Phage-inducible Chromosomal Islands Research Articles

Phage-inducible chromosomal minimalist islands (PICMIs), a novel family of small marine satellites of virulent phages

Phage satellites are bacterial genetic elements that co-opt phage machinery for their own dissemination. Here we identify a family of satellites, named Phage-Inducible Chromosomal Minimalist Islands (PICMIs), that are broadly distributed in marine bacteria of the family Vibrionaceae. A typical PICMI is characterized by reduced gene content, does not encode genes for capsid remodelling, and packages its DNA as a concatemer. PICMIs integrate in the bacterial host genome next to the fis regulator, and encode three core proteins necessary for excision and replication. PICMIs are dependent on virulent phage particles to spread to other bacteria, and protect their hosts from other competitive phages without interfering with their helper phage. Thus, our work broadens our understanding of phage satellites and narrows down the minimal number of functions necessary to hijack a tailed phage.

The heterogenous and diverse population of prophages in Mycobacterium genomes.

Bacterial genomes commonly carry one or more integrated prophages, although prophage prevalence varies greatly in different bacterial genera and species. We have used the recently developed DEPhT software to discover prophages present in sequenced Mycobacterium genomes. Prophages are unevenly distributed among mycobacteria; Mycobacterium tuberculosis strains are devoid of intact prophages, whereas 75% of Mycobacterium abscessus-chelonae strains carry at least one prophage, and some have up to nine. Newly discovered prophage sequences are generally distinct from those of lytic phages isolated using Mycobacterium smegmatis as a host, although assembly of over 3,800 phage and prophage genomes into clusters based on shared sequence similarity yields seven clusters containing both phage and prophage genomes. M. abscessus prophages use a usually large repertoire of attB sites for prophage integration, and new integration-proficient vectors demonstrate the functionality of integration systems identified in silico and provide tools useful for advancing M. abscessus genetics. Genomic analysis reveals additional accessory and likely mobile parts of Mycobacterium genomes including defective prophages, prophage-like regions, and satellite elements proposed to be candidate phage-inducible chromosomal islands (PICIs). M. abscessus prophages and PICIs are unusual in that they often encode phage-encoded ESX-secreted toxin (PEST) systems that are located adjacent to the phage integration functions and are lysogenically expressed and likely provide a fitness advantage to their host. IMPORTANCE Mycobacterium species include several human pathogens and mycobacteriophages show potential for therapeutic use to control Mycobacterium infections. However, phage infection profiles vary greatly among Mycobacterium abscessus clinical isolates and phage therapies must be personalized for individual patients. Mycobacterium phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced Mycobacterium strains. The prophages are numerous and diverse, especially in M. abscessus genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages.

Phage-Inducible Chromosomal Islands as a Diagnostic Platform to Capture and Detect Bacterial Pathogens.

Phage-inducible chromosomal islands (PICIs) are a family of phage satellites that hijack phage components to facilitate their mobility and spread. Recently, these genetic constructs are repurposed as antibacterial drones, enabling a new toolbox for unorthodox applications in biotechnology. To illustrate a new suite of functions, the authors have developed a user-friendly diagnostic system, based upon PICI transduction to selectively enrich bacteria, allowing the detection and sequential recovery of Escherichia coli and Staphylococcus aureus. The system enables high transfer rates and sensitivities in comparison with phages, with detection down to ≈50CFUmL-1 . In contrast to conventional detection strategies, which often rely on nucleic acid molecular assays, and cannot differentiate between dead and live organisms, this approach enables visual sensing of viable pathogens only, through the expression of a reporter gene encoded in the PICI. The approach extends diagnostic sensing mechanisms beyond cell-free synthetic biology strategies, enabling new synthetic biology/biosensing toolkits.

Identification and characterization of thousands of bacteriophage satellites across bacteria.

