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19 páginas, 9 figuras, 1 tabla., Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature., This work was supported by grants MR/V000772/1, MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1, BB/S003835/1 and BB/V002376/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust201531/Z/16/Z, and ERC-ADG-2014 Proposal n◦670932 Dut-signal from EU to J.R.P.; grants PID2019-108541GB-I00 from Spanish Government (Ministerio de Econom´ıa y Competitividad y Ministerio de Ciencia e Innovacion) and PROMETEO ´ /2020/012 from Valencian Government to A.M.; grants MOE2017-T2-2-163 and MOE2019-T2-2-162 from the Ministry of Education to J.C.; and grant NIHR01 AI083255 to G.C. J.T. was supported by NIH IRACDA Grant K12GM093857 to Virginia Commonwealth University. We acknowledge Diamond Light Source for time on Beamline I03 for X-ray crystallography and B21 for SEC-SAXS under Proposal 16258. L.M.-R. was the recipient of a Spanish postdoctoral fellowship from Fundacion Ram ´ on Areces (2018–2020). J.R.P. is ´thankful to the Royal Society and the Wolfson Foundation for providing him support through a Royal Society Wolfson Fellowship. Funding for open access charge: University funds.

Phages can use a small-molecule communication system, termed arbitrium, to coordinate lysis-lysogeny decisions. It was proposed that this system contains three main players. AimR is a transcriptional activator that promotes expression of aimX, a small RNA which favours the lytic cycle of the phage by inhibiting the phage master repressor using an uncharacterised cis-antisense mechanism. The third player, AimP, is a peptide that accumulates after several rounds of phage infection. When this occurs, AimP binds to AimR inhibiting its function, a process that promotes lysogeny, since aimX will not be transcribed. Here we redefined the role for the main players and identified new key ones, explaining the molecular basis of the arbitrium system. Our results demonstrate that AimR is an antiterminator that eliminates the function of a terminator located between aimP and aimX, which are expressed then as part of a bicistronic operon that initiates in the aimP promoter. Importantly, our genetic, biochemical, and structural analyses also demonstrate that aimX is not a regulatory small RNA but encodes a peptide, AimX, which promotes the lytic cycle of the phage by binding to the second repressor of the arbitrium system, YopN, eliminating its function. Our results reveal the mechanism by which the arbitrium system controls lysis/lysogeny.

Bacteriophage-bacteria interactions are affected by phage satellites, elements that exploit phages for transfer between bacterial cells. Satellites can encode defense systems, antibiotic resistance genes, and virulence factors, but their number and diversity are unknown for lack of a tool to identify them. We developed a flexible and updateable program to identify satellites in bacterial genomes – SatelliteFinder – and use it to identify the best described families: P4-like, phage inducible chromosomal islands (PICI), capsid-forming PICI, and phage-inducible chromosomal island-like elements (PLE). We vastly expanded the number of described elements to ∼5000, finding hundreds of bacterial genomes with two different families of satellites, and dozens of Escherichia coli genomes with three. Most satellites were found in Proteobacteria and Firmicutes, but some are in novel taxa such as Actinobacteria. We characterized the gene repertoires of satellites, which are variable in size and composition, and their genomic organization, which is very conserved. With the partial exception of PICI and cfPICI, there are few homologous core genes between families of satellites, and even fewer homologous to phages. Hence, phage satellites are ancient, diverse, and probably evolved multiple times independently. Occasionally, core genes of a given family of satellites are found in another, suggesting gene flow between different satellites. Given the many elements found in spite of our conservative approach, the many bacteria infected by phages that still lack known satellites, and the recent proposals for novel families, we speculate that we are at the beginning of the discovery of massive numbers and types of satellites. SatelliteFinder is accessible for the community as a Galaxy service at https://galaxy.pasteur.fr/root?tool_id=toolshed.pasteur.fr/repos/fmareuil/satellitefinder/SatelliteFinder/0.9

versión pre-print: 30 páginas, 5 figuras, 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., The writing of this review was supported by grants MR/M003876/1 and MR/S00940X/1 from the UK Medical Research Council, grants BB/N002873/1 and BB/S003835/1 from the UK Biotechnology and Biological Sciences Research Council, grant 201531/Z/16/Z from the Wellcome Trust, and ERC-ADG-2014 proposal 670932 “DUT-signal” from the European Research Council to J.R.P., as well as by grants BIO2016-78571-P and PID2019-108541GB-I00 from the Spanish government and PROMETEO/2020/012 from the Valencian Government to A.M. A.F-R. is a recipient of a PhD fellowship from the FPU program of the Spanish Government (reference FPU19/00433). Figures were created with BioRender.com.

