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Viruses that infect bacteria (phages) can influence bacterial community dynamics, bacterial genome evolution and ecosystem biogeochemistry. These influences differ depending on whether phages establish lytic, chronic or lysogenic infections. Although the first two produce virion progeny, with lytic infections resulting in cell destruction, phages undergoing lysogenic infections replicate with cells without producing virions. The impacts of lysogeny are numerous and well-studied at the cellular level, but ecosystem-level consequences remain underexplored compared to those of lytic infections. Here, we review lysogeny from molecular mechanisms to ecological patterns to emerging approaches of investigation. Our goal is to highlight both its diversity and importance in complex communities. Altogether, using a combined viral ecology toolkit that is applied across broad model systems and environments will help us understand more of the diverse lifestyles and ecological impacts of lysogens in nature.

Bacteriophages are the main vehicle for gene swapping in bacteria, notoriously of pathogenicity islands and antibiotic resistance genes. Chen et al. noticed that the Staphylococcus aureus prophages do not excise from their host's genome until very late in their life cycles (see the Perspective by Davidson). Thus, the phage DNA is amplified while embedded in the bacterial chromosome. The resulting concatemers are processively packed into virus capsules while still integrated in the host chromosome. Each virion is only set loose when the capsule has reached physical capacity—a process called “headful” packaging. In situ amplification maximizes viral replication, and the headful mechanism means adjacent bacterial-host DNA also gets grabbed to fill the capsule. This process ensures that host genes are transmitted along with the phage. Science , this issue p. 207 ; see also p. 152 Staphylococcus aureus phages amplify and package while chromosomally integrated such that host DNA becomes incorporated in the virus particle. Genetic transduction is a major evolutionary force that underlies bacterial adaptation. Here we report that the temperate bacteriophages of Staphylococcus aureus engage in a distinct form of transduction we term lateral transduction. Staphylococcal prophages do not follow the previously described excision-replication-packaging pathway but instead excise late in their lytic program. Here, DNA packaging initiates in situ from integrated prophages, and large metameric spans including several hundred kilobases of the S. aureus genome are packaged in phage heads at very high frequency. In situ replication before DNA packaging creates multiple prophage genomes so that lateral-transducing particles form during normal phage maturation, transforming parts of the S. aureus chromosome into hypermobile regions of gene transfer.

In this Opinion article, Herskovits and colleagues introduce an emerging class of bacteria–phage symbiotic interaction — which they term 'active lysogeny' — in which phages regulate the expression of bacterial genes by precise insertion and excision events. Unlike lytic phages, temperate phages that enter lysogeny maintain a long-term association with their bacterial host. In this context, mutually beneficial interactions can evolve that support efficient reproduction of both phages and bacteria. Temperate phages are integrated into the bacterial chromosome as large DNA insertions that can disrupt gene expression, and they may pose a fitness burden on the cell. However, they have also been shown to benefit their bacterial hosts by providing new functions in a bacterium–phage symbiotic interaction termed lysogenic conversion. In this Opinion article, we discuss another type of bacterium–phage interaction, active lysogeny, in which phages or phage-like elements are integrated into the bacterial chromosome within critical genes or operons and serve as switches that regulate bacterial genes via genome excision.

Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI. Phage P1 is a temperate phage which makes the lytic or lysogenic decision upon infecting bacteria. During the lytic cycle, progeny phages are produced and the cell lyses, and in the lysogenic cycle, P1 DNA exists as a low-copy-number plasmid and replicates autonomously. Previous studies at the bulk level showed that P1 lysogenization was independent of m ultiplicity o f i nfection (MOI; the number of phages infecting a cell), whereas lysogenization probability of the paradigmatic phage λ increases with MOI. However, the mechanism underlying the P1 behavior is unclear. In this work, using a fluorescent reporter system, we demonstrated this P1 MOI-independent lysogenic response at the single-cell level. We further observed that the activity of the major repressor of lytic functions (C1) is a determining factor for the final cell fate. Specifically, the repression activity of P1, which arises from a combination of C1, the anti-repressor Coi, and the corepressor Lxc, remains constant for different MOI, which results in the MOI-independent lysogenic response. Additionally, by increasing the distance between phages that infect a single cell, we were able to engineer a λ-like, MOI-dependent lysogenization upon P1 infection. This suggests that the large separation of coinfecting phages attenuates the effective communication between them, allowing them to make decisions independently of each other. Our work establishes a highly quantitative framework to describe P1 lysogeny establishment. This system plays an important role in disseminating antibiotic resistance by P1-like plasmids and provides an alternative to the lifestyle of phage λ. IMPORTANCE Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI. In this work, we built a simple genetic model to elucidate this MOI independency based on the gene-regulatory circuitry of P1. We also proposed that the effective communication between coinfecting phages contributes to the lysis-lysogeny decision-making of P1 and highlighted the significance of spatial organization in the process of cell fate determination in a single-cell environment. Finally, our work provides new insights into different strategies acquired by viruses to interact with their bacterial hosts in different scenarios for their optimal survival.

Bacteriophages 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.

On infection of their host, temperate viruses that infect bacteria (bacteriophages; hereafter referred to as phages) enter either a lytic or a lysogenic cycle. The former results in lysis of bacterial cells and phage release (resulting in horizontal transmission), whereas lysogeny is characterized by the integration of the phage into the host genome, and dormancy (resulting in vertical transmission)(1). Previous co-culture experiments using bacteria and mutants of temperate phages that are locked in the lytic cycle have shown that CRISPR-Cas systems can efficiently eliminate the invading phages(2,3). Here we show that, when challenged with wild-type temperate phages (which can become lysogenic), type I CRISPR-Cas immune systems cannot eliminate the phages from the bacterial population. Furthermore, our data suggest that, in this context, CRISPR-Cas immune systems are maladaptive to the host, owing to the severe immunopathological effects that are brought about by imperfect matching of spacers to the integrated phage sequences (prophages). These fitness costs drive the loss of CRISPR-Cas from bacterial populations, unless the phage carries anti-CRISPR (acr) genes that suppress the immune system of the host. Using bioinformatics, we show that this imperfect targeting is likely to occur frequently in nature. These findings help to explain the patchy distribution of CRISPR-Cas immune systems within and between bacterial species, and highlight the strong selective benefits of phage-encoded acr genes for both the phage and the host under these circumstances.CRISPR-Cas systems cannot eliminate temperate bacteriophages from bacterial populations and-in this context-the systems impose immunopathological costs on the host, creating selective pressures that may explain their patchy distribution in bacteria.

Most bacteria in the biosphere are predicted to be polylysogens harbouring multiple prophages 1 – 5 . In studied systems, prophage induction from lysogeny to lysis is near-universally driven by DNA-damaging agents 6 . 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.  Prophage lysogeny-to-lysis transitions are controlled by regulatory modules consisting of transcription factors and partner small proteins that are activated through DNA-damage-independent pathways, including by quorum sensing, and these modules determine inter-prophage competition outcomes.

Background Active lysogeny is a newly characterized mechanism that the dynamic integration and excision of prophages serve as molecular switches to coordinately regulate bacterial gene expression without generating progeny virions. The Sa3int family phages, the most prevalent prophages in Staphylococcus aureus , specifically integrate into the β-toxin-coding gene hlb . While infection conditions favor the loss of Sa3int phages and the emergence of Hlb-producing variants, highlighting their potential for active lysogeny, the environmental cues and underlying mechanisms controlling the peculiar life cycle of Sa3int phages remain largely unexplored. Methods In this study, we identified a Sa3int phage, designated ΦSa3XN, from the methicillin-resistant S. aureus strain XN108. The active lysogeny feature of ΦSa3XN was analyzed by combinational PCR, plaque assay, transmission electron microscopy, and DNase protection assay. Additionally, glucose-induced active lysogeny of ΦSa3XN and its impact on S. aureus virulence were evaluated via reporter assay, electrophoretic mobility shift assay, hemolytic assay, and mouse infection models. Results ΦSa3XN acts as a genuine molecular switch, capable of excision without producing progeny phages. Glucose serves as an environmental cue that triggers ΦSa3XN excision and reinstates hlb expression, wherein the catabolite control protein A (CcpA) directly binds to the promoter region of cI and suppresses the expression of CI repressor, thus switching the phage life cycle. Moreover, glucose-induced active lysogeny of ΦSa3XN significantly enhances bacterial hemolytic activity, exacerbating skin inflammation and subcutaneous abscess formation in hyperglycemic mice. Conclusion This study illustrates a novel example of active lysogeny for Sa3int phages and elucidates a glucose-responsive CcpA pathway that regulates ΦSa3XN excision to augment S. aureus virulence, advancing our understanding of the sophisticated interactions between S. aureus and phages.

Temperate phage can transmit both horizontally (lytic cycle) and vertically (lysogenic cycle). Many temperate phage have the ability to modify their lysis/lysogeny decisions based on various environmental cues. For instance, many prophage are known to reactivate when SOS stress responses of their host are triggered. Temperate phage infecting Bacilli can also use peptide signals (“arbitrium”) to control their lysis/lysogeny decisions. However, information from the arbitrium and SOS systems can be potentially conflicting, and it is unclear how phage integrate information carried by these two different signals when making lysis–lysogeny decisions. Here, we use evolutionary epidemiology theory to explore how phage could evolve to use both systems to modulate lysis/lysogeny decisions in a fluctuating environment. Our model predicts that it can be adaptive for phage to respond to both host SOS systems and arbitrium signaling, as they provide complementary information on the quality of the infected host and the availability of alternative hosts. Using the phage phi3T and its host Bacillus subtilis , we show that during lytic infection and as prophage, lysis–lysogeny decisions rely on the integration of information on host condition and arbitrium signal concentrations. For example, free-phage are more likely to lysogenise a stressed host, and prophage are less likely to abandon a stressed host, when high arbitrium concentrations suggest susceptible hosts are unavailable. These experimental results are consistent with our theoretical predictions and demonstrate that phage can evolve plastic life-history strategies to adjust their infection dynamics to account for both the within-host environment (host quality) and the external environment that exists outside of their host (availability of susceptible hosts in the population). More generally, our work yields a new theoretical framework to study the evolution of viral plasticity under the influence of multiple environmental cues.

Bacteriophage lambda infection of Escherichia coli can result in distinct cell fate outcomes. For example, some cells lyse whereas others survive as lysogens. A quantitative biophysical model of lambda infection supports the hypothesis that spontaneous differences in the timing of individual molecular events during lambda infection leads to variation in the selection of cell fates. Building from this analysis, the lambda lysis-lysogeny decision now serves as a paradigm for how intrinsic molecular noise can influence cellular behavior, drive developmental processes, and produce population heterogeneity. Here, we report experimental evidence that warrants reconsidering this framework. By using cell fractioning, plating, and single-cell fluorescent microscopy, we find that physical differences among cells present before infection bias lambda developmental outcomes. Specifically, variation in cell volume at the time of infection can be used to help predict cell fate: a [almost equal to]2-fold increase in cell volume results in a 4- to 5-fold decrease in the probability of lysogeny. Other cell fate decisions now thought to be stochastic might also be determined by pre-existing variation.