Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12. In eukaryotes and their viruses, TnpB homologs occur as two distinct types, Fanzor1 and Fanzor2. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, revealing that a clade of IS607 TnpBs with unusual active site arrangement found primarily in Cyanobacteriota likely gave rise to both types of Fanzors. The wide-spread nature of Fanzors imply that the properties of this particular group of IS607 TnpBs were particularly suited to adaptation and evolution in eukaryotes and their viruses. Experimental characterization of a prokaryotic IS607 TnpB and virally encoded Fanzor1s uncovered features that may have fostered coevolution between TnpBs/Fanzors and their cognate transposases. Our results provide insight into the evolutionary origins of a ubiquitous family of RNA-guided proteins that shows remarkable conservation across domains of life.

Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes.

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2-4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

O2 partitioning of sulfur oxidizing bacteria drives acidity and thiosulfate distributions in mining waters.

The acidification of water in mining areas is a global environmental issue primarily catalyzed by sulfur-oxidizing bacteria (SOB). Little is known about microbial sulfur cycling in circumneutral pH mine tailing impoundment waters. Here we investigate biological sulfur oxidation over four years in a mine tailings impoundment water cap, integrating aqueous sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. The microbial community is consistently dominated by neutrophilic, chemolithoautotrophic SOB (relative abundances of ~76% in 2015, ~55% in 2016/2017 and ~60% in 2018). Results reveal two SOB strategies alternately dominate across the four years, influencing acid generation and sulfur speciation. Under oxic conditions, novel Halothiobacillus drive lower pH conditions (as low as 4.3) and lower [S2O32-] via the complete Sox pathway coupled to O2. Under anoxic conditions, Thiobacillus spp. dominate in activity, via the incomplete Sox and rDSR pathways coupled to NO3-, resulting in higher [S2O32-] and no net significant acidity generation. This study provides genomic evidence explaining acidity generation and thiosulfate accumulation patterns in a circumneutral mine tailing impoundment and has significant environmental applications in preventing the discharge of sulfur compounds that can impact downstream environments. These insights illuminate opportunities for in situ biotreatment of reduced sulfur compounds and prediction of acidification events using gene-based monitoring and in situ RNA detection.

Long-Term Incubation of Lake Water Enables Genomic Sampling of Consortia Involving Planctomycetes and Candidate Phyla Radiation Bacteria.

Microbial communities in lakes can profoundly impact biogeochemical processes through their individual activities and collective interactions. However, the complexity of these communities poses challenges, particularly for studying rare organisms such as Candidate Phyla Radiation bacteria (CPR) and enigmatic entities such as aster-like nanoparticles (ALNs). Here, a reactor was inoculated with water from Lake Fargette, France, and maintained under dark conditions at 4°C for 31 months and enriched for ALNs, diverse Planctomycetes, and CPR bacteria. We reconstructed draft genomes and predicted metabolic traits for 12 diverse Planctomycetes and 9 CPR bacteria, some of which are likely representatives of undescribed families or genera. One CPR genome representing the little-studied lineage "Candidatus Peribacter" was curated to completion (1.239 Mbp) and unexpectedly encodes the full gluconeogenesis pathway. Metatranscriptomic data indicate that some planctomycetes and CPR bacteria were active under the culture conditions, accounting for ∼30% and ∼1% of RNA reads mapping to the genome set, respectively. We also reconstructed genomes and obtained transmission electron microscope images for numerous viruses, including one with a >300-kbp genome and several predicted to infect Planctomycetes. Together, our analyses suggest that freshwater Planctomycetes are central players in a subsystem that includes ALNs, symbiotic CPR bacteria, and viruses. IMPORTANCE Laboratory incubations of natural microbial communities can aid in the study of member organisms and their networks of interaction. This is particularly important for understudied lineages for which key elements of basic biology are still emerging. Using genomics and microscopy, we found that members of the bacterial lineage Planctomycetes may be central players in a subset of a freshwater lake microbiome that includes other bacteria, archaea, viruses, and mysterious entities, called aster-like nanoparticles (ALNs), whose origin is unknown. Our results help constrain the possible origins of ALNs and provide insight into possible interactions within a complex lake ecosystem.

Alum Addition Triggers Hypoxia in an Engineered Pit Lake.

