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.

Infant microbiome cultivation and metagenomic analysis reveal Bifidobacterium 2'-fucosyllactose utilization can be facilitated by coexisting species.

The early-life gut microbiome development has long-term health impacts and can be influenced by factors such as infant diet. Human milk oligosaccharides (HMOs), an essential component of breast milk that can only be metabolized by some beneficial gut microorganisms, ensure proper gut microbiome establishment and infant development. However, how HMOs are metabolized by gut microbiomes is not fully elucidated. Isolate studies have revealed the genetic basis for HMO metabolism, but they exclude the possibility of HMO assimilation via synergistic interactions involving multiple organisms. Here, we investigate microbiome responses to 2'-fucosyllactose (2'FL), a prevalent HMO and a common infant formula additive, by establishing individualized microbiomes using fecal samples from three infants as the inocula. Bifidobacterium breve, a prominent member of infant microbiomes, typically cannot metabolize 2'FL. Using metagenomic data, we predict that extracellular fucosidases encoded by co-existing members such as Ruminococcus gnavus initiate 2'FL breakdown, thus critical for B. breve's growth. Using both targeted co-cultures and by supplementation of R. gnavus into one microbiome, we show that R. gnavus can promote extensive growth of B. breve through the release of lactose from 2'FL. Overall, microbiome cultivation combined with genome-resolved metagenomics demonstrates that HMO utilization can vary with an individual's microbiome.

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile.

IMPORTANCE: Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.

Energetic Electron Precipitation Driven by Electromagnetic Ion Cyclotron Waves from ELFIN's Low Altitude Perspective.

We review comprehensive observations of electromagnetic ion cyclotron (EMIC) wave-driven energetic electron precipitation using data collected by the energetic electron detector on the Electron Losses and Fields InvestigatioN (ELFIN) mission, two polar-orbiting low-altitude spinning CubeSats, measuring 50-5000 keV electrons with good pitch-angle and energy resolution. EMIC wave-driven precipitation exhibits a distinct signature in energy-spectrograms of the precipitating-to-trapped flux ratio: peaks at >0.5 MeV which are abrupt (bursty) (lasting ∼17 s, or ΔL∼0.56) with significant substructure (occasionally down to sub-second timescale). We attribute the bursty nature of the precipitation to the spatial extent and structuredness of the wave field at the equator. Multiple ELFIN passes over the same MLT sector allow us to study the spatial and temporal evolution of the EMIC wave - electron interaction region. Case studies employing conjugate ground-based or equatorial observations of the EMIC waves reveal that the energy of moderate and strong precipitation at ELFIN approximately agrees with theoretical expectations for cyclotron resonant interactions in a cold plasma. Using multiple years of ELFIN data uniformly distributed in local time, we assemble a statistical database of ∼50 events of strong EMIC wave-driven precipitation. Most reside at L∼5-7 at dusk, while a smaller subset exists at L∼8-12 at post-midnight. The energies of the peak-precipitation ratio and of the half-peak precipitation ratio (our proxy for the minimum resonance energy) exhibit an L-shell dependence in good agreement with theoretical estimates based on prior statistical observations of EMIC wave power spectra. The precipitation ratio's spectral shape for the most intense events has an exponential falloff away from the peak (i.e., on either side of ∼1.45 MeV). It too agrees well with quasi-linear diffusion theory based on prior statistics of wave spectra. It should be noted though that this diffusive treatment likely includes effects from nonlinear resonant interactions (especially at high energies) and nonresonant effects from sharp wave packet edges (at low energies). Sub-MeV electron precipitation observed concurrently with strong EMIC wave-driven >1 MeV precipitation has a spectral shape that is consistent with efficient pitch-angle scattering down to ∼ 200-300 keV by much less intense higher frequency EMIC waves at dusk (where such waves are most frequent). At ∼100 keV, whistler-mode chorus may be implicated in concurrent precipitation. These results confirm the critical role of EMIC waves in driving relativistic electron losses. Nonlinear effects may abound and require further investigation.

Youth organizations, social mobility and health in middle age: evidence from a Scottish 1950s prospective cohort study.

BACKGROUND: Informal educational programmes focused on youth development appear to improve health and well-being at time of involvement. Less is known about long-term effects. We investigate their impact on self-reported general health in mid-life using the Aberdeen Children of the 1950s (ACONF) cohort. METHODS: We use a subset (n = 1333) of the ACONF cohort, born 1950-56, in Aberdeen Scotland, who took part in Family and Reading Surveys in 1964 and a follow-up questionnaire in 2001. We explore exposure to youth development focused clubs in childhood on self-reported general health around age 50 mediated by adult socioeconomic position. Logistic regression and mediation analysis were used to report odds ratios and natural direct and indirect effects, respectively, on multiply imputed data. RESULTS: Being a member of the Scouts/Guides (G&S) was associated with a 53% (95% confidence interval 1.03-2.27) higher odds of 'excellent' general health in adulthood compared to children attending 'other clubs'. Indirect effects of G&S and Boys'/Girls' Brigade (B&GB) on general health acting via higher socioeconomic position show positive associations; 12% and 6% higher odds of 'excellent' general health in adulthood compared to children attending 'other clubs', respectively. Comparison of indirect with direct effects suggests 27% of this association is mediated through a higher adult socioeconomic position in adulthood. CONCLUSIONS: These results suggest a beneficial association between attending G&S and B&GB clubs in childhood and adult general health. As these organizations are volunteer-led, this may represent a cost-effective method for improving population health.

Experimental validation that human microbiome phages use alternative genetic coding.

Previous bioinformatic analyses of metagenomic data have indicated that bacteriophages can use genetic codes different from those of their host bacteria. In particular, reassignment of stop codon TAG to glutamine (a variation known as 'genetic code 15') has been predicted. Here, we use LC-MS/MS-based metaproteomics of human fecal samples to provide experimental evidence of the use of genetic code 15 in two crAss-like phages. Furthermore, the proteomic data from several phage structural proteins supports the reassignment of the TAG stop codon to glutamine late in the phage infection cycle. Thus, our work experimentally validates the expression of genetic code 15 in human microbiome phages.

Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes.

Bacteriophages (phages) are obligate parasites that use host bacterial translation machinery to produce viral proteins. However, some phages have alternative genetic codes with reassigned stop codons that are predicted to be incompatible with bacterial translation systems. We analysed 9,422 phage genomes and found that stop-codon recoding has evolved in diverse clades of phages that infect bacteria present in both human and animal gut microbiota. Recoded stop codons are particularly over-represented in phage structural and lysis genes. We propose that recoded stop codons might function to prevent premature production of late-stage proteins. Stop-codon recoding has evolved several times in closely related lineages, which suggests that adaptive recoding can occur over very short evolutionary timescales.

Species- and site-specific genome editing in complex bacterial communities.

Understanding microbial gene functions relies on the application of experimental genetics in cultured microorganisms. However, the vast majority of bacteria and archaea remain uncultured, precluding the application of traditional genetic methods to these organisms and their interactions. Here, we characterize and validate a generalizable strategy for editing the genomes of specific organisms in microbial communities. We apply environmental transformation sequencing (ET-seq), in which nontargeted transposon insertions are mapped and quantified following delivery to a microbial community, to identify genetically tractable constituents. Next, DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-seq are used to enable organism- and locus-specific genetic manipulation in a community context. Using a combination of ET-seq and DART in soil and infant gut microbiota, we conduct species- and site-specific edits in several bacteria, measure gene fitness in a nonmodel bacterium and enrich targeted species. These tools enable editing of microbial communities for understanding and control.

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.

Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa.

Bacteria deploy multiple defenses to prevent mobile genetic element (MGEs) invasion. CRISPR-Cas immune systems use RNA-guided nucleases to target MGEs, which counter with anti-CRISPR (Acr) proteins. Our understanding of the biology and co-evolutionary dynamics of the common Type I-C CRISPR-Cas subtype has lagged because it lacks an in vivo phage-host model system. Here, we show the anti-phage function of a Pseudomonas aeruginosa Type I-C CRISPR-Cas system encoded on a conjugative pKLC102 island, and its Acr-mediated inhibition by distinct MGEs. Seven genes with anti-Type I-C function (acrIC genes) were identified, many with highly acidic amino acid content, including previously described DNA mimic AcrIF2. Four of the acr genes were broad spectrum, also inhibiting I-E or I-F P. aeruginosa CRISPR-Cas subtypes. Dual inhibition comes at a cost, however, as simultaneous expression of Type I-C and I-F systems renders phages expressing the dual inhibitor AcrIF2 more sensitive to targeting. Mutagenesis of numerous acidic residues in AcrIF2 did not impair anti-I-C or anti-I-F function per se but did exacerbate inhibition defects during competition, suggesting that excess negative charge may buffer DNA mimics against competition. Like AcrIF2, five of the Acr proteins block Cascade from binding DNA, while two function downstream, likely preventing Cas3 recruitment or activity. One such inhibitor, AcrIC3, is found in an 'anti-Cas3' cluster within conjugative elements, encoded alongside bona fide Cas3 inhibitors AcrIF3 and AcrIE1. Our findings demonstrate an active battle between an MGE-encoded CRISPR-Cas system and its diverse MGE targets.

Differential influences of early growth and social factors on young children's cognitive performance in four low-and-middle-income birth cohorts (Brazil, Guatemala, Philippines, and South Africa).

