Dynamics of CRISPR-mediated virus-host interactions in the human gut microbiome.

Arms races between mobile genetic elements and prokaryotic hosts are major drivers of ecological and evolutionary change in microbial communities. Prokaryotic defense systems such as CRISPR-Cas have the potential to regulate microbiome composition by modifying the interactions among bacteria, plasmids, and phages. Here, we used longitudinal metagenomic data from 130 healthy and diseased individuals to study how the interplay of genetic parasites and CRISPR-Cas immunity reflects on the dynamics and composition of the human gut microbiome. Based on the coordinated study of 80 000 CRISPR-Cas loci and their targets, we show that CRISPR-Cas immunity effectively modulates bacteriophage abundances in the gut. Acquisition of CRISPR-Cas immunity typically leads to a decrease in the abundance of lytic phages but does not necessarily cause their complete disappearance. Much smaller effects are observed for lysogenic phages and plasmids. Conversely, phage-CRISPR interactions shape bacterial microdiversity by producing weak selective sweeps that benefit immune host lineages. We also show that distal (and chronologically older) regions of CRISPR arrays are enriched in spacers that are potentially functional and target crass-like phages and local prophages. This suggests that exposure to reactivated prophages and other endemic viruses is a major selective pressure in the gut microbiome that drives the maintenance of long-lasting immune memory.

CI:Mor interactions in the lysogeny switches of Lactococcus lactis TP901-1 and Staphylococcus aureus φ13 bacteriophages.

Aim: To structurally characterize in detail the interactions between the phage repressor (CI) and the antirepressor (Mor) in the lysis-lysogeny switches of two Gram-positive bacteriophages, the lactococcal TP901-1 and staphylococcal φ13. Methods: We use crystallographic structure determination, computational structural modeling, and analysis, as well as biochemical methods, to elucidate similarities and differences in the CI:Mor interactions for the two genetic switches. Results: By comparing a newly determined and other available crystal structures for the N-terminal domain of CI (CI-NTD), we show that the CI interface involved in Mor binding undergoes structural changes upon binding in TP901-1. Most importantly, we show experimentally for the first time the direct interaction between CI and Mor for φ13, and model computationally the interaction interface. The computational modeling supports similar side chain rearrangements in TP901-1 and φ13. Conclusion: This study ascertains experimentally that, like in the TP901-1 lysogeny switch, staphylococcal φ13 CI and Mor interact with each other. The structural basis of the interaction of φ13 CI and Mor was computationally modeled and is similar to the interaction demonstrated experimentally between TP901-1 CI-NTD and Mor, likely involving similar rearrangement of residue side chains during the formation of the complex. The study identifies one CI residue, Glu69, which unusually interacts primarily through its aliphatic chain with an aromatic residue on Mor after changing its conformation compared to the un-complexed structure. This and other residues at the interface are suggested for investigation in future studies.

Lysogeny destabilizes computationally simulated microbiomes.

Microbiomes are ecosystems, and their stability can impact the health of their hosts. Theory predicts that predators influence ecosystem stability. Phages are key predators of bacteria in microbiomes, but phages are unusual predators because many have lysogenic life cycles. It has been hypothesized that lysogeny can destabilize microbiomes, but lysogeny has no direct analog in classical ecological theory, and no formal theory exists. We studied the stability of computationally simulated microbiomes with different numbers of temperate (lysogenic) and virulent (obligate lytic) phage species. Bacterial populations were more likely to fluctuate over time when there were more temperate phages species. After disturbances, bacterial populations returned to their pre-disturbance densities more slowly when there were more temperate phage species, but cycles engendered by disturbances dampened more slowly when there were more virulent phage species. Our work offers the first formal theory linking lysogeny to microbiome stability.

Coinfecting phages impede each other's entry into the cell.

