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.

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.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

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 a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

Factors influencing the therapeutic effect of hyperopic correction on esotropia in patients with partially accommodative esotropia.

OBJECTIVES: To collate data on partially accommodative esotropia (PAET) to better understand this condition's aetiology and to evaluate and predict the therapeutic effect of a hyperopic correction on PAET. METHODS: Eighty-nine consecutive patients diagnosed with PAET with a spherical equivalent (SE) refractive error >+2.50 D were included in this retrospective review. Clinical characteristics, including gender, age, SE, angle of esodeviation, accommodative convergence/accommodation (AC/A) ratio, near-distance disparity (NDD) and anatomical features of the rectus muscles were compared among different PAET subgroups. Multiple linear regression was used to identify independent factors that influenced the therapeutic effect of a hyperopic correction on esotropia. RESULTS: No significant differences were observed for the angle of esodeviation as a function of age in individuals with PAET. The incidence of SE in PAET participants >9 years old was significantly greater than in those <5 and 6-8 years of age. The therapeutic effect of hyperopic correction on esotropia was positively associated with SE both at distance and near. In addition, the limbus insertion distance (LID) of the lateral rectus (LR) muscle was positively associated with NDD at distance, but negatively associated at near. CONCLUSION: A greater incidence of hyperopia was observed in older (>9 years old) PAET patients. A hyperopic correction had a greater effect on esotropia in individuals with a higher SE, larger LID of the LR muscle and a smaller NDD.

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.

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.

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.

Three-Dimensional Eye Shape, Myopic Maculopathy, and Visual Acuity: The Zhongshan Ophthalmic Center-Brien Holden Vision Institute High Myopia Cohort Study.

PURPOSE: To investigate the relationship among eye shape, myopic maculopathy, and visual acuity in highly myopic eyes using 3-dimensional (3D) magnetic resonance imaging (MRI). DESIGN: Observational case series. PARTICIPANTS: The study included 190 eyes of 95 participants with bilateral high myopia from the Zhongshan Ophthalmic Center-Brien Holden Vision Institute High Myopia Cohort Study, with spherical power ≤-6.00 diopters (D) in both eyes. METHODS: The participants underwent best-corrected visual acuity (BCVA), fundus photography, cycloplegic refraction, and ocular biometry. Posterior staphyloma was identified with 3D MRI, and ocular shapes were categorized into spheroidal, ellipsoidal, conical, nasally distorted, temporally distorted, and barrel shapes according to the inferior view from T2-weighted 3D MRI (Achieva 3.0T; Philips Medical Systems, Best, the Netherlands). Myopic maculopathy was graded as C0 to C4 according to the International Photographic Classification and Grading System using fundus photography. The statistical significance of the differences in distribution of myopic maculopathy and BCVA in eyes with different ocular shapes was determined using Pearson's chi-square tests. MAIN OUTCOME MEASURES: Distributions of myopic maculopathy and BCVA in relation to different eye shapes. RESULTS: The mean spherical equivalent and axial length were -11.74±4.10 D and 28.18±1.73 mm in the right eyes, respectively. The same ocular shapes were observed in both eyes in 69 participants (72.6%). The predominant shape was spheroidal (53.7%), followed by nasally distorted and conical types (both 14.7%). C2 and above myopic maculopathy was observed in all barrel-shaped eyes, in 75% temporally distorted eyes, and in 71.4% nasally distorted and conical eyes. In eyes with posterior staphyloma (n = 22), 18 (81.8%) had C2 and above myopic maculopathy compared with 40 eyes (54.8%) without posterior staphyloma (n = 73). Eyes with temporal and nasal distortion, and eyes with staphyloma were more likely to have BCVA <20/40. CONCLUSIONS: Not all highly myopic eyes are deformed. Spheroid was the predominant ocular shape in this series of young patients with high myopia bilaterally. Barrel-shaped and temporally distorted eyes present significant myopic maculopathy, whereas eyes with posterior staphyloma display more severe chorioretinal atrophy. Eyes of more deformed shapes tend to have more severe myopic maculopathy and worse BCVA.

Correlating microbial diversity patterns with geochemistry in an extreme and heterogeneous environment of mine tailings.

Recent molecular surveys have advanced our understanding of the forces shaping the large-scale ecological distribution of microbes in Earth's extreme habitats, such as hot springs and acid mine drainage. However, few investigations have attempted dense spatial analyses of specific sites to resolve the local diversity of these extraordinary organisms and how communities are shaped by the harsh environmental conditions found there. We have applied a 16S rRNA gene-targeted 454 pyrosequencing approach to explore the phylogenetic differentiation among 90 microbial communities from a massive copper tailing impoundment generating acidic drainage and coupled these variations in community composition with geochemical parameters to reveal ecological interactions in this extreme environment. Our data showed that the overall microbial diversity estimates and relative abundances of most of the dominant lineages were significantly correlated with pH, with the simplest assemblages occurring under extremely acidic conditions and more diverse assemblages associated with neutral pHs. The consistent shifts in community composition along the pH gradient indicated that different taxa were involved in the different acidification stages of the mine tailings. Moreover, the effect of pH in shaping phylogenetic structure within specific lineages was also clearly evident, although the phylogenetic differentiations within the Alphaproteobacteria, Deltaproteobacteria, and Firmicutes were attributed to variations in ferric and ferrous iron concentrations. Application of the microbial assemblage prediction model further supported pH as the major factor driving community structure and demonstrated that several of the major lineages are readily predictable. Together, these results suggest that pH is primarily responsible for structuring whole communities in the extreme and heterogeneous mine tailings, although the diverse microbial taxa may respond differently to various environmental conditions.