BACKGROUND: Studies relating childhood cognitive development to poor linear growth seldom take adequate account of social conditions related to both, leading to a focus on nutrition interventions. We aimed to assess the roles of both biological and social conditions in determining early childhood cognition, mediated by birthweight and early linear growth. METHODS: After exploratory structural equation modelling to identify determining factors, we tested direct and indirect paths to cognitive performance through birthweight and child height-for-age at 2 years, assessed between 4 and 8.5 years of age among 2448 children in four birth cohort studies in low-and-middle-income countries (Brazil, Guatemala, Philippines and South Africa). Determinants were compared across the cohorts. FINDINGS: Three factors yielded excellent fit, comprising birth endowment (primarily maternal age and birth order), household resources (crowding, dependency) and parental capacity (parental education). We estimated their strength together with maternal height in determining cognitive performance. Percentage shares of total effects of the four determinants show a marked transition from mainly biological determinants of birth weight (birth endowment 34%) and maternal height (30%) compared to household resources (25%) and parental capacity (11%), through largely economic determinants of height at 2 years (household resources (60%) to cognitive performance being predominantly determined by parental capacity (64%) followed by household resources (29%). The largely biological factor, birth endowment (maternal age and birth order) contributed only 7% to childhood cognitive performance and maternal height was insignificant. In summary, the combined share of social total effects (household resources and parental capacity) rises from 36∙2% on birth weight, to 78∙2% on height for age at 24 m, and 93∙4% on cognitive functioning. INTERPRETATION: Across four low- and middle-income contexts, cognition in childhood is influenced more by the parental capacity of families and their economic resources than by birth weight and early linear growth. Improving children's cognitive functioning requires multi-sectoral interventions to improve parental education and enhance their economic wellbeing, interventions that are known to improve also early childhood growth.

Machine-learning approach expands the repertoire of anti-CRISPR protein families.

The CRISPR-Cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. We present a machine-learning approach for comprehensive Acr prediction. The model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate Acr families. Experimental validation of top candidates revealed two unknown Acrs (AcrIC9, IC10) and three other top candidates were coincidentally identified and found to possess anti-CRISPR activity. These results substantially expand the repertoire of predicted Acrs and provide a resource for experimental Acr discovery.

Bacterial alginate regulators and phage homologs repress CRISPR-Cas immunity.

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection1. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases2. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB-AlgB), previously characterized in the regulation of Pseudomonas aeruginosa alginate biosynthesis3,4, as a regulator of the expression and activity of the P. aeruginosa Type I-F CRISPR-Cas system. Downstream of KinB-AlgB, activators of alginate production AlgU (a σE orthologue) and AlgR repress CRISPR-Cas activity during planktonic and surface-associated growth5. AmrZ, another alginate regulator6, is triggered to repress CRISPR-Cas immunity upon surface association. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme and carry hijacked homologs of AmrZ that repress CRISPR-Cas expression and activity. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

Author Correction: Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth's biomes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Type IV-A CRISPR-Cas System in Pseudomonas aeruginosa Mediates RNA-Guided Plasmid Interference In Vivo.

Bacteria and archaea use CRISPR-Cas adaptive immune systems to destroy complementary nucleic acids using RNAs derived from CRISPR loci. Here, we provide the first functional evidence for type IV CRISPR-Cas, demonstrating that the system from Pseudomonas aeruginosa strain PA83 mediates RNA-guided interference against a plasmid in vivo, both clearing the plasmid and inhibiting its uptake. This interference depends on the putative NTP-dependent helicase activity of Csf4/DinG.

Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription.

Phages express anti-CRISPR (Acr) proteins to inhibit CRISPR-Cas systems that would otherwise destroy their genomes. Most acr genes are located adjacent to anti-CRISPR-associated (aca) genes, which encode proteins with a helix-turn-helix DNA-binding motif. The conservation of aca genes has served as a signpost for the identification of acr genes, but the function of the proteins encoded by these genes has not been investigated. Here we reveal that an acr-associated promoter drives high levels of acr transcription immediately after phage DNA injection and that Aca proteins subsequently repress this transcription. Without Aca activity, this strong transcription is lethal to a phage. Our results demonstrate how sufficient levels of Acr proteins accumulate early in the infection process to inhibit existing CRISPR-Cas complexes in the host cell. They also imply that the conserved role of Aca proteins is to mitigate the deleterious effects of strong constitutive transcription from acr promoters.

Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth's biomes.

Bacteriophages from the Inoviridae family (inoviruses) are characterized by their unique morphology, genome content and infection cycle. One of the most striking features of inoviruses is their ability to establish a chronic infection whereby the viral genome resides within the cell in either an exclusively episomal state or integrated into the host chromosome and virions are continuously released without killing the host. To date, a relatively small number of inovirus isolates have been extensively studied, either for biotechnological applications, such as phage display, or because of their effect on the toxicity of known bacterial pathogens including Vibrio cholerae and Neisseria meningitidis. Here, we show that the current 56 members of the Inoviridae family represent a minute fraction of a highly diverse group of inoviruses. Using a machine learning approach leveraging a combination of marker gene and genome features, we identified 10,295 inovirus-like sequences from microbial genomes and metagenomes. Collectively, our results call for reclassification of the current Inoviridae family into a viral order including six distinct proposed families associated with nearly all bacterial phyla across virtually every ecosystem. Putative inoviruses were also detected in several archaeal genomes, suggesting that, collectively, members of this supergroup infect hosts across the domains Bacteria and Archaea. Finally, we identified an expansive diversity of inovirus-encoded toxin-antitoxin and gene expression modulation systems, alongside evidence of both synergistic (CRISPR evasion) and antagonistic (superinfection exclusion) interactions with co-infecting viruses, which we experimentally validated in a Pseudomonas model. Capturing this previously obscured component of the global virosphere may spark new avenues for microbial manipulation approaches and innovative biotechnological applications.