Bacteriophages avoid autoimmunity from cognate immune systems as an intrinsic part of their life cycles.

Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned off during prophage induction, balancing immunity and autoimmunity. Our results show that the fine regulation of these processes is essential for the correct development of prophages' life cycle.

Antagonistic interactions between phage and host factors control arbitrium lysis-lysogeny decision.

Phages can use a small-molecule communication arbitrium system to coordinate lysis-lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin-antitoxin system MazE-MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis-lysogeny by domesticating and fine-tuning a phage-defence mechanism.

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.

Insights into the mechanism of action of the arbitrium communication system in SPbeta phages.

The arbitrium system is employed by phages of the SPbeta family to communicate with their progeny during infection to decide either to follow the lytic or the lysogenic cycle. The system is controlled by a peptide, AimP, that binds to the regulator AimR, inhibiting its DNA-binding activity and expression of aimX. Although the structure of AimR has been elucidated for phages SPß and phi3T, there is still controversy regarding the molecular mechanism of AimR function, with two different proposed models for SPß. In this study, we deepen our understanding of the system by solving the structure of an additional AimR that shows chimerical characteristics with the SPß receptor. The crystal structures of this AimR (apo, AimP-bound and DNA-bound) together with in vitro and in vivo analyses confirm a mechanism of action by AimP-induced conformational restriction, shedding light on peptide specificity and cross regulation with relevant biological implications.

Bacterial chromosomal mobility via lateral transduction exceeds that of classical mobile genetic elements.

It is commonly assumed that the horizontal transfer of most bacterial chromosomal genes is limited, in contrast to the frequent transfer observed for typical mobile genetic elements. However, this view has been recently challenged by the discovery of lateral transduction in Staphylococcus aureus, where temperate phages can drive the transfer of large chromosomal regions at extremely high frequencies. Here, we analyse previously published as well as new datasets to compare horizontal gene transfer rates mediated by different mechanisms in S. aureus and Salmonella enterica. We find that the horizontal transfer of core chromosomal genes via lateral transduction can be more efficient than the transfer of classical mobile genetic elements via conjugation or generalized transduction. These results raise questions about our definition of mobile genetic elements, and the potential roles played by lateral transduction in bacterial evolution.

The arbitrium system controls prophage induction.

Some Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium, to coordinate the lysis-lysogeny decision. In this system, the phage produces AimP peptide during the lytic cycle. Once internalized by the host cell, AimP binds to the transcription factor AimR, reducing aimX expression and promoting lysogeny. Although these systems are present in a variety of mobile genetic elements, their role in the phage life cycle has only been characterized in phage phi3T during phage infection. Here, using the B. subtilis SPß prophage, we show that the arbitrium system is also required for normal prophage induction. Deletion of the aimP gene increased phage reproduction, although the aimR deletion significantly reduced the number of phage particles produced after prophage induction. Moreover, our results indicated that AimR is involved in a complex network of regulation and brought forward two new players in the SPß lysis-lysogeny decision system, YopN and the phage repressor YopR. Importantly, these proteins are encoded in an operon, the function of which is conserved across all SPß-like phages encoding the arbitrium system. Finally, we obtained mutant phages in the arbitrium system, which behaved almost identically to the wild-type (WT) phage, indicating that the arbitrium system is not essential in the laboratory but is likely beneficial for phage fitness in nature. In support of this, by possessing a functional arbitrium system, the SPß phage can optimize production of infective particles while also preserving the number of cells that survive after prophage induction, a strategy that increases phage persistence in nature.

Molecular Basis of Lysis-Lysogeny Decisions in Gram-Positive Phages.

Temperate bacteriophages (phages) are viruses of bacteria. Upon infection of a susceptible host, a temperate phage can establish either a lytic cycle that kills the host or a lysogenic cycle as a stable prophage. The life cycle pursued by an infecting temperate phage can have a significant impact not only on the individual host bacterium at the cellular level but also on bacterial communities and evolution in the ecosystem. Thus, understanding the decision processes of temperate phages is crucial. This review delves into the molecular mechanisms behind lysis-lysogeny decision-making in Gram-positive phages. We discuss a variety of molecular mechanisms and the genetic organization of these well-understood systems. By elucidating the strategies used by phages to make lysis-lysogeny decisions, we can improve our understanding of phage-host interactions, which is crucial for a variety of studies including bacterial evolution, community and ecosystem diversification, and phage therapeutics.

Genome hypermobility by lateral transduction.

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.

A novel ejection protein from bacteriophage 80α that promotes lytic growth.

Many staphylococcal bacteriophages encode a minor capsid protein between the genes for the portal and scaffolding proteins. In Staphylococcus aureus bacteriophage 80α, this protein, called gp44, is essential for the production of viable phage, but dispensable for the phage-mediated mobilization of S. aureus pathogenicity islands. We show here that gp44 is not required for capsid assembly, DNA packaging or ejection of the DNA, nor for generalized transduction of plasmids. An 80α Δ44 mutant could be complemented in trans by gp44 expressed from a plasmid, indicating that gp44 plays a post-injection role in the host. Our results show that gp44 is an ejection (pilot) protein that is involved in deciding the fate of the phage DNA after injection. Our data are consistent with a model in which gp44 acts as a regulatory protein that promotes progression to the lytic cycle.

Lysogenization of Staphylococcus aureus RN450 by phages ϕ11 and ϕ80α leads to the activation of the SigB regulon.

Staphylococcus aureus is a major opportunistic pathogen that commonly forms biofilms on various biotic and abiotic surfaces. Also, most isolates are known to carry prophages in their genomes. With this in mind, it seems that acquiring a better knowledge of the impact of prophages on the physiology of S. aureus biofilm cells would be useful for developing strategies to eliminate this pathogen. Here, we performed RNA-seq analysis of biofilm cells formed by S. aureus RN450 and two derived strains carrying prophages ϕ11 and ϕ80α. The lysogenic strains displayed increased biofilm formation and production of the carotenoid pigment staphyloxanthin. These phenotypes could be partly explained by the differences in gene expression displayed by prophage-harboring strains, namely an activation of the alternative sigma factor (SigB) regulon and downregulation of genes controlled by the Agr quorum-sensing system, especially the decreased transcription of genes encoding dispersion factors like proteases. Nonetheless, spontaneous lysis of part of the population could also contribute to the increased attached biomass. Interestingly, it appears that the phage CI protein plays a role in orchestrating these phage-host interactions, although more research is needed to confirm this possibility. Likewise, future studies should examine the impact of these two prophages during the infection.

Phage-inducible islands in the Gram-positive cocci.

The SaPIs are a cohesive subfamily of extremely common phage-inducible chromosomal islands (PICIs) that reside quiescently at specific att sites in the staphylococcal chromosome and are induced by helper phages to excise and replicate. They are usually packaged in small capsids composed of phage virion proteins, giving rise to very high transfer frequencies, which they enhance by interfering with helper phage reproduction. As the SaPIs represent a highly successful biological strategy, with many natural Staphylococcus aureus strains containing two or more, we assumed that similar elements would be widespread in the Gram-positive cocci. On the basis of resemblance to the paradigmatic SaPI genome, we have readily identified large cohesive families of similar elements in the lactococci and pneumococci/streptococci plus a few such elements in Enterococcus faecalis. Based on extensive ortholog analyses, we found that the PICI elements in the four different genera all represent distinct but parallel lineages, suggesting that they represent convergent evolution towards a highly successful lifestyle. We have characterized in depth the enterococcal element, EfCIV583, and have shown that it very closely resembles the SaPIs in functionality as well as in genome organization, setting the stage for expansion of the study of elements of this type. In summary, our findings greatly broaden the PICI family to include elements from at least three genera of cocci.

Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party.

We have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions.

Virus Satellites Drive Viral Evolution and Ecology.

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts.

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages.

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.

Bacteriophage-mediated spread of bacterial virulence genes.

Bacteriophages are types of viruses that infect bacteria. They are the most abundant and diverse entities in the biosphere, and influence the evolution of most bacterial species by promoting gene transfer, sometimes in unexpected ways. Although pac-type phages can randomly package and transfer bacterial DNA by a process called generalized transduction, some mobile genetic elements have developed elegant and sophisticated strategies to hijack the phage DNA-packaging machinery for their own transfer. Moreover, phage-like particles (gene transfer agents) have also evolved, that can package random pieces of the producing cell's genome. The purpose of this review is to give an overview of some of the various ways by which phages and phage-like particles can transfer bacterial genes, driving bacterial evolution and promoting the emergence of novel pathogens.

Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases.

Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. In this report, we have characterized one of the non-terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages--i.e., it has both pac and cos sites--a configuration that has not hitherto been described for any mobile element, phages included--and uses the two different phage-coded TerSs. To our knowledge, this is the first example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.

Unravelling bacteriophage ϕ11 requirements for packaging and transfer of mobile genetic elements in Staphylococcus aureus.

Bacteriophages play a major role in spreading mobile genetic elements (MGEs)-encoded genes among bacterial populations. In spite of this, the molecular requirements for building phage transducing particles have not been completely deciphered. Here, we systematically inactivated each ORF from the packaging and lysis modules of the staphylococcal phage ϕ11, used as a model for the Siphoviridae phages infecting Gram-positive bacteria, and determined their functional role in transferring different MGEs including plasmids, staphylococcal pathogenicity islands (SaPIs) and the phage itself. In a previous report, we identified seven of these ORFs as being required for the production of functional phage or SaPI particles. In this report, we have completed the mutational analysis and have identified and characterized 15 additional phage-encoded proteins required for the production of mature phage, SaPI, or transducing particles. Apart from these, we have not yet ascertained any specific function for the six remaining ϕ11 genes, though they are highly conserved among the staphylococcal bacteriophages. To the best of our knowledge, this study represents the first systematic deletion analysis of all the ORFs comprising the morphogenetic and lysis modules of a phage, clearly defining the molecular requirements involved in phage-mediated MGEs transfer.

The peptidoglycan hydrolase of Staphylococcus aureus bacteriophage 11 plays a structural role in the viral particle.

The role of virion-associated peptidoglycan hydrolases (VAPGHs) in the phage infection cycle is not clear. gp49, the VAPGH from Staphylococcus aureus phage 11, is not essential for phage growth but stabilizes the viral particles. 11Δ49 phages showed a reduced burst size and delayed host lysis. Complementation of gp49 with HydH5 from bacteriophage vB_SauS-phiIPLA88 restored the wild-type phenotype.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria.

Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer.