P2-like Phage Research Articles

Bacteriophage P22 SieA-mediated superinfection exclusion.

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC, sieA, and sieB genes. Here we report that the P22-encoded SieA protein is necessary and sufficient for exclusion by the SieA system and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants that overcome the SieA block were isolated, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single-amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in phage target specificity. Our data strongly suggest a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.IMPORTANCEThe ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called "superinfection exclusion." A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection.

Genomic Analysis of Two Cold-Active Pseudoalteromonas Phages Isolated from the Continental Shelf in the Arctic Ocean.

Cold-active bacteriophages are bacterial viruses that infect and replicate at low temperatures (≤4 °C). Understanding remains limited of how cold-active phage-host systems sustain high viral abundance despite the persistently low temperatures in pelagic sediments in polar seas. In this study, two Pseudoalteromonas phages, ACA1 and ACA2, were isolated from sediment core samples of the continental shelf in the western Arctic Ocean. These phages exhibited successful propagation at a low temperature of 1 °C and displayed typical myovirus morphology with isometric icosahedral heads and contractile tails. The complete genome sequences of phages ACA1 and ACA2 were 36,825 bp and 36,826 bp in size, respectively, sharing almost the same gene content. These are temperate phages encoding lysogeny-related proteins such as anti-repressor, immunity repressor and integrase. The absence of cross-infection between the host strains, which were genomically distinct Pseudoalteromonas species, can likely be attributed to heavy divergence in the anti-receptor apparently mediated by an associated diversity-generating retroelement. HHpred searching identified genes for all of the structural components of a P2-like phage (family Peduoviridae), although the whole of the Peduoviridae family appeared to be divided between two anciently diverged tail modules. In contrast, Blast matching and whole genome tree analysis are dominated by a nonstructural gene module sharing high similarity with Pseudoalteromonas phage C5a (founder of genus Catalunyavirus). This study expands the knowledge of diversity of P2-like phages known to inhabit Peudoalteromonas and demonstrates their presence in the Arctic niche.

Identification and characterization of P2-like bacteriophages of Yersinia pestis

Yersinia pestis is the cause of plague, historically known as the “Black Death”. Marmota himalayana in the Qinghai-Tibet Plateau (QTP) natural plague focus is the primary host in China. Although several phages originating from Y. pestis have been characterized. This is the first report of isolation of P2-like phages of Y. pestis from M. himalayana. In this study, the isolation and characterization of three P2-like phages of Y. pestis were reported, which were named as vB_YpM_22, vB_YpM_46 and vB_YpM_50. Comparative genome analysis revealed that vB_YpM_22, vB_YpM_46 and vB_YpM_50 are members of the nonlambdoid P2 family, and are highly similar and collinear with enterobacteriophage P2, plague diagnostic phage L-413C and enterobacteriophage fiAA91-ss. The role of LPS core structure of Y. pestis in the phages’ receptor was pinpointed. The findings of this study contribute an advance in our current knowledge of Y. pestis phages and will also play a key role in understanding the evolution of Y. pestis phages.

Complete Genome Sequences of Five Bacteriophages That Infect Enterobacteriales Hosts.

ABSTRACTFull genome sequences of five bacteriophages that were isolated from raw sewage samples and infect Enterobacteriales hosts are presented. Brookers is a P22-like Proteus phage, OddieOddie is a 9g-like Escherichia coli phage, Diencephelon is a Kp3-like Klebsiella phage, and Rgz1 and Lilpapawes are classic T4-like and T7-like virulent Proteus phages, respectively.

Phages and their satellites encode hotspots of antiviral systems

SummaryBacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E. coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements.

Introduction: the secret lives of microbial mobile genetic elements.

Introduction: the secret lives of microbial mobile genetic elements.

Lateral transduction is inherent to the life cycle of the archetypical Salmonella phage P22

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.

Genomic Characterization of a Prophage, Smhb1, That Infects Salinivibrio kushneri BNH Isolated from a Namib Desert Saline Spring.