Bacteriophage-bacteria interactions are affected by phage satellites, elements that exploit phages for transfer between bacteria. Satellites can encode defense systems, antibiotic resistance genes, and virulence factors, but their number and diversity are unknown. We developed SatelliteFinder to identify satellites in bacterial genomes, detecting the four best described families: P4-like, phage inducible chromosomal islands (PICI), capsid-forming PICI, and PICI-like elements (PLE). We vastly expanded the number of described elements to ∼5000, finding bacterial genomes with up to three different families of satellites. 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. Phylogenies of core genes in PICI and cfPICI indicate independent evolution of their hijacking modules. There are few other homologous core genes between other families of satellites, and even fewer homologous to phages. Hence, phage satellites are ancient, diverse, and probably evolved multiple times independently. Given 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.

PICI thieves: Molecular piracy and cooperation

Phage-inducible chromosomal islands (PICIs) steal structural proteins from helper phages. In two related studies, Penadés and coworkers reveal that PICIs are not parasites but mutualists. Some PICIs mobilize defense systems that restrict niche competitors, while other PICIs encode their own capsids and steal helper phage tails without affecting their fitness.

A widespread family of phage-inducible chromosomal islands only steals bacteriophage tails to spread in nature

Phage satellites are genetic elements that couple their life cycle to that of helper phages they parasitize, interfering with phage packaging through the production of small capsids, where only satellites are packaged. So far, in all analyzed systems, the satellite-sized capsids are composed of phage proteins. Here, we report that a family of phage-inducible chromosomal islands (PICIs), a type of satellites, encodes all the proteins required for both the production of small-sized capsids and the exclusive packaging of the PICIs into these capsids. Therefore, this new family, named capsid-forming PICIs (cf-PICIs), only requires phage tails to generate PICI particles. Remarkably, the representative cf-PICIs are produced with no cost from their helper phages, suggesting that the relationship between these elements is not parasitic. Finally, our phylogenomic studies indicate that cf-PICIs are present both in gram-positive and gram-negative bacteria and have evolved at least three times independently to spread in nature.

Bacterial immunity: Mobile genetic elements are hotspots for defence systems

A new study reports that phage-inducible chromosomal islands (PICIs) are hotspots of defence systems against phages, other PICIs and plasmids. This discovery highlights how competition between mobile genetic elements shapes bacterial defence gene repertoires and helps to better understand how defence systems are exchanged among bacteria.

Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors

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.

Enteric Chromosomal Islands: DNA Packaging Specificity and Role of λ-like Helper Phage Terminase.

The phage-inducible chromosomal islands (PICIs) of Gram-negative bacteria are analogous to defective prophages that have lost the ability to propagate without the aid of a helper phage. PICIs have acquired genes that alter the genetic repertoire of the bacterial host, including supplying virulence factors. Recent work by the Penadés laboratory elucidates how a helper phage infection or prophage induction induces the island to excise from the bacterial chromosome, replicate, and become packaged into functional virions. PICIs lack a complete set of morphogenetic genes needed to construct mature virus particles. Rather, PICIs hijack virion assembly functions from an induced prophage acting as a helper phage. The hijacking strategy includes preventing the helper phage from packaging its own DNA while enabling PICI DNA packaging. In the case of recently described Gram-negative PICIs, the PICI changes the specificity of DNA packaging. This is achieved by an island-encoded protein (Rpp) that binds to the phage protein (TerS), which normally selects phage DNA for packaging from a DNA pool that includes the helper phage and host DNAs. The Rpp–TerS interaction prevents phage DNA packaging while sponsoring PICI DNA packaging. Our communication reviews published data about the hijacking mechanism and its implications for phage DNA packaging. We propose that the Rpp–TerS complex binds to a site in the island DNA that is positioned analogous to that of the phage DNA but has a completely different sequence. The critical role of TerS in the Rpp–TerS complex is to escort TerL to the PICI cosN, ensuring appropriate DNA cutting and packaging.

Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis.

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.

Staphylococcal phages and pathogenicity islands drive plasmid evolution

Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilisable and non-transmissible plasmids coincides with the average size of phages (~40 kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.

Development and Application of a Prophage Integrase Typing Scheme for Group B Streptococcus.

