Clinical evaluation of a highly multiplexed CRISPR-based diagnostic assay for diagnosing lower respiratory tract infection: a prospective cohort study.

OBJECTIVE: Accurate and rapid identification of causative pathogens is essential to guide the clinical management of lower respiratory tract infections (LRTIs). Here we conducted a single-centre prospective study in 284 patients suspected of lower respiratory tract infections to evaluate the utility of a nucleic acid test based on highly multiplexed polymerase chain reaction (PCR) and CRISPR-Cas12a. METHODS: We determined the analytical and diagnostic performance of the CRISPR assay using a combination of reference standards, including conventional microbiological tests (CMTs), metagenomic Next-Generation Sequencing (mNGS), and clinical adjudication by a panel of experts on infectious diseases and microbiology. RESULTS: The CRISPR assay showed a higher detection rate (63.0%) than conventional microbiological tests (38.4%) and was lower than metagenomic Next-Generation Sequencing (72.9%). In detecting polymicrobial infections, the positivity rate of the CRISPR assay (19.4%) was higher than conventional microbiological tests (3.5%) and lower than metagenomic Next-Generation Sequencing (28.9%). The overall diagnostic sensitivity of the CRISPR assay (67.8%) was higher than conventional microbiological tests (41.8%), and lower than metagenomic Next-Generation Sequencing (93.2%). CONCLUSIONS: Considering the low cost, ease of operation, short turnaround time, and broad range of pathogens detected in a single test, the CRISPR assay has the potential to be implemented as a screening tool for the aetiological diagnosis of lower respiratory tract infections patients, especially in cases where atypical bacteria or coinfections are suspected.

pH and thiosulfate dependent microbial sulfur oxidation strategies across diverse environments.

Sulfur oxidizing bacteria (SOB) play a key role in sulfur cycling in mine tailings impoundment (TI) waters, where sulfur concentrations are typically high. However, our understanding of SOB sulfur cycling via potential S oxidation pathways (sox, rdsr, and S4I) in these globally ubiquitous contexts, remains limited. Here, we identified TI water column SOB community composition, metagenomics derived metabolic repertoires, physicochemistry, and aqueous sulfur concentration and speciation in four Canadian base metal mine, circumneutral-alkaline TIs over four years (2016 - 2019). Identification and examination of genomes from nine SOB genera occurring in these TI waters revealed two pH partitioned, metabolically distinct groups, which differentially influenced acid generation and sulfur speciation. Complete sox (csox) dominant SOB (e.g., Halothiobacillus spp., Thiomonas spp.) drove acidity generation and S2O3 2- consumption via the csox pathway at lower pH (pH ~5 to ~6.5). At circumneutral pH conditions (pH ~6.5 to ~8.5), the presence of non-csox dominant SOB (hosting the incomplete sox, rdsr, and/or other S oxidation reactions; e.g. Thiobacillus spp., Sulfuriferula spp.) were associated with higher [S2O3 2-] and limited acidity generation. The S4I pathway part 1 (tsdA; S2O3 2- to S4O6 2-), was not constrained by pH, while S4I pathway part 2 (S4O6 2- disproportionation via tetH) was limited to Thiobacillus spp. and thus circumneutral pH values. Comparative analysis of low, natural (e.g., hydrothermal vents and sulfur hot springs) and high (e.g., Zn, Cu, Pb/Zn, and Ni tailings) sulfur systems literature data with these TI results, reveals a distinct TI SOB mining microbiome, characterized by elevated abundances of csox dominant SOB, likely sustained by continuous replenishment of sulfur species through tailings or mining impacted water additions. Our results indicate that under the primarily oxic conditions in these systems, S2O3 2- availability plays a key role in determining the dominant sulfur oxidation pathways and associated geochemical and physicochemical outcomes, highlighting the potential for biological management of mining impacted waters via pH and [S2O3 2-] manipulation.

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.

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.

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.

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.

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.

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.

Long-Term Axial Length Shortening in Myopic Orthokeratology: Incident Probability, Time Course, and Influencing Factors.

Purpose: Long-term axial length (AL) shortening in myopia is uncommon but noteworthy. Current understanding on the condition is limited due to difficulties in case collection. The study reported percentage, probability, and time course of long-term AL shortening in myopic orthokeratology based on a large database. Methods: This study reviewed 142,091 medical records from 29,825 subjects in a single-hospital orthokeratology database that were collected over 10 years. Long-term AL shortening was defined as a change in AL of -0.1 mm or less at any follow-up beyond 1 year. Incident probability was calculated based on multivariate logistic regression. Time course was estimated using mixed-effect regression model. Results: A total of 10,093 subjects (mean initial age, 11.70 ± 2.52 years; 58.8% female) with 80,778 visits were included. The number of subjects experienced long-term AL shortening was 1,662 (16.47%; 95% confidence interval, 15.75%-17.21%). Initial age showed significant impact on the incident occurrence (OR, 1.37; 95% confidence interval, 1.34-1.40; P < 0.001). The estimated probability of AL shortening was approximately 2% for subjects with initial age of 6 years and 50% for those aged 18. Among the 1662 AL shortening cases, the median magnitude of the maximum AL reduction was 0.19 mm. The shortening process mostly occurred within the initial 2 years. Subject characteristics had limited associations with the shortening rate. Conclusions: Long-term AL shortening is possible in subjects receiving myopic orthokeratology. Although age notably affect the incident probability, the time course seems to not vary significantly.

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.

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.

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.

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.

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.