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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.

The ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called “superinfection exclusion.” A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection.

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA 1 (msDNA). Despite decades of research on the biosynthesis of msDNA 2 , 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 3 , 4 . 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. Retron-Sen2 of Salmonella Typhimurium encodes a toxin and a reverse transcriptase, which, together with the Sen2 multi-copy single-stranded DNA synthesized by the reverse transcriptase make up a tripartite toxin–antitoxin system that functions in anti-phage defence.

In this study, several different depolymerases encoded in the prophage regions of Acinetobacter baumannii genomes have been bioinformatically predicted and recombinantly produced. The identified depolymerases possessed multi-domain structures and were identical or closely homologous to various proteins encoded in other A. baumannii genomes. This means that prophage-derived depolymerases are widespread, and different bacterial genomes can be the source of proteins with polysaccharide-degrading activities. For two depolymerases, the specificity to capsular polysaccharides (CPSs) of A. baumannii belonging to K1 and K92 capsular types (K types) was determined. The data obtained showed that the prophage-derived depolymerases were glycosidases that cleaved the A. baumannii CPSs by the hydrolytic mechanism to yield monomers and oligomers of the K units. The recombinant proteins with established enzymatic activity significantly reduced the mortality of Galleria mellonella larvae infected with A. baumannii of K1 and K92 capsular types. Therefore, these enzymes can be considered as suitable candidates for the development of new antibacterials against corresponding A. baumannii K types.

Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redβ from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redβ homolog from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA. Specific ssDNA binding proteins promote the annealing of complementary strands. Here the authors reveal an intermediate of annealing in which a duplex DNA is bound in an unusual conformation that is highly extended and unwound for the ssDNA binding protein Redβ from bacteriophage λ.

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ , the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.

Transcriptional regulation of bacterial toxin-antitoxin (TA) systems allows compensation of toxin and antitoxin proteins to maintain a neutral state and avoid cell intoxication unless TA genes are lost. Such models have been primarily studied in plasmids, but TAs are equally present in other mobile genetic elements, such as transposons and prophages. Type II toxin-antitoxin (TA) systems are classically composed of two genes that encode a toxic protein and a cognate antitoxin protein. Both genes are organized in an operon whose expression is autoregulated at the level of transcription by the antitoxin-toxin complex, which binds operator DNA through the antitoxin’s DNA-binding domain. Here, we investigated the transcriptional regulation of a particular TA system located in the immunity region of a cryptic lambdoid prophage in the Escherichia coli O157:H7 EDL933 strain. This noncanonical paaA2-parE2 TA operon contains a third gene, paaR2 , that encodes a transcriptional regulator that was previously shown to control expression of the TA. We provide direct evidence that the PaaR2 is a transcriptional regulator which shares functional similarities to the lambda CI repressor. Expression of the paaA2-parE2 TA operon is regulated by two other transcriptional regulators, YdaS and YdaT, encoded within the same region. We argue that YdaS and YdaT are analogous to lambda Cro and CII and that they do not constitute a TA system, as previously debated. We show that PaaR2 primarily represses the expression of YdaS and YdaT, which in turn controls the expression of paaR2 - paaA2-parE2 operon. Overall, our results show that the paaA2-parE2 TA is embedded in an intricate lambdoid prophage-like regulation network. Using single-cell analysis, we observed that the entire locus exhibits bistability, which generates diversity of expression in the population. Moreover, we confirmed that paaA2-parE2 is addictive and propose that it could limit genomic rearrangements within the immunity region of the CP-933P cryptic prophage. IMPORTANCE Transcriptional regulation of bacterial toxin-antitoxin (TA) systems allows compensation of toxin and antitoxin proteins to maintain a neutral state and avoid cell intoxication unless TA genes are lost. Such models have been primarily studied in plasmids, but TAs are equally present in other mobile genetic elements, such as transposons and prophages. Here, we demonstrate that the expression of a TA system located in a lambdoid cryptic prophage is transcriptionally coupled to the prophage immunity region and relies on phage transcription factors. Moreover, competition between transcription factors results in bistable expression, which generates cell-to-cell heterogeneity in the population, but without, however, leading to any detectable phenotype, even in cells expressing the TA system. We show that despite the lack of protein sequence similarity, this locus retains major lambda prophage regulation features.

Extracellular contractile injection systems (eCISs) are structurally similar to headless phages and are versatile nanomachines conserved among diverse classes of bacteria. Herein, Streptomyces species, which comprise filamentous Gram-positive bacteria and are ubiquitous in soil, were shown to produce Streptomyces phage tail-like particles (SLPs) from eCIS-related genes that are widely conserved among Streptomyces species. In some Streptomyces species, these eCIS-related genes are regulated by a key regulatory gene, which is essential for Streptomyces life cycle and is involved in morphological differentiation and antibiotic production. Deletion mutants of S. lividans of the eCIS-related genes appeared phenotypically normal in terms of morphological differentiation and antibiotic production, suggesting that SLPs are involved in other aspects of Streptomyces life cycle. Using co-culture method, we found that colonies of SLP-deficient mutants of S. lividans were more severely invaded by fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe . In addition, microscopic and transcriptional analyses demonstrated that SLP expression was elevated upon co-culture with the fungi. In contrast, co-culture with Bacillus subtilis markedly decreased SLP expression and increased antibiotic production. Our findings demonstrate that in Streptomyces, eCIS-related genes affect microbial competition, and the patterns of SLP expression can differ depending on the competitor species.

The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes.