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Nature Sep 2022Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA (msDNA). Despite decades of research on the...
Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA (msDNA). Despite decades of research on the biosynthesis of msDNA, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.
Topics: Antitoxins; Bacteriophages; DNA, Bacterial; DNA, Single-Stranded; Nucleic Acid Conformation; Prophages; RNA-Directed DNA Polymerase; Retroelements; Salmonella typhimurium; Toxin-Antitoxin Systems
PubMed: 35850148
DOI: 10.1038/s41586-022-05091-4 -
Nature Nov 2023Many bacteria use CRISPR-Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids. In turn, these invasive elements have evolved anti-CRISPR...
Many bacteria use CRISPR-Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity. Here we unveil a distinct type of CRISPR-Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR-Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR-Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes. Functional testing of nine candidates spanning the two CRISPR-Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications.
Topics: Bacteria; Bacteriophages; Biotechnology; CRISPR-Associated Proteins; CRISPR-Cas Systems; Molecular Mimicry; Plasmids; Prophages; RNA, Viral
PubMed: 37853129
DOI: 10.1038/s41586-023-06612-5 -
Cell Host & Microbe May 2022Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites...
Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E. coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements.
Topics: Antiviral Agents; Bacteria; Bacteriophages; Escherichia coli; Prophages
PubMed: 35316646
DOI: 10.1016/j.chom.2022.02.018 -
Nature Microbiology Oct 2022The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including...
The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species.
Topics: Antiviral Agents; Bacteriophages; CRISPR-Cas Systems; Escherichia coli; Prophages
PubMed: 36123438
DOI: 10.1038/s41564-022-01219-4 -
MSphere Apr 2022Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In...
Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In natural systems, bacteria simultaneously possess or encounter multiple MGEs, yet their combined influences on microbial communities are poorly understood. Here, we investigate interactions among MGEs in the marine bacterium Sulfitobacter pontiacus. Two related strains, CB-D and CB-A, each harbor a single prophage. These prophages share high sequence identity with one another and an integration site within the host genome, yet these strains exhibit differences in "spontaneous" prophage induction (SPI) and consequent fitness. To better understand mechanisms underlying variation in SPI between these lysogens, we closed their genomes, which revealed that in addition to harboring different prophage genotypes, CB-A lacks two of the four large, low-copy-number plasmids possessed by CB-D. To assess the relative roles of plasmid content versus prophage genotype on host physiology, a panel of derivative strains varying in MGE content were generated. Characterization of these derivatives revealed a robust link between plasmid content and SPI, regardless of prophage genotype. Strains possessing all four plasmids had undetectable phage in cell-free lysates, while strains lacking either one plasmid (pSpoCB-1) or a combination of two plasmids (pSpoCB-2 and pSpoCB-4) produced high (>10 PFU/mL) phage titers. Homologous plasmid sequences were identified in related bacteria, and plasmid and phage genes were found to be widespread in Oceans metagenomic data sets. This suggests that plasmid-dependent stabilization of prophages may be commonplace throughout the oceans. The consequences of prophage induction on the physiology of microbial populations are varied and include enhanced biofilm formation, conferral of virulence, and increased opportunity for horizontal gene transfer. These traits lead to competitive advantages for lysogenized bacteria and influence bacterial lifestyles in a variety of niches. However, biological controls of "spontaneous" prophage induction, the initiation of phage replication and phage-mediated cell lysis without an overt stressor, are not well understood. In this study, we observed a novel interaction between plasmids and prophages in the marine bacterium Sulfitobacter pontiacus. We found that loss of one or more distinct plasmids-which we show carry genes ubiquitous in the world's oceans-resulted in a marked increase in prophage induction within lysogenized strains. These results demonstrate cross talk between different mobile genetic elements and have implications for our understanding of the lysogenic-lytic switches of prophages found not only in marine environments, but throughout all ecosystems.