Bacteriophages (phages) are the predominant biological entities on the planet and play an important role in the spread of bacterial virulence, pathogenicity, and antimicrobial resistance. After infection, temperate phages can integrate in the bacterial chromosome thanks to the expression of the prophage-encoded CI master repressor. Upon SOS induction, and promoted by RecA*, CI auto-cleaves generating two fragments, one containing the N-terminal domain (NTD), which retains strong DNA-binding capacity, and other corresponding to the C-terminal part of the protein. However, it is unknown how the CI NTD is removed, a process that is essential to allow prophage induction. Here we identify for the first time that the specific interaction of the ClpX protease with the CI NTD repressor fragment is essential and sufficient for prophage activation after SOS-mediated CI autocleavage, defining the final stage in the prophage induction cascade. Our results provide unexpected roles for the bacterial protease ClpX in phage biology.

Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution.

15 páginas, 7 figuras, 1 tabla, 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 SPβ and phi3T, there is still controversy regarding the molecular mechanism of AimR function, with two different proposed models for SPβ. In this study, we deepen our understanding of the system by solving the structure of an additional AimR that shows chimerical characteristics with the SPβ 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., This work was supported by grants BIO2016-78571-P and PID2019-108541GB-I00 from Spanish Government (Ministerio de Economía y Competitividad y Ministerio de Ciencia e Innovación) and PROMETEO/2020/012 by Valencian Government to A.M, and grant MR/M003876/1, MR/V000772/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1, BB/V002376/1 and BB/S003835/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), ERC-ADG-2014 Proposal n° 670932 Dut-signal (from EU), and Wellcome Trust 201531/Z/16/Z to J.R.P.

Funding: This work was supported by the following grants awarded to JPR: MR/M003876/1, MR/S00940X/1 and MR/V000772/1 from the Medical Research Council (MRC, UK; https://mrc.ukri.org); BB/N002873/1, BB/S003835/1 and BB/V002376/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK; https://bbsrc.ukri.org); 201531/Z/16/Z from the Wellcome Trust (https://wellcome.org). Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution. Publisher PDF

Summary 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 SPβ 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 SPβ 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 SPβ-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 SPβ 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., Graphical abstract, Highlights • The arbitrium system controls prophage induction in B. subtilis • An operon downstream of the arbitrium system is involved in controlling lysogeny • The operon is functionally conserved in SPβ-like phages encoding arbitrium systems • YopR acts as the phage repressor in SPβ, Bacillus subtilis phages from the SPβ family use the arbitrium system to communicate during infection of the host. Brady et al. show that this system is also required for induction of the resident prophage after activation of the host SOS response and identify a key operon involved in the control of the lytic/lysogenic cycle.

Staphylococcus aureus pathogenicity islands (SaPIs) are molecular parasites that hijack helper phages for their transfer. SaPIbov5, the prototypical member of a family of cos type SaPIs, redirects the assembly of ϕ12 helper capsids from prolate to isometric. This size and shape shift is dependent on the SaPIbov5-encoded protein Ccm, a homolog of the ϕ12 capsid protein (CP). Using cryo-electron microscopy, we have determined structures of prolate ϕ12 procapsids and isometric SaPIbov5 procapsids. ϕ12 procapsids have icosahedral end caps with Tend = 4 architecture and a Tmid = 14 cylindrical midsection, whereas SaPIbov5 procapsids have T = 4 icosahedral architecture. We built atomic models for CP and Ccm, and show that Ccm occupies the pentameric capsomers in the isometric SaPIbov5 procapsids, suggesting that preferential incorporation of Ccm pentamers prevents the cylindrical midsection from forming. Our results highlight that pirate elements have evolved diverse mechanisms to suppress phage multiplication, including the acquisition of phage capsid protein homologs., Phage-inducible chromosomal islands (PICIs) are a group of mobile genetic elements that hijack the replication and assembly machinery of helper bacteriophages. Here the authors describe a mechanism by which a group of PICIs from Staphylococcus aureus re-direct the assembly pathway of their helpers using a capsid protein homolog.