Here, we examine the geobiological response to a whole-lake alum (aluminum sulfate) treatment (2016) of Base Mine Lake (BML), the first pilot-scale pit lake established in the Alberta oil sands region. The rationale for trialing this management amendment was based on its successful use to reduce internal phosphorus loading to eutrophying lakes. Modest increases in water cap epilimnetic oxygen concentrations, associated with increased Secchi depths and chlorophyll-a concentrations, were co-incident with anoxic waters immediately above the fluid fine tailings (FFT) layer post alum. Decreased water cap nitrate and detectable sulfide concentrations, as well as increased hypolimnetic phospholipid fatty acid abundances, signaled greater anaerobic heterotrophic activity. Shifts in microbial community to groups associated with greater organic carbon degradation (i.e., SAR11-LD12 subclade) and the SRB group Desulfuromonodales emerged post alum and the loss of specialist groups associated with carbon-limited, ammonia-rich restricted niches (i.e., MBAE14) also occurred. Alum treatment resulted in additional oxygen consumption associated with increased autochthonous carbon production, watercap anoxia and sulfide generation, which further exacerbate oxygen consumption associated with on-going FFT mobilized reductants. The results illustrate the importance of understanding the broader biogeochemical implications of adaptive management interventions to avoid unanticipated outcomes that pose greater risks and improve tailings reclamation for oil sands operations and, more broadly, the global mining sector.

Detection and enumeration of Lak megaphages in microbiome samples by endpoint and quantitative PCR.

Lak megaphages are prevalent across diverse gut microbiomes and may potentially impact animal and human health through lysis of Prevotella. Given their large genome size (up to 660 kbp), Lak megaphages are difficult to culture, and their identification relies on molecular techniques. Here, we present optimized protocols for identifying Lak phages in various microbiome samples, including procedures for DNA extraction, followed by detection and quantification of genes encoding Lak structural proteins using diagnostic endpoint and SYBR green-based quantitative PCR, respectively. For complete details on the use and execution of this protocol, please refer to Crisci et al., (2021).

Phage-encoded ribosomal protein S21 expression is linked to late-stage phage replication.

The ribosomal protein S21 (bS21) gene has been detected in diverse viruses with a large range of genome sizes, yet its in situ expression and potential significance have not been investigated. Here, we report five closely related clades of bacteriophages (phages) represented by 47 genomes (8 curated to completion and up to 331 kbp in length) that encode a bS21 gene. The bS21 gene is on the reverse strand within a conserved region that encodes the large terminase, major capsid protein, prohead protease, portal vertex proteins, and some hypothetical proteins. Based on CRISPR spacer targeting, the predominance of bacterial taxonomic affiliations of phage genes with those from Bacteroidetes, and the high sequence similarity of the phage bS21 genes and those from Bacteroidetes classes of Flavobacteriia, Cytophagia and Saprospiria, these phages are predicted to infect diverse Bacteroidetes species that inhabit a range of depths in freshwater lakes. Thus, bS21 phages have the potential to impact microbial community composition and carbon turnover in lake ecosystems. The transcriptionally active bS21-encoding phages were likely in the late stage of replication when collected, as core structural genes and bS21 were highly expressed. Thus, our analyses suggest that the phage bS21, which is involved in translation initiation, substitutes into the Bacteroidetes ribosomes and selects preferentially for phage transcripts during the late-stage replication when large-scale phage protein production is required for assembly of phage particles.

Closely related Lak megaphages replicate in the microbiomes of diverse animals.

Lak phages with alternatively coded ∼540 kbp genomes were recently reported to replicate in Prevotella in microbiomes of humans that consume a non-Western diet, baboons, and pigs. Here, we explore Lak phage diversity and broader distribution using diagnostic polymerase chain reaction and genome-resolved metagenomics. Lak phages were detected in 13 animal types, including reptiles, and are particularly prevalent in pigs. Tracking Lak through the pig gastrointestinal tract revealed significant enrichment in the hindgut compared to the foregut. We reconstructed 34 new Lak genomes, including six curated complete genomes, all of which are alternatively coded. An anomalously large (∼660 kbp) complete genome reconstructed for the most deeply branched Lak from a horse microbiome is also alternatively coded. From the Lak genomes, we identified proteins associated with specific animal species; notably, most have no functional predictions. The presence of closely related Lak phages in diverse animals indicates facile distribution coupled to host-specific adaptation.