Recent years have seen the classification and reclassification of many viruses related to the model enterobacterial phage P2. Here, we report the identification of a prophage (Smhb1) that infects Salinivibrio kushneri BNH isolated from a Namib Desert salt pan (playa). Analysis of the genome revealed that it showed the greatest similarity to P2-like phages that infect Vibrio species and showed no relation to any of the previously described Salinivibrio-infecting phages. Despite being distantly related to these Vibrio infecting phages and sharing the same modular gene arrangement as seen in most P2-like viruses, the nucleotide identity to its closest relatives suggest that, for now, Smhb1 is the lone member of the Peduovirus genus Playavirus. Although host range testing was not extensive and no secondary host could be identified for Smhb1, genomic evidence suggests that the phage is capable of infecting other Salinivibrio species, including Salinivibrio proteolyticus DV isolated from the same playa. Taken together, the analysis presented here demonstrates how adaptable the P2 phage model can be.

Complete Genome Sequence of Burkholderia gladioli Myophage Mana.

Burkholderia gladioli is known to cause respiratory tract infections in cystic fibrosis patients. Here, we describe the annotation of the 38,038-bp genome sequence of Mana, a P2-like phage of B. gladioli Understanding the genomic characteristics of phages infecting pathogens like B. gladioli can lead to advancements in phage therapy.

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI–40 13–am43 and L cII–101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 72 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (immI: ant, arc) or known genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ∼35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

Complete genomic sequence of a novel phytopathogenic Burkholderia phage isolated from fallen leaf compost

In contrast to most Burkholderia species, which affect humans or animals, Burkholderia glumae is a bacterial pathogen of plants that causes panicle blight disease in rice seedlings, resulting in serious damage to rice cultivation. Attempts to combat this disease would benefit from research involving a phage known to attack this type of bacterium. Some Burkholderia phages have been isolated from soil or bacterial species in the order Burkholderiales, but so far there has been no report of a complete genome nucleotide sequence of a phage of B. glumae. In this study, a novel phage, FLC5, of the phytopathogen B. glumae was isolated from leaf compost, and its complete genome nucleotide sequence was determined. The genome consists of a 32,090-bp circular DNA element and exhibits a phylogenetic relationship to members of the genus Peduovirus, with closest similarity to B. multivorans phage KS14. In addition to B. glumae, FLC5 was also able to lyse B. plantarii, a pathogen causing rice bacterial damping-off disease. This is the first report of isolation of a P2-like phage from phytopathogenic Burkholderia, determination of its complete genomic sequence, and the finding of its potential to infect two Burkholderia species: B. glumae and B. plantarii.

R-type bacteriocins of Xenorhabdus bovienii determine the outcome of interspecies competition in a natural host environment.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii (Xb-Sj), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath (xbpS1) or tail fibre (xbpH1) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii (Xsz-Sr), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj. Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr. To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj, suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta, while in competition with the xenorhabdicin-deficient strain (Xb-Sj:S1), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

Draft Genome Sequence of Escherichia coli UMB1353, Isolated from the Female Urinary Tract.

Here, we present the draft genome sequence of Escherichia coli UMB1353, isolated from a patient with a urinary tract infection. The sequence of this antibiotic-resistant E. coli strain contains one intact P2-like phage.

Characteristics of two myoviruses induced from the coastal photoheterotrophic bacterium Porphyrobacter sp. YT40.

In this study, we characterized two induced myoviruses from one marine photoheterotrophic bacterium Porphyrobacter sp. YT40 belonging to the Sphingomonadales family in Alphaproteobacteria. The genome sequence of prophage A is ∼36.9kb with an average GC content of 67.1%, and its core or functional genes are homologous to Mu or Mu-like phages. Furthermore, induced viral particles from prophage A show a knob-like neck structure, which is only found in bacteriophage Mu. The genome size of prophage B is ∼36.8kb with an average GC content of 65.3%. Prophage B contains a conserved gene cluster Q-P-O-N-M-L, which is unique in P2 phages. Induced viral particles from prophage B display an icosahedral head with a diameter of ∼55nm and a 130±5nm long contractile tail. To our knowledge, this is the first report that characterizes the induced P2-like phage in marine Alphaproteobacteria. Phylogeny analyses suggest that these two types of prophages are commonly found in sequenced bacteria of the Sphingomonadales family. This study sheds light on the ongoing interaction between marine bacteria and phages, and improves our understanding of bacterial genomic plasticity and evolution.

R18C is a new viable P2-like bacteriophage of rabbit origin infecting Citrobacter rodentium and Shigella sonnei strains

Here, we report a novel virulent P2-like bacteriophage, R18C, isolated from rabbit faeces, which, in addition to Escherichia coli K-12 strains, was able to be propagated on Citrobacter rodentium strain ICC169 and a range of Shigella sonnei strains with high efficiency of plating (EOP). It represents the first lytic bacteriophage originating from rabbit and the first infectious P2-like phage of animal origin. In the three characteristic moron-containing regions of P2-like phages, R18C contains genes with unknown function that have so far only been found in cryptic P2-like prophages.

A Hydrophobic Network: Intersubunit and Intercapsomer Interactions Stabilizing the Bacteriophage P22 Capsid.

Double-stranded DNA (dsDNA) tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here, we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold.IMPORTANCE Similarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra- and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra- and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.

The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy.

The major coat proteins of dsDNA tailed phages (order Caudovirales) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary 'decoration' (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

Lysogenic Conversion of the Phytopathogen Ralstonia solanacearum by the P2virus ϕRSY1.

A P2-like phage ϕRSY1 infecting the phytopathogen Ralstonia solanacearum was isolated and characterized. The 40-kb genome of ϕRSY1 showed high sequence similarity to the Ralstonia phage ϕRSA1 and the GMI1000 prophage ϕRSX. The major genomic differences between these phages were the different orientation of the int gene and the gene content close to the cosL. ϕRSY1 and ϕRSX use a 15-base 3′ portion of the serine tRNA(GGA) gene as attB, while ϕRSA1 uses a 45-base 3′ portion of the arginine tRNA(CCG) gene. The different orientation of int in the genomes means that the gene arrangements in the prophage states are reversed in ϕRSY1 and ϕRSA1. Several putative gene products of ϕRSY1 may affect the bacterium’s fitness. ϕRSY1 contains an open reading frame (ORF) that seems to encode a protein similar to Vgr in the type VI secretion system of various bacterial species. ϕRSY1 lysogens showed phenotypic changes including enhanced twitching motility, large colony formation, and easy aggregation of cells, suggesting involvement of this ORF in the changes. In view of these phage gene arrangements, we surveyed prophages in the genomes of various R. solanacearum strains and found that the P2-like phages of R. solanacearum (14 phages) consist of two major groups: the ϕRSY1-type and the ϕRSA1-type. The relationships and evolution of these P2-like phages inferred from our data are discussed in detail.

Complete Genome Sequence of Bacteriophage SEN8, a Temperate Phage Isolated from Salmonella enterica subsp. salamae.

ABSTRACTA temperate phage, SEN8, having a broad activity against pathogenic Salmonella serovars, was isolated from Salmonella enterica subsp. salamae strain Sen8. The complete genome sequence of phage SEN8 was determined (35,203 bp) and showed relatedness to P2-like phages (Salmonella phages Fels-2 and RE-2010).

Inducible Prophage Mutant of Escherichia coli Can Lyse New Host and the Key Sites of Receptor Recognition Identification.

The use of bacteriophages as therapeutic agents is hindered by their narrow and specific host range, and by a lack of the knowledge concerning the molecular mechanism of receptor recognition. Two P2-like coliphages, named P88 and pro147, were induced from Escherichia coli strains K88 and DE147, respectively. A comparison of the genomes of these two and other P2-like coliphages obtained from GenBank showed that the tail fiber protein genes, which are the key genes for receptor recognition in other myoviridae phages, showed more diversity than the conserved lysin, replicase, and terminase genes. Firstly, replacing hypervariable region 2 (HR2: amino acids 716–746) of the tail fiber protein of P88 with that of pro147 changed the host range of P88. Then, replacing six amino acids in HR2 with the corresponding residues from pro147 altered the host range only in these mutants with changes at position 730 (leucine) and 744 (glutamic acid). Thus, we predicted that these amino acids are vital to establish the host range of P88. This study provided a vector of lysogenic bacteria that could be used to change or expand the phage host range of P88. These results illustrated that, in P2-like phage P88, the tail fiber protein determined the receptor recognition. Amino acids 716–746 and the amino acids at positions 730 and 744 were important for receptor recognition.