Group B Streptococcus (GBS) is a gram-positive pathogen mainly affecting humans, cattle, and fishes. Mobile genetic elements play an important role in the evolution of GBS, its adaptation to host species and niches, and its pathogenicity. In particular, lysogenic prophages have been associated with a high virulence of certain strains and with their ability to cause invasive infections in humans. It is therefore important to be able to accurately detect and classify prophages in GBS genomes. Several bioinformatic tools for the identification of prophages in bacterial genomes are available on-line. However, genome searches for most of these programs are affected by the composition of their reference database. Lack of databases specific to GBS results in failure to recognize all prophages in the species. Additionally, performance of these programs is affected by genome fragmentation in the case of draft genomes, leading to underestimation of the number of phages. They also prove impractical when dealing with large genome datasets and they do not offer a quick way of classifying bacteriophages. We developed a GBS-specific method to screen genome assemblies for the presence of prophages and to classify them based on a reproducible typing scheme. This was achieved through an extensive search of a vast number of high-quality GBS sequences (n = 572) originating from different host species and countries in order to build a database of phage integrase types, on which the scheme is based. The proposed typing scheme comprises 12 integration sites and sixteen prophage integrase types, including multiple subtypes per integration site and integrase genes that were not site-specific. Two putative phage-inducible chromosomal islands (PICI) and their insertion sites were also identified during the course of these analyses. Phages were common and diverse in all major clonal complexes associated with human disease and detected in isolates from every animal species and continent included in the study. This database will facilitate further work on the prevalence and role of prophages in GBS evolution, and identifies the roles of PICIs in GBS and of prophage in hypervirulent ST283 as areas for further research.

Assembly-free single-molecule sequencing recovers complete virus genomes from natural microbial communities.

Viruses are the most abundant biological entities on Earth and play key roles in host ecology, evolution, and horizontal gene transfer. Despite recent progress in viral metagenomics, the inherent genetic complexity of virus populations still poses technical difficulties for recovering complete virus genomes from natural assemblages. To address these challenges, we developed an assembly-free, single-molecule nanopore sequencing approach, enabling direct recovery of complete virus genome sequences from environmental samples. Our method yielded thousands of full-length, high-quality draft virus genome sequences that were not recovered using standard short-read assembly approaches. Additionally, our analyses discriminated between populations whose genomes had identical direct terminal repeats versus those with circularly permuted repeats at their termini, thus providing new insight into native virus reproduction and genome packaging. Novel DNA sequences were discovered, whose repeat structures, gene contents, and concatemer lengths suggest they are phage-inducible chromosomal islands, which are packaged as concatemers in phage particles, with lengths that match the size ranges of co-occurring phage genomes. Our new virus sequencing strategy can provide previously unavailable information about the genome structures, population biology, and ecology of naturally occurring viruses and viral parasites.

Rebooting Synthetic Phage-Inducible Chromosomal Islands: One Method to Forge Them All.

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.

The structure of a polygamous repressor reveals how phage-inducible chromosomal islands spread in nature

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.

Vibrio cholerae Outer Membrane Vesicles Inhibit Bacteriophage Infection.

Novel preventatives could help in efforts to limit Vibrio cholerae infection and the spread of cholera. Bacteriophage (phage) treatment has been proposed as an alternative intervention, given the rapid replication of virulent phages, prey specificity, and relative ease of finding new virulent phages. Phage tropism is dictated in part by the presence of phage receptors on the bacterial surface. While many phages that can kill V. cholerae have been isolated, whether this pathogen is able to defend itself by neutralizing phage binding is unknown. Here, we show that secreted outer membrane vesicles (OMVs) act as a defense mechanism that confers protection to V. cholerae against phage predation and that this OMV-mediated inhibition is phage receptor dependent. Our results suggest that phage therapy or prophylaxis should take into consideration the production of OMVs as a bacterial decoy mechanism that could influence the outcome of phage treatment.IMPORTANCE Phages have been increasingly recognized for the significance of their interactions with bacterial cells in multiple environments. Bacteria use myriad strategies to defend against phage infection, including restriction modification, abortive infection, phase variation of cell surface receptors, phage-inducible chromosomal islands, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems. The data presented here suggest that the apparently passive process of OMV release can also contribute to phage defense. By considering the effect of OMVs on V. cholerae infection by three unique virulent phages, ICP1, ICP2, and ICP3, we show that, in vitro, a reproducible reduction in bacterial killing is both dose and phage receptor dependent. This work supports a role for OMVs as natural decoys to defend bacteria from phage predation.