Biogeochemical processes governing natural pyrite oxidation and release of acid metalliferous drainage.

The oxidative dissolution of sulfide minerals (principally pyrite) is responsible for the majority of acid metalliferous drainage from mine sites, which represents a significant environmental problem worldwide. Understanding the complex biogeochemical processes governing natural pyrite oxidation is critical not only for solving this problem but also for understanding the industrial bioleaching of sulfide minerals. To this end, we conducted a simulated experiment of natural pyrite oxidative dissolution. Pyrosequencing analysis of the microbial community revealed a distinct succession across three stages. At the early stage, a newly proposed genus, Tumebacillus (which can use sodium thiosulfate and sulfite as the sole electron donors), dominated the microbial community. At the midstage, Alicyclobacillus (the fifth most abundant genus at the early stage) became the most dominant genus, whereas Tumebacillus was still ranked as the second most abundant. At the final stage, the microbial community was dominated by Ferroplasma (the tenth most abundant genus at the early stage). Our geochemical and mineralogical analyses indicated that exchangeable heavy metals increased as the oxidation progressed and that some secondary sulfate minerals (including jarosite and magnesiocopiapite) were formed at the final stage of the oxidation sequence. Additionally, we propose a comprehensive model of biogeochemical processes governing the oxidation of sulfide minerals.

COBRA improves the completeness and contiguity of viral genomes assembled from metagenomes.

Viruses are often studied using metagenome-assembled sequences, but genome incompleteness hampers comprehensive and accurate analyses. Contig Overlap Based Re-Assembly (COBRA) resolves assembly breakpoints based on the de Bruijn graph and joins contigs. Here we benchmarked COBRA using ocean and soil viral datasets. COBRA accurately joined the assembled sequences and achieved notably higher genome accuracy than binning tools. From 231 published freshwater metagenomes, we obtained 7,334 bacteriophage clusters, ~83% of which represent new phage species. Notably, ~70% of these were circular, compared with 34% before COBRA analyses. We expanded sampling of huge phages (≥200 kbp), the largest of which was curated to completion (717 kbp). Improved phage genomes from Rotsee Lake provided context for metatranscriptomic data and indicated the in situ activity of huge phages, whiB-encoding phages and cysC- and cysH-encoding phages. COBRA improves viral genome assembly contiguity and completeness, thus the accuracy and reliability of analyses of gene content, diversity and evolution.

Adaptive strategies and ecological roles of phages in habitats under physicochemical stress.

Bacteriophages (phages) play a vital role in ecosystem functions by influencing the composition, genetic exchange, metabolism, and environmental adaptation of microbial communities. With recent advances in sequencing technologies and bioinformatics, our understanding of the ecology and evolution of phages in stressful environments has substantially expanded. Here, we review the impact of physicochemical environmental stress on the physiological state and community dynamics of phages, the adaptive strategies that phages employ to cope with environmental stress, and the ecological effects of phage-host interactions in stressful environments. Specifically, we highlight the contributions of phages to the adaptive evolution and functioning of microbiomes and suggest that phages and their hosts can maintain a mutualistic relationship in response to environmental stress. In addition, we discuss the ecological consequences caused by phages in stressful environments, encompassing biogeochemical cycling. Overall, this review advances an understanding of phage ecology in stressful environments, which could inform phage-based strategies to improve microbiome performance and ecosystem resilience and resistance in natural and engineering systems.

Methanotrophic Methanoperedens archaea host diverse and interacting extrachromosomal elements.

Methane emissions are mitigated by anaerobic methane-oxidizing archaea, including Methanoperedens. Some Methanoperedens host huge extrachromosomal genetic elements (ECEs) called Borgs that may modulate their activity, yet the broader diversity of Methanoperedens ECEs is understudied. Here we report small enigmatic linear ECEs, circular viruses and unclassified ECEs that are predicted to replicate within Methanoperedens. Linear ECEs have inverted terminal repeats, tandem repeats and coding patterns that are strongly reminiscent of Borgs, but they are only 52-145 kb in length. As they share proteins with Borgs and Methanoperedens, we refer to them as mini-Borgs. Mini-Borgs are genetically diverse and can be assigned to at least five family-level groups. We identify eight families of Methanoperedens viruses, some of which encode multi-haem cytochromes, and circular ECEs encoding transposon-associated TnpB genes with proximal population-heterogeneous CRISPR arrays. These ECEs exchange genetic information with each other and with Methanoperedens, probably impacting their archaeal host activity and evolution.