Postprint version:25 páginas, 6 figuras, 1 tabla. Free en PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611864/pdf/EMS136588.pdf, Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage-encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA-immR-str'. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl-str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites., This work was supported by grants MR/V000772/1, MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1, BB/S003835/1 and BB/V002376/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust 201531/Z/16/Z, and ERC-ADG-2014 Proposal n° 670932 Dut-signal from EU to J.R.P. J.R.P. is thankful to the Royal Society and the Wolfson Foundation for providing him support through a Royal Society Wolfson Fellowship. The authors thank Dr Leandro LemgruberSoares of the Glasgow Imaging Facility for their support & assistance in this work.

Mobile genetic elements (MGEs) control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here we describe an unprecedented repression-derepression mechanism involved in the transfer of the Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation, never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterise a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.SignificanceWhile most repressors controlling the transfer of mobile genetic elements are dimers, we demonstrate here that the Staphylococcal pathogenicity island 1 (SaPI1) is repressed by two tetramers, which have a novel structural fold in their body that has never been seen before in other proteins. Moreover, by solving the structure of the SaPI1 repressor in complex with its inducing protein Sri, we have demonstrated that Sri forces the SaPI1 repressor to adopt a conformation that is incompatible with DNA binding, explaining how SaPI1 is induced. Finally, our results demonstrate that this repression system is not exclusive of the SaPIs but widespread in nature. Our studies provide important insights understanding how SaPIs spread in nature.

Lysogenic induction ends the stable association between a bacteriophage and its host, and the transition to the lytic cycle begins with prophage excision followed by DNA replication and packaging (ERP) – a temporal program that is considered universal for most temperate phages. Here we report that the long-standing ERP program is an artefact of the experimentally favoured Salmonella phage P22 tsc229 heat-inducible mutant, and that wildtype P22 actually follows a replication-packaging-excision (RPE) program. We found that unlike P22 tsc229, P22 delayed excision to just before it was detrimental to phage production. Thus, at minimal expense to itself, P22 has tuned the timing of excision to balance propagation with lateral transduction, powering the evolution of its host through gene transfer in the interest of self-preservation.One Sentence SummaryGenetic analyses propose a new life cycle for temperate bacteriophages.

The emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as a switch in host or disease tropism are typically accompanied by genetic diversification. Staphylococcus aureus is a multi-host bacterial species associated with human and livestock infections. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for Morel’s disease, a lymphadenitis restricted to sheep and goats. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown. Population genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages that contain African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes, resulting in a highly fastidious metabolism. Importantly, expansion of ~87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, including a novel mechanism associated with IS-mediated anti-anti-sense decoupling of ancestral gene repression. Our findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of diverse mechanisms in parallel., Author summary The emergence of new pathogens is a major threat to public and veterinary health. Some bacteria such as Staphylococcus aureus, have the capacity to infect many different host species including humans and livestock while others such as the closely-related S. aureus subsp. anaerobius, associated with a single type of pathology called Morel’s disease in small ruminants, are highly niche-restricted. However, our understanding of the genetic basis for such differences in bacterial host-tropism is very limited. Here, we discovered that S. aureus subsp. anaerobius evolved from an S. aureus ancestor and underwent an array of extensive changes to its genome that accompanied the transition to its current restricted lifestyle. We observed genome decay involving loss of function of hundreds of genes, large intra-chromosomal rearrangements affecting most of the genome, acquisition of a pathogenicity island, and expansion of large numbers of insertion sequences that are inserted at intergenic sites around the genome. Importantly, we found that IS elements affect the expression of neighbouring genes in different ways including a novel mechanism of IS-enabled disruption of ancestral gene repression. Taken together, we provide a remarkable example of radical genomic changes associated with evolutionary transition from a multi-host to highly restricted host ecology.