Phage-inducible chromosomal islands are ubiquitous within the bacterial universe

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.

Staphylococcus edaphicus sp. nov., Isolated in Antarctica, Harbors the mecC Gene and Genomic Islands with a Suspected Role in Adaptation to Extreme Environments.

Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources comprising stone fragments and sandy soil in James Ross Island, Antarctica. Here, we describe properties of a novel species of the genus Staphylococcus that has a 16S rRNA gene sequence nearly identical to that of Staphylococcus saprophyticus However, compared to S. saprophyticus and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of <85% and inferred DNA-DNA hybridization of <30%. It forms a separate branch in the S. saprophyticus phylogenetic clade as confirmed by multilocus sequence analysis of six housekeeping genes, rpoB, hsp60, tuf, dnaJ, gap, and sod Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and key biochemical characteristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbors. In contrast to S. saprophyticus subsp. saprophyticus, the novel strains are pyrrolidonyl arylamidase and β-glucuronidase positive and β-galactosidase negative, nitrate is reduced, and acid produced aerobically from d-mannose. Whole-genome sequencing of the 2.69-Mb large chromosome revealed the presence of a number of mobile genetic elements, including the 27-kb pseudo-staphylococcus cassette chromosome mec of strain P5085T (ψSCCmecP5085), harboring the mecC gene, two composite phage-inducible chromosomal islands probably essential to adaptation to extreme environments, and one complete and one defective prophage. Both strains are resistant to penicillin G, ampicillin, ceftazidime, methicillin, cefoxitin, and fosfomycin. We hypothesize that antibiotic resistance might represent an evolutionary advantage against beta-lactam producers, which are common in a polar environment. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus edaphicus sp. nov. The type strain is P5085T (= CCM 8730T = DSM 104441T).IMPORTANCE The description of Staphylococcus edaphicus sp. nov. enables the comparison of multidrug-resistant staphylococci from human and veterinary sources evolved in the globalized world to their geographically distant relative from the extreme Antarctic environment. Although this new species was not exposed to the pressure of antibiotic treatment in human or veterinary practice, mobile genetic elements carrying antimicrobial resistance genes were found in the genome. The genomic characteristics presented here elucidate the evolutionary relationships in the Staphylococcus genus with a special focus on antimicrobial resistance, pathogenicity, and survival traits. Genes encoded on mobile genetic elements were arranged in unique combinations but retained conserved locations for the integration of mobile genetic elements. These findings point to enormous plasticity of the staphylococcal pangenome, shaped by horizontal gene transfer. Thus, S. edaphicus can act not only as a reservoir of antibiotic resistance in a natural environment but also as a mediator for the spread and evolution of resistance genes.

Analysis of the CRISPR-Cas system in bacteriophages active on epidemic strains of Vibrio cholerae in Bangladesh

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) are microbial nuclease systems involved in defense against phages. Bacteria also resist phages by hosting phage-inducible chromosomal islands (PICI) which prevent phage reproduction. Vibrio cholerae which causes cholera epidemics, interacts with numerous phages in the environment and in cholera patients. Although CRISPR-Cas systems are usually carried by bacteria and archea, recently V. cholerae specific ICP1 phages were found to host a CRISPR-Cas system that inactivates PICI-like elements (PLE) in V. cholerae. We analyzed a collection of phages and V. cholerae isolated during seasonal cholera epidemics in Bangladesh, to study the distribution, and recent evolution of the phage-encoded CRISPR-Cas system. Five distinct but related phages carrying the CRISPR-Cas system, and possible CRISPR-Cas negative progenitor phages were identified. Furthermore, CRISPR arrays in the phages were found to have evolved by acquisition of new spacers targeting diverse regions of PLEs carried by the V. cholerae strains, enabling the phages to efficiently grow on PLE positive strains. Our results demonstrate a continuing arms-race involving genetic determinants of phage-resistance in V. cholerae, and the phage-encoded CRISPR-Cas system in the co-evolution of V. cholerae and its phages, presumably fostered by their enhanced interactions during seasonal epidemics of cholera.

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.