bp genome with 26 predicted ORFs. Based on multilocus phylogenetic analysis, YSLV6 shows a close evolutionary relationship with YSLV1 to -4, whereas YSLV5 and YSLV7 are distantly related to the others, and YSLV7 represents the fourth novel virophage lineage. In addition, the genome of YSLV5 has a G+C content of 51.1% that is much higher than all other known virophages, indicating a unique host range for YSLV5. These results suggest that virophages are abundant and have diverse genotypes that likely mirror diverse giant viral and eukaryotic hosts within the Yellowstone Lake ecosystem. IMPORTANCE: This study discovered novel virophages present within the Yellowstone Lake ecosystem using a conserved major capsid protein as a phylogenetic anchor for assembly of sequence reads from Yellowstone Lake metagenomic samples. The three novel virophage genomes (YSLV5 to -7) were completed by identifying specific environmental samples containing these respective virophages, and closing gaps by targeted PCR and sequencing. Most of the YSLV genotypes were associated primarily with photic-zone and nonhydrothermal samples; however, YSLV5 had a unique distribution with an occurrence in vent samples similar to that in photic-zone samples and with a higher GC content that suggests a distinct host and habitat compared to other YSLVs. In addition, genome content and phylogenetic analyses indicate that YSLV5 and YSLV7 are distinct from known virophages and that additional as-yet-uncharacterized virophages are likely present within the Yellowstone Lake ecosystem.

Diversity of virophages in metagenomic data sets.

Virophages, e.g., Sputnik, Mavirus, and Organic Lake virophage (OLV), are unusual parasites of giant double-stranded DNA (dsDNA) viruses, yet little is known about their diversity. Here, we describe the global distribution, abundance, and genetic diversity of virophages based on analyzing and mapping comprehensive metagenomic databases. The results reveal a distinct abundance and worldwide distribution of virophages, involving almost all geographical zones and a variety of unique environments. These environments ranged from deep ocean to inland, iced to hydrothermal lakes, and human gut- to animal-associated habitats. Four complete virophage genomic sequences (Yellowstone Lake virophages [YSLVs]) were obtained, as was one nearly complete sequence (Ace Lake Mavirus [ALM]). The genomes obtained were 27,849 bp long with 26 predicted open reading frames (ORFs) (YSLV1), 23,184 bp with 21 ORFs (YSLV2), 27,050 bp with 23 ORFs (YSLV3), 28,306 bp with 34 ORFs (YSLV4), and 17,767 bp with 22 ORFs (ALM). The homologous counterparts of five genes, including putative FtsK-HerA family DNA packaging ATPase and genes encoding DNA helicase/primase, cysteine protease, major capsid protein (MCP), and minor capsid protein (mCP), were present in all virophages studied thus far. They also shared a conserved gene cluster comprising the two core genes of MCP and mCP. Comparative genomic and phylogenetic analyses showed that YSLVs, having a closer relationship to each other than to the other virophages, were more closely related to OLV than to Sputnik but distantly related to Mavirus and ALM. These findings indicate that virophages appear to be widespread and genetically diverse, with at least 3 major lineages.

Methylphosphonate Degradation and Salt-Tolerance Genes of Two Novel Halophilic Marivita Metagenome-Assembled Genomes from Unrestored Solar Salterns.

Aerobic bacteria that degrade methylphosphonates and produce methane as a byproduct have emerged as key players in marine carbon and phosphorus cycles. Here, we present two new draft genome sequences of the genus Marivita that were assembled from metagenomes from hypersaline former industrial salterns and compare them to five other Marivita reference genomes. Phylogenetic analyses suggest that both of these metagenome-assembled genomes (MAGs) represent new species in the genus. Average nucleotide identities to the closest taxon were <85%. The MAGs were assembled with SPAdes, binned with MetaBAT, and curated with scaffold extension and reassembly. Both genomes contained the phnCDEGHIJLMP suite of genes encoding the full C-P lyase pathway of methylphosphonate degradation and were significantly more abundant in two former industrial salterns than in nearby reference and restored wetlands, which have lower salinity levels and lower methane emissions than the salterns. These organisms contain a variety of compatible solute biosynthesis and transporter genes to cope with high salinity levels but harbor only slightly acidic proteomes (mean isoelectric point of 6.48).