Topics: Bacteriophages; Ecosystem; Plasmids; Prophages; Rhodobacteraceae
PubMed: 35311569
DOI: 10.1128/msphere.00930-21 -
Yi Chuan = Hereditas Mar 2021As the most abundant biological entities on earth, bacteriophages (phages) were considered as the antagonists of bacteria. With the rapid development of genomics and... (Review)
Review
As the most abundant biological entities on earth, bacteriophages (phages) were considered as the antagonists of bacteria. With the rapid development of genomics and molecular biology technologies, a subtle and complex relationship between phages and their host bacteria has been uncovered. Prophage refers to an intracellular form of a bacteriophage, which is usually integrated into the hereditary material of the host. Prophage is ubiquitously distributed in bacterial genomes. It reproduces when the host does and can affect important biological properties of their bacterial hosts, such as virulence, biofilm formation and host immunity. Interestingly, prophages were also involved in regulating the lysogeny-lytic state by "monitoring" the quorum sensing of bacteria. Recently, anti-CRISPR proteins encoded by prophages were found, which attracts a lot of attention. In this review, we summarized the prediction, distribution, classification and functions of prophages to lay a foundation for further studying interactions between phages and bacteria.
Topics: Bacteria; Bacteriophages; Genome, Bacterial; Lysogeny; Prophages
PubMed: 33724208
DOI: 10.16288/j.yczz.20-355 -
Viruses Jan 2023Bacteriophages are ubiquitous organisms that can be specific to one or multiple strains of hosts, in addition to being the most abundant entities on the planet. It is... (Review)
Review
Bacteriophages are ubiquitous organisms that can be specific to one or multiple strains of hosts, in addition to being the most abundant entities on the planet. It is estimated that they exceed ten times the total number of bacteria. They are classified as temperate, which means that phages can integrate their genome into the host genome, originating a prophage that replicates with the host cell and may confer immunity against infection by the same type of phage; and lytics, those with greater biotechnological interest and are viruses that lyse the host cell at the end of its reproductive cycle. When lysogenic, they are capable of disseminating bacterial antibiotic resistance genes through horizontal gene transfer. When professionally lytic-that is, obligately lytic and not recently descended from a temperate ancestor-they become allies in bacterial control in ecological imbalance scenarios; these viruses have a biofilm-reducing capacity. Phage therapy has also been advocated by the scientific community, given the uniqueness of issues related to the control of microorganisms and biofilm production when compared to other commonly used techniques. The advantages of using bacteriophages appear as a viable and promising alternative. This review will provide updates on the landscape of phage applications for the biocontrol of pathogens in industrial settings and healthcare.
Topics: Bacteriophages; Prophages; Lysogeny; Biofilms; Biotechnology
PubMed: 36851563
DOI: 10.3390/v15020349 -
Periodontology 2000 Jun 2021Oral bacteriophages (or phages), especially periodontal ones, constitute a growing area of interest, but research on oral phages is still in its infancy. Phages are... (Review)
Review
Oral bacteriophages (or phages), especially periodontal ones, constitute a growing area of interest, but research on oral phages is still in its infancy. Phages are bacterial viruses that may persist as intracellular parasitic deoxyribonucleic acid (DNA) or use bacterial metabolism to replicate and cause bacterial lysis. The microbiomes of saliva, oral mucosa, and dental plaque contain active phage virions, bacterial lysogens (ie, carrying dormant prophages), and bacterial strains containing short fragments of phage DNA. In excess of 2000 oral phages have been confirmed or predicted to infect species of the phyla Actinobacteria (>300 phages), Bacteroidetes (>300 phages), Firmicutes (>1000 phages), Fusobacteria (>200 phages), and Proteobacteria (>700 phages) and three additional phyla (few phages only). This article assesses the current knowledge of the diversity of the oral phage population and the mechanisms by which phages may impact the ecology of oral biofilms. The potential use of phage-based therapy to control major periodontal pathogens is also discussed.
Topics: Bacteria; Bacteriophages; Humans; Microbiota; Prophages; Virome
PubMed: 33690937
DOI: 10.1111/prd.12363 -
MSystems Apr 2022Temperate phages (prophages) are ubiquitous in nature and persist as dormant components of host cells (lysogenic stage) before activating and lysing the host (lytic...
Temperate phages (prophages) are ubiquitous in nature and persist as dormant components of host cells (lysogenic stage) before activating and lysing the host (lytic stage). Actively replicating prophages contribute to central community processes, such as enabling bacterial virulence, manipulating biogeochemical cycling, and driving microbial community diversification. Recent advances in sequencing technology have allowed for the identification and characterization of diverse phages, yet no approaches currently exist for identifying if a prophage has activated. Here, we present PropagAtE (Prophage Activity Estimator), an automated software tool for estimating if a prophage is in the lytic or lysogenic stage of infection. PropagAtE uses statistical analyses of prophage-to-host read coverage ratios to decipher actively replicating prophages, irrespective of whether prophages were induced or spontaneously activated. We demonstrate that PropagAtE is fast, accurate, and sensitive, regardless of sequencing depth. Application of PropagAtE to prophages from 348 complex metagenomes from human gut, murine gut, and soil environments identified distinct spatial and temporal prophage activation signatures, with the highest proportion of active prophages in murine gut samples. In infants treated with antibiotics or infants without treatment, we identified active prophage populations correlated with specific treatment groups. Within time series samples from the human gut, 11 prophage populations, some encoding the sulfur metabolism gene or a -like virulence factor, were consistently present over time but not active. Overall, PropagAtE will facilitate accurate representations of viruses in microbiomes by associating prophages with their active roles in shaping microbial communities in nature. Viruses that infect bacteria are key components of microbiomes and ecosystems. They can kill and manipulate microorganisms, drive planetary-scale processes and biogeochemical cycling, and influence the structures of entire food networks. Prophages are viruses that can exist in a dormant state within the genome of their host (lysogenic stage) before activating in order to replicate and kill the host (lytic stage). Recent advances have allowed for the identification of diverse viruses in nature, but no approaches exist for characterizing prophages and their stages of infection (prophage activity). We develop and benchmark an automated approach, PropagAtE, to identify the stages of infection of prophages from genomic data. We provide evidence that active prophages vary in identity and abundance across multiple environments and scales. Our approach will enable accurate and unbiased analyses of viruses in microbiomes and ecosystems.
Topics: Humans; Animals; Mice; Prophages; Metagenome; Lysogeny; Bacteriophages; Microbiota
PubMed: 35323045
DOI: 10.1128/msystems.00084-22 -
Nature Aug 2023Most bacteria in the biosphere are predicted to be polylysogens harbouring multiple prophages. In studied systems, prophage induction from lysogeny to lysis is...
Most bacteria in the biosphere are predicted to be polylysogens harbouring multiple prophages. In studied systems, prophage induction from lysogeny to lysis is near-universally driven by DNA-damaging agents. Thus, how co-residing prophages compete for cell resources if they respond to an identical trigger is unknown. Here we discover regulatory modules that control prophage induction independently of the DNA-damage cue. The modules bear little resemblance at the sequence level but share a regulatory logic by having a transcription factor that activates the expression of a neighbouring gene that encodes a small protein. The small protein inactivates the master repressor of lysis, which leads to induction. Polylysogens that harbour two prophages exposed to DNA damage release mixed populations of phages. Single-cell analyses reveal that this blend is a consequence of discrete subsets of cells producing one, the other or both phages. By contrast, induction through the DNA-damage-independent module results in cells producing only the phage sensitive to that specific cue. Thus, in the polylysogens tested, the stimulus used to induce lysis determines phage productivity. Considering the lack of potent DNA-damaging agents in natural habitats, additional phage-encoded sensory pathways to lysis likely have fundamental roles in phage-host biology and inter-prophage competition.
Topics: Bacteriophages; Lysogeny; Prophages; Viral Proteins; Virus Activation; Bacteria; DNA Damage; DNA, Viral; Single-Cell Analysis; Transcription Factors; Host-Pathogen Interactions
PubMed: 37495698
DOI: 10.1038/s41586-023-06376-y