The developmental choice made by temperate phages, between cell death (lysis) and viral dormancy (lysogeny), is influenced by the relative abundance of viruses and hosts in the environment. The paradigm for this abundance-driven decision is phage lambda of E. coli, whose propensity to lysogenize increases with the number of viruses coinfecting the same bacterium. It is believed that lambda uses this number to infer whether phages or bacteria outnumber each other. However, this interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). Here, we show this premise to be faulty. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, performed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of phage entry decrease with the number of adsorbed phages. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage attachment, as evidenced by compromised membrane integrity and loss of membrane potential. The dependence of entry dynamics on the surrounding medium results in a strong impact on the infection outcome, while the protracted entry of coinfecting phages increases the heterogeneity in infection outcome at a given MOI. Our findings in lambda, and similar results we obtained for phages T5 and P1, demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection.

Insight into the Mechanism of Lysogeny Control of phiCDKH01 Bacteriophage Infecting Clinical Isolate of Clostridioides difficile.

Clostridioides difficile is a causative agent of antibiotic-associated diarrhea as well as pseudomembranous colitis. So far, all known bacteriophages infecting these bacteria are temperate, which means that instead of prompt lysis of host cells, they can integrate into the host genome or replicate episomally. While C. difficile phages are capable of spontaneous induction and entering the lytic pathway, very little is known about the regulation of their maintenance in the state of lysogeny. In this study, we investigated the properties of a putative major repressor of the recently characterized C. difficile phiCDKH01 bacteriophage. A candidate protein belongs to the XRE family and controls the transcription of genes encoding putative phage antirepressors, known to be involved in the regulation of lytic development. Hence, the putative major phage repressor is likely to be responsible for maintenance of the lysogeny.

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some ß-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

Prophage maintenance is determined by environment-dependent selective sweeps rather than mutational availability.

Prophages, viral sequences integrated into bacterial genomes, can be beneficial and costly. Despite the risk of prophage activation and subsequent bacterial death, active prophages are present in most bacterial genomes. However, our understanding of the selective forces that maintain prophages in bacterial populations is limited. Combining experimental evolution with stochastic modeling, we show that prophage maintenance and loss are primarily determined by environmental conditions that alter the net fitness effect of a prophage on its bacterial host. When prophages are too costly, they are rapidly lost through environment-specific sequences of selective sweeps. Conflicting selection pressures that select against the prophage but for a prophage-encoded accessory gene can maintain prophages. The dynamics of prophage maintenance additionally depend on the sociality of this accessory gene. Prophage-encoded genes that exclusively benefit the lysogen maintain prophages at higher frequencies compared with genes that benefit the entire population. That is because the latter can protect phage-free "cheaters," reducing the benefit of maintaining the prophage. Our simulations suggest that environmental variation plays a larger role than mutation rates in determining prophage maintenance. These findings highlight the complexity of selection pressures that act on mobile genetic elements and challenge our understanding of the role of environmental factors relative to random chance events in shaping the evolutionary trajectory of bacterial populations. By shedding light on the key factors that shape microbial populations in the face of environmental changes, our study significantly advances our understanding of the complex dynamics of microbial evolution and diversification.

Phage phylogeny, molecular signaling, and auxiliary antimicrobial resistance in aerobic and anaerobic membrane bioreactors.

Phage emit communication signals that inform their lytic and lysogenic life cycles. However, little is known regarding the abundance and diversity of the genes associated with phage communication systems in wastewater treatment microbial communities. This study focused on phage communities within two distinct biochemical wastewater environments, specifically aerobic membrane bioreactors (AeMBRs) and anaerobic membrane bioreactors (AnMBRs) exposed to varying antibiotic concentrations. Metagenomic data from the bench-scale systems were analyzed to explore phage phylogeny, life cycles, and genetic capacity for antimicrobial resistance and quorum sensing. Two dominant phage families, Schitoviridae and Peduoviridae, exhibited redox-dependent dynamics. Schitoviridae prevailed in anaerobic conditions, while Peduoviridae dominated in aerobic conditions. Notably, the abundance of lytic and lysogenic proteins varied across conditions, suggesting the coexistence of both life cycles. Furthermore, the presence of antibiotic resistance genes (ARGs) within viral contigs highlighted the potential for phage to transfer ARGs in AeMBRs. Finally, quorum sensing genes in the virome of AeMBRs indicated possible molecular signaling between phage and bacteria. Overall, this study provides insights into the dynamics of viral communities across varied redox conditions in MBRs. These findings shed light on phage life cycles, and auxiliary genetic capacity such as antibiotic resistance and bacterial quorum sensing within wastewater treatment microbial communities.