Lysogenic induction ends the stable association between a bacteriophage and its host, and the transition to the lytic cycle begins with early prophage excision followed by DNA replication and packaging (ERP). This temporal program is considered universal for P22-like temperate phages, though there is no direct evidence to support the timing and sequence of these events. Here we report that the long-standing ERP program is an observation of the experimentally favored Salmonella phage P22 tsc229 heat-inducible mutant, and that wild-type P22 actually follows the replication-packaging-excision (RPE) program. We find that P22 tsc229 excises early after induction, but P22 delays excision to just before it is detrimental to phage production. This allows P22 to engage in lateral transduction. Thus, at minimal expense to itself, P22 has tuned the timing of excision to balance propagation with lateral transduction, powering the evolution of its host through gene transfer in the interest of self-preservation., During the transition from lysogeny (a stable association between a phage and its bacterial host) to the lytic cycle, prophage excision can be followed or preceded by DNA replication and packaging. Here, the authors show that prophage excision is delayed in Salmonella phage P22, thus allowing the packaging and transfer of large fragments of host DNA via lateral transduction.

BackgroundThe emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as those associated with a switch in host or disease tropism are often accompanied by genetic adaptation. Staphylococcus aureus is a multi-host bacterial species comprising strains with distinct tropisms for human and livestock species. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for outbreaks of Morel’s disease, a lymphadenitis in small ruminants. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown.ResultsEvolutionary genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages representing African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, several large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes resulting in a highly fastidious metabolism. Importantly, expansion of ∼87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, representing a novel concept of the IS regulon.ConclusionsOur findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of parallel mechanisms.

This work was supported by grants MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1 and BB/S003835/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust 201531/Z/16/Z, and ERC-ADG-2014 Proposal no. 670932 Dut-signal from EU to J.R.P. P.D-M. is a recipient of a FPI fellowship to grant BIO2014-53530-R from the Spanish Ministry of Science, Innovation and Universities. Work in the Laboratory of Microbial Pathogenesis is funded by grant BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union). J.R.P. is thankful to the Royal Society and the Wolfson Foundation for providing him support through a Royal Society Wolfson Fellowship. Phage-inducible chromosomal islands (PICIs) are a widespread family of mobile genetic elements, which have an important role in bacterial pathogenesis. These elements mobilize among bacterial species at extremely high frequencies, representing an attractive tool for the delivery of synthetic genes. However, tools for their genetic manipulation are limited and timing consuming. Here, we have adapted a synthetic biology approach for rapidly editing of PICIs in Saccharomyces cerevisiae based on their ability to excise and integrate into the bacterial chromosome of their cognate host species. As proof of concept, we engineered several PICIs from Staphylococcus aureus and Escherichia coli and validated this methodology for the study of the biology of these elements by generating multiple and simultaneous mutations in different PICI genes. For biotechnological purposes, we also synthetically constructed PICIs as Trojan horses to deliver different CRISPR-Cas9 systems designed to either cure plasmids or eliminate cells carrying the targeted genes. Our results demonstrate that the strategy developed here can be employed universally to study PICIs and enable new approaches for diagnosis and treatment of bacterial diseases. Publisher PDF

7 páginas, 2 figuras, Phage satellites are genetic elements that depend on helper phages for induction, packaging and transfer. To promote their lifestyles, they have evolved elegant and sophisticated strategies to inhibit phage reproduction, which will be reviewed here. We will principally focus on the convergent interference mechanisms used by phage-inducible chromosomal islands (PICIs), which are a family of satellite phages present in both Gram-positive and Gram-negative bacteria. While some PICI elements have been extensively studied for their roles in virulence and antibiotic resistance, recent studies have highlighted their relevance in controlling phage ecology and diversity. In many cases, these interference mechanisms are complemented by additional strategies that promote the preferential PICI packaging and dissemination of these elements in nature. Since the PICI-encoded mechanisms target conserved phage mechanisms, we propose here that the PICIs form part of the initial innate immune system that phages must overcome to infect their bacterial host., This work was supported by grants MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1 and BB/S003835/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust201531/Z/16/Z, and ERC-ADG-2014 Proposal n 670932 Dut-signal from EU to J.R.P. L.M-R is the recipient of a Spanish postdoctoral fellowship from Fundacio´n Ramo´n Areces. J.R.P. is thankful to the Royal Society and the Wolfson Foundation for providing him support through a Royal Society Wolfson Fellowship.

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.

Staphylococcus aureus is a serious human pathogen with remarkable adaptive powers. Antibiotic-resistant clones rapidly emerge mainly by acquisition of antibiotic-resistance genes from other S. aureus strains or even from other genera. Transfer is mediated by a diverse complement of mobile genetic elements and occurs primarily by conjugation or bacteriophage transduction, with the latter traditionally being perceived as the primary route. Recent work on conjugation and transduction suggests that transfer by these mechanisms may be more extensive than previously thought, in terms of the range of plasmids that can be transferred by conjugation and the efficiency with which transduction occurs. Here, we review the main routes of antibiotic resistance gene transfer in S. aureus in the context of its biology as a human commensal and a life-threatening pathogen.

Infections caused by Staphylococcus aureus pose a serious and sometimes fatal health issue. With the aim of exploring a novel therapeutic approach, we chose GraXRS, a Two-Component System (TCS) that determines bacterial resilience against host innate immune barriers, as an alternative target to disarm S. aureus. Following a drug repurposing methodology, and taking advantage of a singular staphylococcal strain that lacks the whole TCS machinery but the target one, we screened 1.280 offpatent FDA-approved drug for GraXRS inhibition. Reinforcing the connection between this signaling pathway and redox sensing, we found that antioxidant and redox-active molecules were capable of reducing the expression of the GraXRS regulon. Among all the compounds, verteporfin (VER) was really efficient in enhancing PMN-mediated bacterial killing, while topical administration of such drug in a murine model of surgical wound infection significantly reduced the bacterial load. Experiments relying on the chemical mimicry existing between VER and heme group suggest that redox active residue C227 of GraS participates in the inhibition exerted by this FDA-approved drug. Based on these results, we propose VER as a promising candidate for sensitizing S. aureus that could be helpful to combat persistent or antibiotic-resistant infections. This study was supported by Centre for the Development of Industrial Technology (CDTI), (NEO16RECOMBINA; EXP 00112635/SNEO-20161233). Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities grant BIO2017-83035-R (AEI/FEDER, EU).

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes., CRISPR technology is emerging as a potential antimicrobial against antimicrobial-resistant bacteria. Here the authors develop a bacteriophage delivered Cas13a system for killing target bacteria and detecting bacterial genes.

Emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. We established a series of CRISPR-Cas13a-based antibacterial nucleocapsid, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus through promiscuous RNA cleavage after recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs were generated by packaging CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibited strong bacterial killing activities upon recognizing target genes regardless of their location. The antimicrobials’ treatment efficacy was confirmed using a Galleria mellonella larvae model. Further, we demonstrated that the CapsidCas13a(s) can assist in bacterial gene detection without employing nucleic acid amplification and optical devices.

A model of bacterial host switching provides a window into the population dynamics of host adaptation., While many bacterial pathogens are restricted to single host species, some have the capacity to undergo host switches, leading to the emergence of new clones that are a threat to human and animal health. However, the bacterial traits that underpin a multihost ecology are not well understood. Following transmission to a new host, bacterial populations are influenced by powerful forces such as genetic drift that reduce the fixation rate of beneficial mutations, limiting the capacity for host adaptation. Here, we implement a novel experimental model of bacterial host switching to investigate the ability of the multihost pathogen Staphylococcus aureus to adapt to new species under continuous population bottlenecks. We demonstrate that beneficial mutations accumulated during infection can overcome genetic drift and sweep through the population, leading to host adaptation. Our findings highlight the remarkable capacity of some bacteria to adapt to distinct host niches in the face of powerful antagonistic population forces.

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16 páginas, 7 figuras, 2 tablas. Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 6H48, 6H4B, 6H49, 6H4C., Stl is a master repressor encoded by Staphylococcus aureus pathogenicity islands (SaPIs) that maintains integration of these elements in the bacterial chromosome. After infection or induction of a resident helper phage, SaPIs are de-repressed by specific interactions of phage proteins with Stl. SaPIs have evolved a fascinating mechanism to ensure their promiscuous transfer by targeting structurally unrelated proteins performing identically conserved functions for the phage. Here we decipher the molecular mechanism of this elegant strategy by determining the structure of SaPIbov1 Stl alone and in complex with two structurally unrelated dUTPases from different S. aureus phages. Remarkably, SaPIbov1 Stl has evolved different domains implicated in DNA and partner recognition specificity. This work presents the solved structure of a SaPI repressor protein and the discovery of a modular repressor that acquires multispecificity through domain recruiting. Our results establish the mechanism that allows widespread dissemination of SaPIs in nature., This work was supported by grant BIO2016-78571-P from the Ministerio de Economia y Competitividad (Spain) and grant Prometeo II/2014/029 from Valencian Government (Spain) to A.M., and grants MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1 and BB/S003835/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust 201531/Z/16/Z, and ERC-ADG-2014 Proposal no. 670932 Dut-signal from EU to J.R.P. C.A. and J.R.C were supported by FPI BES-2014-068617 and FPU13/02880 predoctoral fellowships respectively. X-ray diffraction data collection was supported by Diamond Light Source block allocation group (BAG) Proposal MX14739 and MX16258 and Spanish Synchrotron Radiation Facility ALBA Proposal 2016071762 and 2017072262. J.R.P. is thankful to the Royal Society and the Wolfson Foundation for providing him support through a Royal Society Wolfson Fellowship.

Staphylococcus aureus subsp. anaerobius is a microaerophilic, catalase-negative bacterium responsible for abscess pathology (Morel’s disease) in small ruminants. We performed whole-genome sequencing to a collection of isolates taken in Europe and Africa over the last 30 years, and carried out an evolutionary genomic analysis to understand the molecular bases of its host adaptation and restricted metabolism. Phylodynamic analyses showed that S. anaerobius emerged from a Staphylococcus aureus progenitor about 1000 years ago (716–1184), with an evolutionary rate of ∼1.2 SNPs/year (approximately 10-fold slower than S. aureus clones), before differentiating into two distinct lineages separating African and European isolates. The S. anaerobius genome displays signatures of extreme adaptation to a highly specific niche, with 205 pseudogenes that together represent over 10 % of the genome and affect many metabolic and pathogenic pathways. In addition, S. anaerobius contains 87 highly similar insertion sequences (IS) located in intergenic regions. Our functional analysis suggests that the IS transcription could affect the expression of their flanking genes, using at least two complementary strategies: antisense RNA or modification of promoter regions. We propose that the IS-mediated control of gene expression underpins an orchestrated mechanism of host adaptation. Remarkably, we also identified 6 large genomic transversions (size range: 70–346 kb) flanked by IS, presumably the result of homologous recombination. In summary, S. anaerobius evolved from S. aureus undergoing restrictive host specialisation, which shaped its genome through widespread pseudogenisation, accumulation of IS that modulate gene expression, and large chromosomal rearrangements.

Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival., Author summary Viruses (phages) that only attack bacteria are highly common. Some of these phages naturally reside within the bacterial chromosome for extended periods of time. Upon release and propagation on phage susceptible cells, new phage particles are made but occasionally bacterial rather than phage DNA is packaged into phage heads. Transfer of random pieces of bacterial DNA between cells by such transducing particles has been termed generalized transduction and has long been used as a tool for genetic engineering. We show here that bacteria and generalized transducing phages cooperate to survive adverse conditions such as the presence of antibiotics by phage integration and transduction of antibiotic resistance genes from neighboring cells. The resulting cells are resistant to phage attack due to immunity provided by the integrated prophage and they are able to exchange bacterial DNA with other cells containing a similar prophage via the transducing particles. Previously, transduction has been considered an accident in phage biology. Here we propose that rather than being an accident, generalized transduction is an evolved trait selected by some temperate phages to persist in rapidly-changing environments.

14 páginas, 7 figuras, 1 tabla. Contiene material suplementario., Bacillus phages use a communication system, termed "arbitrium," to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system., This work was supported by grant BIO2016-78571-P from the Ministerio de Economia y Competitividad (Spain) to A.M. and grants MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/S003835/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust 201531/Z/16/Z, and ERC-ADG-2014 Proposal n 670932 Dut-signal from European Union to J.R.P. X-ray diffraction data collection was supported by Diamond Light Source Block Allocation Group (BAG) proposal MX14739 and MX20229, and Spanish Synchrotron Radiation Facility ALBA proposal 2017072262 and 2018072901.