Infant gut DNA bacteriophage strain persistence during the first 3 years of life.

Bacteriophages are key components of gut microbiomes, yet the phage colonization process in the infant gut remains uncertain. Here, we establish a large phage sequence database and use strain-resolved analyses to investigate DNA phage succession in infants throughout the first 3 years of life. Analysis of 819 fecal metagenomes collected from 28 full-term and 24 preterm infants and their mothers revealed that early-life phageome richness increases over time and reaches adult-like complexity by age 3. Approximately 9% of early phage colonizers, which are mostly maternally transmitted and infect Bacteroides, persist for 3 years and are more prevalent in full-term than in preterm infants. Although rare, phages with stop codon reassignment are more likely to persist than non-recoded phages and generally display an increase in in-frame reassigned stop codons over 3 years. Overall, maternal seeding, stop codon reassignment, host CRISPR-Cas locus prevalence, and diverse phage populations contribute to stable viral colonization.

Shifts in microbial community composition and function in the acidification of a lead/zinc mine tailings.

In an attempt to link the microbial community composition and function in mine tailings to the generation of acid mine drainage, we simultaneously explored the geochemistry and microbiology of six tailings collected from a lead/zinc mine, i.e. primary tailings (T1), slightly acidic tailings (T2), extremely acidic tailings (T3, T4 and T5) and orange-coloured oxidized tailings (T6). Geochemical results showed that the six tailings (from T1 to T6) likely represented sequential stages of the acidification process of the mine tailings. 16S rRNA pyrosequencing revealed a contrasting microbial composition between the six tailings: Proteobacteria-related sequences dominated T1-T3 with relative abundance ranging from 56 to 93%, whereas Ferroplasma-related sequences dominated T4-T6 with relative abundance ranging from 28 to 58%. Furthermore, metagenomic analysis of the microbial communities of T2 and T6 indicated that the genes encoding key enzymes for microbial carbon fixation, nitrogen fixation and sulfur oxidation in T2 were largely from Thiobacillus and Acidithiobacillus, Methylococcus capsulatus, and Thiobacillus denitrificans respectively; while those in T6 were mostly identified in Acidithiobacillus and Leptospirillum, Acidithiobacillus and Leptospirillum, and Acidithiobacillus respectively. The microbial communities in T2 and T6 harboured more genes suggesting diverse metabolic capacities for sulfur oxidation/heavy metal detoxification and tolerating low pH respectively.

Myopic progression in school-aged children with moderate intermittent exotropia.

Objective: It is still controversial whether intermittent exotropia (IXT) affects myopic progression during the critical period of visual development. This study retrospectively analyzed the long-term myopic changes and the impact of IXT surgery on myopic progression in school-aged children with moderate IXT. Methods: The medical records of 65 children from 5 to 13 years old with or without IXT between 2015 and 2021 were retrospectively reviewed. Patients whose spherical equivalent refraction (SER) were less than -3.00 diopter (D) were included and divided into three groups: IXT surgery group (Group A), which comprised 22 IXT patients who received IXT surgery, IXT observation group (Group B), which comprises 19 IXT patients who only received long-term observational follow-up; and normal control group (Group C), which comprised 24 normal controls without IXT. The main outcome measurement was the rate of myopic progression, which was defined as the mean myopic shift in SER per year. Results: The 3- and 5-years long-term follow-up rates of myopic progression were -0.47 ± 0.28 D per year and -0.48 ± 0.23 D per year respectively in Group B, and those were significantly slower than that in Group C (-0.73 ± 0.32 D per year and -0.76 ± 0.19 D per year respectively). However, there was no significant difference in the rate of myopic progression between Group A and B or between Group A and C. Conclusion: Moderate IXT may have lower rate of myopic progression in school-aged children. Whether IXT surgery influence the rate of myopic progression still needs further study.

Host translation machinery is not a barrier to phages that interact with both CPR and non-CPR bacteria.

IMPORTANCE: Here, we profiled putative phages of Saccharibacteria, which are of particular importance as Saccharibacteria influence some human oral diseases. We additionally profiled putative phages of Gracilibacteria and Absconditabacteria, two Candidate Phyla Radiation (CPR) lineages of interest given their use of an alternative genetic code. Among the phages identified in this study, some are targeted by spacers from both CPR and non-CPR bacteria and others by both bacteria that use the standard genetic code as well as bacteria that use an alternative genetic code. These findings represent new insights into possible phage replication strategies and have relevance for phage therapies that seek to manipulate microbiomes containing CPR bacteria.

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.

Deep-branching evolutionary intermediates reveal structural origins of form I rubisco.

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme-found in plants, algae, and cyanobacteria-forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I') has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I' rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I'' rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I'' rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution.