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Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

Bacteriophage L, a P22-like phage ofSalmonella entericasv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems ofSalmonella. We report the complete genome sequences of bacteriophage LcI−4013−am43 and LcII−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 71 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 genes for superinfection exclusion (sieAandsieB) 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-convertinggtrABCoperon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

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).

Linking morphological studies with molecular phylogeny is important to understanding cryptic speciation and the evolution of host-parasite relationships. Haemosporidian parasites of the Australo-Papuan bird family Artamidae are relatively unstudied. Only one parasite species from the subfamily Cracticinae has been described, and this was based solely on morphological description. This is despite many Cracticinae species being easily observed and abundant over large ranges and in close proximity to human populations. We used morphological and molecular methods to describe a new Haemoproteus species (H. bukaka sp. nov.) from an endemic Butcherbird host (Cracticus louisiadensis) in a relatively unstudied insular area of high avian endemism (Papua New Guinea's Louisiade Archipelago). Phylogenetic reconstructions using parasite cyt-b gene sequences placed the proposed Haemoproteus bukaka sp. nov. close to other host-specialist Haemoproteus species that infect meliphagid honeyeater hosts in the region, e.g. H. ptilotis. Distinct morphological characters of this haemosporidian include macrogametocytes with characteristic large vacuoles opposing a subterminal nucleus on the host cell envelope. Among 27 sampled individuals, prevalence of H. bukaka sp.nov. was high (74 % infection rate) but strongly variable across four islands in the archipelago (ranging from 0 to 100 % prevalence). Parasitaemia levels were low across all infected individuals (0.1-0.6 %). We suspect host density may play a role in maintaining high prevalence given the close proximity and similar physical environments across islands. The findings are discussed in the context of the host genus Cracticus and theory relating to parasite-host evolution and its conservation implications in Papua New Guinea.

Bacteriophage P22 infects Salmonella enterica by injecting its genetic material through the cell envelope. During infection, a specialized tail needle, gp26, is injected into the host, likely piercing a hole in the host cell envelope. The 2.1-Å crystal structure of gp26 reveals a 240-Å elongated protein fiber formed by two trimeric coiled-coil domains interrupted by a triple β-helix.The N terminus of gp26 plugs the portal protein channel, retaining the genetic material inside the virion. The C-terminal tip of the fiber exposes β-hairpins with hydrophobic tips similar to those seen in class II fusion peptides. The α-helical core connecting these two functionally polarized tips presents four trimerization octads with consensus sequence IXXLXXXV. The slender conformation of the gp26 fiber minimizes the surface exposed to solvent, which is consistent with the idea that gp26 traverses the cell envelope lipid bilayers.

The tail needle, gp26, is a highly stable homo-trimeric fiber found in the tail apparatus of bacteriophage P22. In the mature virion, gp26 is responsible for plugging the DNA exit channel, and likely plays an important role in penetrating the host cell envelope. In this article, we have determined the 1.98 A resolution crystal structure of gp26 bound to xenon gas. The structure led us to identify a calcium and a chloride ion intimately bound at the interior of alpha-helical core, as well as seven small cavities occupied by xenon atoms. The two ions engage in buried polar interactions with gp26 side chains that provide specificity and register to gp26 helical core, thus enhancing its stability. Conversely, the distribution of xenon accessible cavities correlates well with the flexibility of the fiber observed in solution and in the crystal structure. We suggest that small internal cavities in gp26 between the helical core and the C-terminal tip allow for flexible swinging of the latter, without affecting the overall stability of the protein. The C-terminal tip may be important in scanning the bacterial surface in search of a cell-envelope penetration site, or for recognition of a yet unidentified receptor on the surface of the host.

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of geneXXXIIresulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32−particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.

Reinfection of Escherichia coli with the bacterial virus T4 causes modifications of the properties of the host cell envelope during the preeplicative phase of the lytic cycle. These changes include altered densities cell enveloped and their subfractions, morphological modifications of membrane vesicles, and association of newly synthesized proteins with the host cell envelope. Polypeptide analysis by high resolution electrophoresis on polyacrylamide slab gels in dodecyl sulfate revealed that most of some 30 prereplicative phage-coded polypeptides are attached to this structure. Different means of cell disruption and selective extraction procedures, such as variations of ionic strength, removal of divalent cations, and the addition of chaotropic agents or detergents were used to study the characteristics of these attachments. Many proteins appeared to be artifactually absorbed or weakly bound to the envelope, Separation of cell walls from plasma membranes showed that all of the tightly bound proteins were associated withthe cell membrane fraction. The partitioning of phage proteins between the different fractions was monitored using 12 polypeptides which were identified as products of distinct phage genes. Of these, 8 were eliminated as potential membrane markers. Four polypeptides, the products of genes rIIA, rIIB, 39, and 52 were operationally defined as membrane proteins. The number of molecules of the 12 identified phage gene products, synthesized during a single lytic cycle, was determined. The results allowed the estimation of the concentration in the membrane of those proteins which were found to be quantitatively associated with that structure. Association of phage proteins with the cell envelope was found to be unaffected by mutations in any of the identified phage polypeptides.