Correction: Bueno de Mesquita et al. Methylphosphonate Degradation and Salt-Tolerance Genes of Two Novel Halophilic Marivita Metagenome-Assembled Genomes from Unrestored Solar Salterns. Genes 2022, 13, 148.

The authors have requested that the following change be made to their paper [...].

Microbial drivers of methane emissions from unrestored industrial salt ponds.

Wetlands are important carbon (C) sinks, yet many have been destroyed and converted to other uses over the past few centuries, including industrial salt making. A renewed focus on wetland ecosystem services (e.g., flood control, and habitat) has resulted in numerous restoration efforts whose effect on microbial communities is largely unexplored. We investigated the impact of restoration on microbial community composition, metabolic functional potential, and methane flux by analyzing sediment cores from two unrestored former industrial salt ponds, a restored former industrial salt pond, and a reference wetland. We observed elevated methane emissions from unrestored salt ponds compared to the restored and reference wetlands, which was positively correlated with salinity and sulfate across all samples. 16S rRNA gene amplicon and shotgun metagenomic data revealed that the restored salt pond harbored communities more phylogenetically and functionally similar to the reference wetland than to unrestored ponds. Archaeal methanogenesis genes were positively correlated with methane flux, as were genes encoding enzymes for bacterial methylphosphonate degradation, suggesting methane is generated both from bacterial methylphosphonate degradation and archaeal methanogenesis in these sites. These observations demonstrate that restoration effectively converted industrial salt pond microbial communities back to compositions more similar to reference wetlands and lowered salinities, sulfate concentrations, and methane emissions.

Methanogenesis and Salt Tolerance Genes of a Novel Halophilic Methanosarcinaceae Metagenome-Assembled Genome from a Former Solar Saltern.

Anaerobic archaeal methanogens are key players in the global carbon cycle due to their role in the final stages of organic matter decomposition in anaerobic environments such as wetland sediments. Here we present the first draft metagenome-assembled genome (MAG) sequence of an unclassified Methanosarcinaceae methanogen phylogenetically placed adjacent to the Methanolobus and Methanomethylovorans genera that appears to be a distinct genus and species. The genome is derived from sediments of a hypersaline (97-148 ppt chloride) unrestored industrial saltern that has been observed to be a significant methane source. The source sediment is more saline than previous sources of Methanolobus and Methanomethylovorans. We propose a new genus name, Methanosalis, to house this genome, which we designate with the strain name SBSPR1A. The MAG was binned with CONCOCT and then improved via scaffold extension and reassembly. The genome contains pathways for methylotrophic methanogenesis from trimethylamine and dimethylamine, as well as genes for the synthesis and transport of compatible solutes. Some genes involved in acetoclastic and hydrogenotrophic methanogenesis are present, but those pathways appear incomplete in the genome. The MAG was more abundant in two former industrial salterns than in a nearby reference wetland and a restored wetland, both of which have much lower salinity levels, as well as significantly lower methane emissions than the salterns.

Microbial Community Field Surveys Reveal Abundant Pseudomonas Population in Sorghum Rhizosphere Composed of Many Closely Related Phylotypes.

While the root-associated microbiome is typically less diverse than the surrounding soil due to both plant selection and microbial competition for plant derived resources, it typically retains considerable complexity, harboring many hundreds of distinct bacterial species. Here, we report a time-dependent deviation from this trend in the rhizospheres of field grown sorghum. In this study, 16S rRNA amplicon sequencing was used to determine the impact of nitrogen fertilization on the development of the root-associated microbiomes of 10 sorghum genotypes grown in eastern Nebraska. We observed that early rhizosphere samples exhibit a significant reduction in overall diversity due to a high abundance of the bacterial genus Pseudomonas that occurred independent of host genotype in both high and low nitrogen fields and was not observed in the surrounding soil or associated root endosphere samples. When clustered at 97% identity, nearly all the Pseudomonas reads in this dataset were assigned to a single operational taxonomic unit (OTU); however, exact sequence variant (ESV)-level resolution demonstrated that this population comprised a large number of distinct Pseudomonas lineages. Furthermore, single-molecule long-read sequencing enabled high-resolution taxonomic profiling revealing further heterogeneity in the Pseudomonas lineages that was further confirmed using shotgun metagenomic sequencing. Finally, field soil enriched with specific carbon compounds recapitulated the increase in Pseudomonas, suggesting a possible connection between the enrichment of these Pseudomonas species and a plant-driven exudate profile.

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library.

Soil microorganisms historically have been a rich resource for natural product discovery, yet the majority of these microbes remain uncultivated and their biosynthetic capacity is left underexplored. To identify the biosynthetic potential of soil microorganisms using a culture-independent approach, we constructed a large-insert metagenomic library in Escherichia coli from a topsoil sampled from the Cullars Rotation (Auburn, AL, United States), a long-term crop rotation experiment. Library clones were screened for biosynthetic gene clusters (BGCs) using either PCR or a NGS (next generation sequencing) multiplexed pooling strategy, coupled with bioinformatic analysis to identify contigs associated with each metagenomic clone. A total of 1,015 BGCs were detected from 19,200 clones, identifying 223 clones (1.2%) that carry a polyketide synthase (PKS) and/or a non-ribosomal peptide synthetase (NRPS) cluster, a dramatically improved hit rate compared to PCR screening that targeted type I polyketide ketosynthase (KS) domains. The NRPS and PKS clusters identified by NGS were distinct from known BGCs in the MIBiG database or those PKS clusters identified by PCR. Likewise, 16S rRNA gene sequences obtained by NGS of the library included many representatives that were not recovered by PCR, in concordance with the same bias observed in KS amplicon screening. This study provides novel resources for natural product discovery and circumvents amplification bias to allow annotation of a soil metagenomic library for a more complete picture of its functional and phylogenetic diversity.

Diversity, evolution, and classification of virophages uncovered through global metagenomics.

BACKGROUND: Virophages are small viruses with double-stranded DNA genomes that replicate along with giant viruses and co-infect eukaryotic cells. Due to the paucity of virophage reference genomes, a collective understanding of the global virophage diversity, distribution, and evolution is lacking. RESULTS: Here we screened a public collection of over 14,000 metagenomes using the virophage-specific major capsid protein (MCP) as "bait." We identified 44,221 assembled virophage sequences, of which 328 represent high-quality (complete or near-complete) genomes from diverse habitats including the human gut, plant rhizosphere, and terrestrial subsurface. Comparative genomic analysis confirmed the presence of four core genes in a conserved block. We used these genes to establish a revised virophage classification including 27 clades with consistent genome length, gene content, and habitat distribution. Moreover, for eight high-quality virophage genomes, we computationally predicted putative eukaryotic virus hosts. CONCLUSION: Overall, our approach has increased the number of known virophage genomes by 10-fold and revealed patterns of genome evolution and global virophage distribution. We anticipate that the expanded diversity presented here will provide the backbone for further virophage studies.

Novel archaeal thermostable cellulases from an oil reservoir metagenome.

Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.

Four novel algal virus genomes discovered from Yellowstone Lake metagenomes.

Phycodnaviruses are algae-infecting large dsDNA viruses that are widely distributed in aquatic environments. Here, partial genomic sequences of four novel algal viruses were assembled from a Yellowstone Lake metagenomic data set. Genomic analyses revealed that three Yellowstone Lake phycodnaviruses (YSLPVs) had genome lengths of 178,262 bp, 171,045 bp, and 171,454 bp, respectively, and were phylogenetically closely related to prasinoviruses (Phycodnaviridae). The fourth (YSLGV), with a genome length of 73,689 bp, was related to group III in the extended family Mimiviridae comprising Organic Lake phycodnaviruses and Phaeocystis globosa virus 16 T (OLPG). A pair of inverted terminal repeats was detected in YSLPV1, suggesting that its genome is nearly complete. Interestingly, these four putative YSL giant viruses also bear some genetic similarities to Yellowstone Lake virophages (YSLVs). For example, they share nine non-redundant homologous genes, including ribonucleotide reductase small subunit (a gene conserved in nucleo-cytoplasmic large DNA viruses) and Organic Lake virophage OLV2 (conserved in the majority of YSLVs). Additionally, putative multidrug resistance genes (emrE) were found in YSLPV1 and YSLPV2 but not in other viruses. Phylogenetic trees of emrE grouped YSLPVs with algae, suggesting that horizontal gene transfer occurred between giant viruses and their potential algal hosts.