Interaction of bacteriophage P1 with an epiphytic Pantoea agglomerans strain-the role of the interplay between various mobilome elements.

P1 is a model, temperate bacteriophage of the 94 kb genome. It can lysogenize representatives of the Enterobacterales order. In lysogens, it is maintained as a plasmid. We tested P1 interactions with the biocontrol P. agglomerans L15 strain to explore the utility of P1 in P. agglomerans genome engineering. A P1 derivative carrying the Tn9 (cmR) transposon could transfer a plasmid from Escherichia coli to the L15 cells. The L15 cells infected with this derivative formed chloramphenicol-resistant colonies. They could grow in a liquid medium with chloramphenicol after adaptation and did not contain prophage P1 but the chromosomally inserted cmR marker of P1 Tn9 (cat). The insertions were accompanied by various rearrangements upstream of the Tn9 cat gene promoter and the loss of IS1 (IS1L) from the corresponding region. Sequence analysis of the L15 strain genome revealed a chromosome and three plasmids of 0.58, 0.18, and 0.07 Mb. The largest and the smallest plasmid appeared to encode partition and replication incompatibility determinants similar to those of prophage P1, respectively. In the L15 derivatives cured of the largest plasmid, P1 with Tn9 could not replace the smallest plasmid even if selected. However, it could replace the smallest and the largest plasmid of L15 if its Tn9 IS1L sequence driving the Tn9 mobility was inactivated or if it was enriched with an immobile kanamycin resistance marker. Moreover, it could develop lytically in the L15 derivatives cured of both these plasmids. Clearly, under conditions of selection for P1, the mobility of the P1 selective marker determines whether or not the incoming P1 can outcompete the incompatible L15 resident plasmids. Our results demonstrate that P. agglomerans can serve as a host for bacteriophage P1 and can be engineered with the help of this phage. They also provide an example of how antibiotics can modify the outcome of horizontal gene transfer in natural environments. Numerous plasmids of Pantoea strains appear to contain determinants of replication or partition incompatibility with P1. Therefore, P1 with an immobile selective marker may be a tool of choice in curing these strains from the respective plasmids to facilitate their functional analysis.

Insights into the genomic features and lifestyle of B1 subcluster mycobacteriophages.

Bacteriophages infecting Mycobacterium smegmatis mc2155 are numerous and, hence, are classified into clusters based on nucleotide sequence similarity. Analyzing phages belonging to clusters/subclusters can help gain deeper insights into their biological features and potential therapeutic applications. In this study, for genomic characterization of B1 subcluster mycobacteriophages, a framework of online tools was developed, which enabled functional annotation of about 55% of the previously deemed hypothetical proteins in B1 phages. We also studied the phenotype, lysogeny status, and antimycobacterial activity of 10 B1 phages against biofilm and an antibiotic-resistant M. smegmatis strain (4XR1). All 10 phages belonged to the Siphoviridae family, appeared temperate based on their spontaneous release from the putative lysogens and showed antibiofilm activity. The highest inhibitory and disruptive effects on biofilm were 64% and 46%, respectively. This systematic characterization using a combination of genomic and experimental tools is a promising approach to furthering our understanding of viral dark matter.

CRISPRpi: Inducing and Curing Prophage Using the CRISPR Interference.

We present here a CRISPR-interference-based protocol to trigger prophage induction, even for non-inducible prophages. This method can also be used to cure the prophage from the bacterial host. The method is based on silencing of the phage's repressor transcription, thanks to CRISPR interference. Plasmid electroporation is used to bring the CRISPRi system into the bacteria, specifically on a plasmid carrying spacers targeting the prophage repressor. This method enables prophage induction and curation in a week or two with a high efficiency.

Bacteriophage tRNA-dependent lysogeny: requirement of phage-encoded tRNA genes for establishment of lysogeny.

Bacteriophages are large and diverse components of the biosphere, and many phages are temperate. Upon infection, temperate phages can establish lysogeny in which a prophage is typically integrated into the bacterial chromosome. Here, we describe the phenomenon of tRNA-dependent lysogeny, a previously unrecognized behavior of some temperate phages. tRNA-dependent lysogeny is characterized by two unusual features. First, a phage-encoded tyrosine family integrase mediates site-specific recombination between a phage attP site and a bacterial attB site overlapping a host tRNA gene. However, attP and attB share only a short (~10 bp) common core such that a functional tRNA is not reconstructed upon integration. Second, the phage encodes a tRNA of the same isotype as the disrupted but essential host tRNA, complementing its loss, and consequently is required for the survival of lysogenic progeny. As expected, an integrase-defective phage mutant forms turbid plaques, and bacterial progeny are immune to superinfection, but they lack stability, and the prophage is rapidly lost. In contrast, a tRNA-defective phage mutant forms clear plaques and more closely resembles a repressor mutant, and lysogens are recovered only at very low frequency through the use of secondary attachment sites elsewhere in the host genome. Integration-proficient plasmids derived from these phages must also carry a cognate phage tRNA gene for efficient integration, and these may be useful tools for mycobacterial genetics. We show that tRNA-dependent lysogeny is used by phages within multiple different groups of related viruses and may be prevalent elsewhere in the broader phage community.IMPORTANCEBacteriophages are the most numerous biological entities in the biosphere, and a substantial proportion of phages are temperate, forming stable lysogens in which a prophage copy of the genome integrates into the bacterial chromosome. Many phages encode a variety of tRNA genes whose roles are poorly understood, although it has been proposed that they enhance translational efficiencies in lytic growth or that they counteract host defenses that degrade host tRNAs. Here, we show that phage-encoded tRNAs play key roles in the establishment of lysogeny of some temperate phages. They do so by compensating for the loss of tRNA function when phages integrate at an attB site overlapping a tRNA gene but fail to reconstruct the tRNA at the attachment junction. In this system of tRNA-dependent lysogeny, the phage-encoded tRNA is required for lysogeny, and deletion of the phage tRNA gives rise to a clear plaque phenotype and obligate lytic growth.

Seasonal trends in lysogeny in an Appalachian oak-hickory forest soil.

Since 1989, investigations into viral ecology have revealed how bacteriophages can influence microbial dynamics within ecosystems at global scales. Most of the information we know about temperate phages, which can integrate themselves into the host genome and remain dormant via a process called lysogeny, has come from research in aquatic ecosystems. Soil environments remain under-studied, and more research is necessary to fully understand the range of impacts phage infections have on the soil bacteria they infect. The aims of this study were to compare the efficacy of different prophage-inducing agents and to elucidate potential temporal trends in lysogeny within a soil bacterial community. In addition to mitomycin C and acyl-homoserine lactones, our results indicated that halosulfuron methyl herbicides may also be potent inducing agents. In optimizing chemical induction assays, we determined that taking steps to reduce background virus particles and starve cells was critical in obtaining consistent results. A clear seasonal trend in inducible lysogeny was observed in an Appalachian oak-hickory forest soil. The average monthly air temperature was negatively correlated with inducible fraction and burst size, supporting the idea that lysogeny provides a mechanism for phage persistence when temperatures are low and host metabolism is slower. Furthermore, the inducible fraction was negatively correlated with both soil bacterial and soil viral abundance, supporting the idea that lysogeny provides a mechanism for temperate phage persistence when host density is lower. The present study is the first of its kind to reveal clear seasonal trends in inducible lysogeny in any soil.IMPORTANCELysogeny is a relationship in which certain viruses that infect bacteria (phages) may exist within their bacterial host cell as a segment of nucleic acid. In this state, the phage genome is protected from environmental damage and retains the potential to generate progeny particles in the future. It is thought that lysogeny provides a mechanism for long-term persistence for phages when host density is low or hosts are starved-two conditions likely to be found in soils. In the present study, we provide the first known evidence for a seasonal trend in lysogeny in a forest soil. Based on clear relationships observed between lysogeny, temperature, and soil microbial abundance, we find support for previous hypotheses regarding the factors governing lysogeny.

Viral metagenomics reveals diverse virus-host interactions throughout the soil depth profile.

IMPORTANCE: Soil viruses can moderate the roles that their host microbes play in global carbon cycling. However, given that most studies investigate the surface layer (i.e., top 20 cm) of soil, the extent to which this occurs in subsurface soil (i.e., below 20 cm) is unknown. Here, we leveraged public sequencing data to investigate the interactions between viruses and their hosts at soil depth intervals, down to 115 cm. While most viruses were detected throughout the soil depth profile, their adaptation to host microbes varied. Nonetheless, we uncovered evidence for the potential of soil viruses to encourage their hosts to recycle plant-derived carbon in both surface and subsurface soils. This work reasons that our understanding of soil viral functions requires us to continue to dig deeper and compare viruses existing throughout soil ecosystems.

Phage Milagro: a platform for engineering a broad host range virulent phage for Burkholderia.

IMPORTANCE: Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.

Characterization of a unique repression system present in arbitrium phages of the SPbeta family.

Arbitrium-coding phages use peptides to communicate and coordinate the decision between lysis and lysogeny. However, the mechanism by which these phages establish lysogeny remains unknown. Here, focusing on the SPbeta phage family's model phages phi3T and SPß, we report that a six-gene operon called the "SPbeta phages repressor operon" (sro) expresses not one but two master repressors, SroE and SroF, the latter of which folds like a classical phage integrase. To promote lysogeny, these repressors bind to multiple sites in the phage genome. SroD serves as an auxiliary repressor that, with SroEF, forms the repression module necessary for lysogeny establishment and maintenance. Additionally, the proteins SroABC within the operon are proposed to constitute the transducer module, connecting the arbitrium communication system to the activity of the repression module. Overall, this research sheds light on the intricate and specialized repression system employed by arbitrium SPß-like phages in making lysis-lysogeny decisions.

An Eco-evolutionary Model on Surviving Lysogeny Through Grounding and Accumulation of Prophages.

Temperate phages integrate into the bacterial genomes propagating along with the bacterial genomes. Multiple phage elements, representing diverse prophages, are present in most bacterial genomes. The evolutionary events and the ecological dynamics underlying the accumulation of prophage elements in bacterial genomes have yet to be understood. Here, we show that the local wastewater had 7% of lysogens (hosting mitomycin C-inducible prophages), and they showed resistance to superinfection by their corresponding lysates. Genomic analysis of four lysogens and four non-lysogens revealed the presence of multiple prophages (belonging to Myoviridae and Siphoviridae) in both lysogens and non-lysogens. For large-scale comparison, 2180 Escherichia coli genomes isolated from various sources across the globe and 523 genomes specifically isolated from diverse wastewaters were analyzed. A total of 15,279 prophages were predicted among 2180 E. coli genomes and 2802 prophages among 523 global wastewater isolates, with a mean of ~ 5 prophages per genome. These observations indicate that most putative prophages are relics of past bacteria-phage conflicts; they are "grounded" prophages that cannot excise from the bacterial genome. Prophage distribution analysis based on the sequence homology suggested the random distribution of E. coli prophages within and between E. coli clades. The independent occurrence pattern of these prophages indicates extensive horizontal transfers across the genomes. We modeled the eco-evolutionary dynamics to reconstruct the events that could have resulted in the prophage accumulation accounting for infection, superinfection immunity, and grounding. In bacteria-phage conflicts, the bacteria win by grounding the prophage, which could confer superinfection immunity.

SVep1, a temperate phage of human oral commensal Streptococcus vestibularis.