Brockarchaeota, a novel archaeal phylum with unique and versatile carbon cycling pathways.

Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, 'Brockarchaeota', named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome.

INTRODUCTION: Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and intriguing patterns of site specificity amidst the lack of dispersal limitation. However, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among taxa that belong to recently discovered branches of microbial life. RESULTS: Here, we assemble metagenomes from tongue and dental plaque samples from multiple individuals and reconstruct 790 non-redundant genomes, 43 of which resolve to TM7, a member of the Candidate Phyla Radiation, forming six monophyletic clades that distinctly associate with either plaque or tongue. Both pangenomic and phylogenomic analyses group tongue-specific clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 group with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and mobilome of cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of microbes. Additionally, we report that prophages are widespread among oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment. CONCLUSIONS: Our data illuminate niche partitioning of enigmatic members of the oral cavity, including TM7, SR1, and GN02, and provide genomes for poorly characterized yet prevalent members of this biome, such as uncultivated Flavobacteriaceae.

Large freshwater phages with the potential to augment aerobic methane oxidation.

There is growing evidence that phages with unusually large genomes are common across various microbiomes, but little is known about their genetic inventories or potential ecosystem impacts. In the present study, we reconstructed large phage genomes from freshwater lakes known to contain bacteria that oxidize methane. Of manually curated genomes, 22 (18 are complete), ranging from 159 kilobase (kb) to 527 kb in length, were found to encode the pmoC gene, an enzymatically critical subunit of the particulate methane monooxygenase, the predominant methane oxidation catalyst in nature. The phage-associated PmoC sequences show high similarity to (>90%), and affiliate phylogenetically with, those of coexisting bacterial methanotrophs, including members of Methyloparacoccus, Methylocystis and Methylobacter spp. In addition, pmoC-phage abundance patterns correlate with those of the coexisting bacterial methanotrophs, supporting host-phage relationships. Future work is needed to determine whether phage-associated PmoC has similar functions to additional copies of PmoC encoded in bacterial genomes, thus contributing to growth on methane. Transcriptomics data from Lake Rotsee (Switzerland) showed that some phage-associated pmoC genes were highly expressed in situ and, of interest, that the most rapidly growing methanotroph was infected by three pmoC-phages. Thus, augmentation of bacterial methane oxidation by pmoC-phages during infection could modulate the efflux of this potent greenhouse gas into the environment.

Groundwater Elusimicrobia are metabolically diverse compared to gut microbiome Elusimicrobia and some have a novel nitrogenase paralog.

Currently described members of Elusimicrobia, a relatively recently defined phylum, are animal-associated and rely on fermentation. However, free-living Elusimicrobia have been detected in sediments, soils and groundwater, raising questions regarding their metabolic capacities and evolutionary relationship to animal-associated species. Here, we analyzed 94 draft-quality, non-redundant genomes, including 30 newly reconstructed genomes, from diverse animal-associated and natural environments. Genomes group into 12 clades, 10 of which previously lacked reference genomes. Groundwater-associated Elusimicrobia are predicted to be capable of heterotrophic or autotrophic lifestyles, reliant on oxygen or nitrate/nitrite-dependent respiration, or a variety of organic compounds and Rhodobacter nitrogen fixation (Rnf) complex-dependent acetogenesis with hydrogen and carbon dioxide as the substrates. Genomes from two clades of groundwater-associated Elusimicrobia often encode a new group of nitrogenase paralogs that co-occur with an extensive suite of radical S-Adenosylmethionine (SAM) proteins. We identified similar genomic loci in genomes of bacteria from the Gracilibacteria phylum and the Myxococcales order and predict that the gene clusters reduce a tetrapyrrole, possibly to form a novel cofactor. The animal-associated Elusimicrobia clades nest phylogenetically within two free-living-associated clades. Thus, we propose an evolutionary trajectory in which some Elusimicrobia adapted to animal-associated lifestyles from free-living species via genome reduction.

Genome-Resolved Metagenomics and Detailed Geochemical Speciation Analyses Yield New Insights into Microbial Mercury Cycling in Geothermal Springs.