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., This work was funded by grants AGL2015-65673-R (Program of Science, Technology and Innovation 2013–2017), Proyecto Intramural CSIC 201770E016, and GRUPIN14-139 (FEDER EU funds, Principado de Asturias, Spain). L.F. was awarded a “Marie Curie Clarin-Cofund” grant. PG, BM and AR are members of the bacteriophage network FAGOMA and the FWO Vlaanderen funded “Phagebiotics” research community (WO.016.14).

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.

12 Páginas, 7 Figuras. Contiene información suplementaria en: http://dx.doi.org/10.1038/s41467-018-02949-y, Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches., This work was supported by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, BIO2014-53530-R, SAF2014-56716-REDT , and RTC-2015-3184-1. J.V. was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness.

En The ISME Journal. Heidelberg (Germany): Springer. Vol. 12, n. 9, pp. 2114-2128 (2018). Este artículo se encuentra disponible en la página web de la revista en la siguiente URL: https://www.nature.com/articles/s41396-018-0156-3 Phage-inducible chromosomal islands (PICIs) are a recently discovered family of pathogenicity islands that contribute substantively to horizontal gene transfer, host adaptation and virulence in Gram-positive cocci. Here we report that similar elements also occur widely in Gram-negative bacteria. As with the PICIs from Gram-positive cocci, their uniqueness is defined by a constellation of features: unique and specific attachment sites, exclusive PICI genes, a phage-dependent mechanism of induction, conserved replication origin organization, convergent mechanisms of phage interference, and specific packaging of PICI DNA into phage-like infectious particles, resulting in very high transfer frequencies. We suggest that the PICIs represent two or more distinct lineages, have spread widely throughout the bacterial world, and have diverged much more slowly than their host organisms or their prophage cousins. Overall, these findings represent the discovery of a universal class of mobile genetic elements.

12 páginas, 5 figuras, 3 tablas, The trimeric staphylococcal phage-encoded dUTPases (Duts) are signalling molecules that induce the cycle of some Staphylococcal pathogenicity islands (SaPIs) by binding to the SaPI-encoded Stl repressor. To perform this regulatory role, these Duts require an extra motif VI, as well as the Dut conserved motifs IV and V. While the apo form of Dut is required for the interaction with the Stl repressor, usually only those Duts with normal enzymatic activity can induce the SaPI cycle. To understand the link between the enzymatic activities and inducing capacities of the Dut protein, we analysed the structural, biochemical and physiological characteristics of the Dut80α D95E mutant, which loses the SaPI cycle induction capacity despite retaining enzymatic activity. Asp95 is located at the threefold central channel of the trimeric Dut where it chelates a divalent ion. Here, using state-of-the-art techniques, we demonstrate that D95E mutation has an epistatic effect on the motifs involved in Stl binding. Thus, ion binding in the central channel correlates with the capacity of motif V to twist and order in the SaPI-inducing disposition, while the tip of motif VI is disturbed. These alterations in turn reduce the affinity for the Stl repressor and the capacity to induce the SaPI cycle., This work was supported by grant BIO2013-42619-P and BIO2016-78571-P from the Ministerio de Economia y Competitividad (Spain) and grant Prometeo II/2014/029 from Valencian Government (Spain) to A.M., and grants MR/M003876/1 from the Medical Research Council (UK), BB/N002873/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), and ERC-ADG-2014 Proposal n° 670932 Dut-signal from EU to J.R.P. C.A. and J.R.C were supported by FPI BES-2014-068617] and FPU13/02880 predoctoral fellowships respectively. E.M. was supported by CSIC JAE-Doc postdoctoral contract (Programa ≪Junta para la Ampliación de Estudios≫) co-funded by the European Social Fund. X-ray diffraction data collection was supported by Diamond Light Source block allocation group (BAG) Proposal MX14739 and Spanish Synchrotron Radiation Facility ALBA Proposal 2015071314 and 2016071762.