Introduction: Bacteriophages play a vital role in the human oral microbiome, yet their precise impact on bacterial physiology and microbial communities remains relatively understudied due to the limited isolation and characterization of oral phages. To address this gap, the current study aimed to isolate and characterize novel oral phages. Methods: To achieve this, oral bacteria were isolated using a culture-omics method from 30 samples collected from healthy individuals. These bacteria were then cultured in three different types of media under both aerobic and anaerobic conditions. The samples were subsequently subjected to full-length 16S rRNA gene sequencing for analysis. Subsequently, we performed the isolation of lytic and lysogenic phages targeting all these bacteria. Results: In the initial step, a total of 75 bacterial strains were successfully isolated, representing 30 species and 9 genera. Among these strains, Streptococcus was found to have the highest number of species. Using a full-length 16S rRNA gene similarity threshold of 98.65%, 14 potential novel bacterial species were identified. In the subsequent phase, a temperate phage, which specifically targets the human oral commensal bacterium S. vestibularis strain SVE8, was isolated. The genome of S. vestibularis SVE8 consists of a 1.96-megabase chromosome, along with a 43,492-base pair prophage designated as SVep1. Annotation of SVep1 revealed the presence of 62 open reading frames (ORFs), with the majority of them associated with phage functions. However, it is worth noting that no plaque formation was observed in S. vestibularis SVE8 following lytic induction using mitomycin C. Phage particles were successfully isolated from the supernatant of mitomycin C-treated cultures of S. vestibularis SVE8, and examination using transmission electron microscopy confirmed that SVep1 is a siphovirus. Notably, phylogenetic analysis suggested a common ancestral origin between phage SVep1 and the cos-type phages found in S. thermophilus. Discussion: The presence of SVep1 may confer immunity to S. vestibularis against infection by related phages and holds potential for being engineered as a genetic tool to regulate oral microbiome homeostasis and oral diseases.

An extended catalog of integrated prophages in the infant and adult fecal microbiome shows high prevalence of lysogeny.

Background and aims: The acquisition and gradual maturation of gut microbial communities during early childhood is central to an individual's healthy development. Bacteriophages have the potential to shape the gut bacterial communities. However, the complex ecological interactions between phages and their bacterial host are still poorly characterized. In this study, we investigated the abundance and diversity of integrated prophages in infant and adult gut bacteria by detecting integrated prophages in metagenome assembled genomes (MAGs) of commensal bacteria. Methods: Our study included 88 infants sampled at 3 weeks, 3 months, 6 months, and 12 months (n = 323 total samples), and their parents around delivery time (n = 138 total samples). Fecal DNA was extracted and characterized by using shotgun metagenomic sequencing, and a collection of prokaryotic MAGs was generated. The MAG collection was screened for the presence of integrated bacteriophage sequences, allowing their taxonomic and functional characterization. Results: A large collection of 6,186 MAGs from infant and adult gut microbiota was obtained and screened for integrated prophages, allowing the identification of 7,165 prophage sequences longer than 10 kb. Strikingly, more than 70% of the near-complete MAGs were identified as lysogens. The prevalence of prophages in MAGs varied across bacterial families, with a lower prevalence observed among Coriobacteriaceae, Eggerthellaceae, Veillonellaceae and Burkholderiaceae, while a very high prevalence of lysogen MAGs were observed in Oscillospiraceae, Enterococcaceae, and Enterobacteriaceae. Interestingly for several bacterial families such as Bifidobacteriaceae and Bacteroidaceae, the prevalence of prophages in MAGs was higher in early infant time point (3 weeks and 3 months) than in later sampling points (6 and 12 months) and in adults. The prophage sequences were clustered into 5,616 species-like vOTUs, 77% of which were novel. Finally, we explored the functional repertoire of the potential auxiliary metabolic genes carried by these prophages, encoding functions involved in carbohydrate metabolism and degradation, amino acid metabolism and carbon metabolism. Conclusion: Our study provides an enhanced understanding of the diversity and prevalence of lysogens in infant and adult gut microbiota and suggests a complex interplay between prophages and their bacterial hosts.

Ecotype formation and prophage domestication during gut bacterial evolution.

How much bacterial evolution occurs in our intestines and which factors control it are currently burning questions. The formation of new ecotypes, some of which capable of coexisting for long periods of time, is highly likely in our guts. Horizontal gene transfer driven by temperate phages that can perform lysogeny is also widespread in mammalian intestines. Yet, the roles of mutation and especially lysogeny as key drivers of gut bacterial adaptation remain poorly understood. The mammalian gut contains hundreds of bacterial species, each with many strains and ecotypes, whose abundance varies along the lifetime of a host. A continuous high input of mutations and horizontal gene transfer events mediated by temperate phages drives that diversity. Future experiments to study the interaction between mutations that cause adaptation in microbiomes and lysogenic events with different costs and benefits will be key to understand the dynamic microbiomes of mammals. Also see the video abstract here: https://youtu.be/Zjqsiyb5Pk0.