Geothermal systems emit substantial amounts of aqueous, gaseous, and methylated mercury, but little is known about microbial influences on mercury speciation. Here, we report results from genome-resolved metagenomics and mercury speciation analysis of acidic warm springs in the Ngawha Geothermal Field (<55°C, pH <4.5), Northland Region, Aotearoa New Zealand. Our aim was to identify the microorganisms genetically equipped for mercury methylation, demethylation, or Hg(II) reduction to volatile Hg(0) in these springs. Dissolved total and methylated mercury concentrations in two adjacent springs with different mercury speciation ranked among the highest reported from natural sources (250 to 16,000 ng liter-1 and 0.5 to 13.9 ng liter-1, respectively). Total solid mercury concentrations in spring sediments ranged from 1,274 to 7,000 µg g-1 In the context of such ultrahigh mercury levels, the geothermal microbiome was unexpectedly diverse and dominated by acidophilic and mesophilic sulfur- and iron-cycling bacteria, mercury- and arsenic-resistant bacteria, and thermophilic and acidophilic archaea. By integrating microbiome structure and metagenomic potential with geochemical constraints, we constructed a conceptual model for biogeochemical mercury cycling in geothermal springs. The model includes abiotic and biotic controls on mercury speciation and illustrates how geothermal mercury cycling may couple to microbial community dynamics and sulfur and iron biogeochemistry.IMPORTANCE Little is currently known about biogeochemical mercury cycling in geothermal systems. The manuscript presents a new conceptual model, supported by genome-resolved metagenomic analysis and detailed geochemical measurements. The model illustrates environmental factors that influence mercury cycling in acidic springs, including transitions between solid (mineral) and aqueous phases of mercury, as well as the interconnections among mercury, sulfur, and iron cycles. This work provides a framework for studying natural geothermal mercury emissions globally. Specifically, our findings have implications for mercury speciation in wastewaters from geothermal power plants and the potential environmental impacts of microbially and abiotically formed mercury species, particularly where they are mobilized in spring waters that mix with surface or groundwaters. Furthermore, in the context of thermophilic origins for microbial mercury volatilization, this report yields new insights into how such processes may have evolved alongside microbial mercury methylation/demethylation and the environmental constraints imposed by the geochemistry and mineralogy of geothermal systems.

Accurate and complete genomes from metagenomes.

Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes.

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.

Putative Mixotrophic Nitrifying-Denitrifying Gammaproteobacteria Implicated in Nitrogen Cycling Within the Ammonia/Oxygen Transition Zone of an Oil Sands Pit Lake.

Anthropogenically-impacted environments offer the opportunity to discover novel microbial species and metabolisms, which may be undetectable in natural systems. Here, a combined metagenomic and geochemical study in Base Mine Lake, Alberta, Canada, which is the only oil sands end pit lake to date, revealed that nitrification was performed by members from Nitrosomonadaceae, Chloroflexi and unclassified Gammaproteobacteria "MBAE14." While Nitrosomonadaceae and Chloroflexi groups were relatively abundant in the upper oxygenated zones, MBAE14 dominated the hypoxic hypolimnetic zones (approximately 30% of total microbial communities); MBAE14 was not detected in the underlying anoxic tailings. Replication rate analyses indicate that MBAE14 grew in metalimnetic and hypolimnetic water cap regions, most actively at the metalimnetic, ammonia/oxygen transition zone consistent with it putatively conducting nitrification. Detailed genomic analyses of MBAE14 evidenced both ammonia oxidation and denitrification into dinitrogen capabilities. However, the absence of known CO2-fixation genes suggests a heterotrophic denitrifying metabolism. Functional marker genes of ammonia oxidation (amo and hao) in the MBAE14 genome are homologous with those conserved in autotrophic nitrifiers, but not with those of known heterotrophic nitrifiers. We propose that this novel MBAE14 inhabits the specific ammonia-rich, oxygen and labile organic matter-limited conditions occurring in Base Mine Lake which selectively favors mixotrophic coupled nitrifier denitrification metabolism. Our results highlight the opportunities to better constrain biogeochemical cycles from the application of metagenomics to engineered systems associated with extractive resource sectors.

Wide Distribution of Phage That Infect Freshwater SAR11 Bacteria.

Fonsibacter (LD12 subclade) is among the most abundant bacterioplankton in freshwater ecosystems. These bacteria belong to the order Pelagibacterales (SAR11) and are related to Pelagibacter (marine SAR11), which dominates many marine habitats. Although a few Pelagibacter phage (Pelagiphage) have been described, no phage that infect Fonsibacter have been reported. In this study, we describe two groups of Podoviridae phage that infect Fonsibacter A complete Fonsibacter genome containing a prophage was reconstructed from metagenomic data. A circularized and complete genome related to the prophage, referred to as uv-Fonsiphage-EPL (lysogenic strategy), shows high similarity to marine Pelagiphage HTVC025P. Additionally, we reconstructed three complete genomes and one draft genome of phage related to marine Pelagiphage HTVC010P and predicted a lytic strategy. The similarity in codon usage and cooccurrence patterns of HTVC010P-related phage and Fonsibacter suggested that these phage infect Fonsibacter Similar phage were detected in Lake Mendota, Wisconsin, where Fonsibacter is also present. A search of related phage revealed the worldwide distribution of some genotypes in freshwater ecosystems, suggesting their substantial role in shaping indigenous microbial assemblages and influence on biogeochemical cycling. However, the uv-Fonsiphage-EPL and one group of HTVC010P-related phage have a more limited distribution in freshwater ecosystems. Overall, the findings provide insights into the genomic features of phage that infect Fonsibacter and expand understanding of the ecology and evolution of these important bacteria.IMPORTANCE Fonsibacter represents a significant microbial group of freshwater ecosystems. Although the genomic and metabolic features of these bacteria have been well studied, no phage infecting them has been reported. In this study, we reconstructed complete genomes of Fonsibacter and infecting phage and revealed their close relatedness to the phage infecting marine SAR11 members. Also, we illustrated that phage that infect Fonsibacter are widely distributed in freshwater habitats. In summary, the results contribute new insights into the ecology and evolution of Fonsibacter and phage.

Candidate Phyla Radiation Roizmanbacteria From Hot Springs Have Novel and Unexpectedly Abundant CRISPR-Cas Systems.

The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria.

Wide diversity of methane and short-chain alkane metabolisms in uncultured archaea.

Methanogenesis is an ancient metabolism of key ecological relevance, with direct impact on the evolution of Earth's climate. Recent results suggest that the diversity of methane metabolisms and their derivations have probably been vastly underestimated. Here, by probing thousands of publicly available metagenomes for homologues of methyl-coenzyme M reductase complex (MCR), we have obtained ten metagenome-assembled genomes (MAGs) belonging to potential methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea. Five of these MAGs represent under-sampled (Verstraetearchaeota, Methanonatronarchaeia, ANME-1 and GoM-Arc1) or previously genomically undescribed (ANME-2c) archaeal lineages. The remaining five MAGs correspond to lineages that are only distantly related to previously known methanogens and span the entire archaeal phylogeny. Comprehensive comparative annotation substantially expands the metabolic diversity and energy conservation systems of MCR-bearing archaea. It also suggests the potential existence of a yet uncharacterized type of methanogenesis linked to short-chain alkane/fatty acid oxidation in a previously undescribed class of archaea ('Candidatus Methanoliparia'). We redefine a common core of marker genes specific to methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea, and propose a possible scenario for the evolutionary and functional transitions that led to the emergence of such metabolic diversity.

Discordance Between Resident and Active Bacterioplankton in Free-Living and Particle-Associated Communities in Estuary Ecosystem.

Bacterioplankton are the major driving force for biogeochemical cycles in estuarine ecosystems, but the communities that mediate these processes are largely unexplored. We sampled in the Pearl River Estuary (PRE) to examine potential differences in the taxonomic composition of resident (DNA-based) and active (RNA-based) bacterioplankton communities in free-living and particle-associated fractions. MiSeq sequencing data showed that the overall bacterial diversity in particle-associated fractions was higher than in free-living communities. Further in-depth analyses of the sequences revealed a positive correlation between resident and active bacterioplankton communities for the particle-associated fraction but not in the free-living fraction. However, a large overlapping of OTUs between free-living and particle-associated communities in PRE suggested that the two fractions may be actively exchanged. We also observed that the positive correlation between resident and active communities is more prominent among the abundant OTUs (relative abundance > 0.2%). Further, the results from the present study indicated that low-abundance bacterioplankton make an important contribution towards the metabolic activity in PRE.