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Viruses SH1 and P23-77, infecting archaeal Haloarcula species and bacterial Thermus species, respectively, were recently designated to form a novel viral lineage. In this study, the lineage is expanded to archaeal Halomicrobium and bacterial Meiothermus species by analysis of five genome-integrated elements that share the core genes with these viruses. doi: 10.1128/JB.00124-10

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TACI25 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.

The temperate double-stranded DNA bacteriophage Bam35 infects gram-positive Bacillus thuringiensis cells. Bam35 has an icosahedral protein coat surrounding the viral membrane that encloses the linear 15-kbp DNA genome. The protein coat of Bam35 uses the same assembly principle as that of PRD1, a lytic bacteriophage infecting gram-negative hosts. In this study, we dissected the process of Bam35 entry into discrete steps: receptor binding, peptidoglycan penetration, and interaction with the plasma membrane (PM). Bam35 very rapidly adsorbs to the cell surface, and N-acetyl-muramic acid is essential for Bam35 binding. Zymogram analysis demonstrated that peptidoglycan-hydrolyzing activity is associated with the Bam35 virion. We showed that the penetration of Bam35 through the PM is a divalent-cation-dependent process, whereas adsorption and peptidoglycan digestion are not.

PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of [K.sup.+] from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the [K.sup.+] efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only ~5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.

Bam35, a temperate double-stranded DNA bacteriophage with a 15-kb linear genome, infects gram-positive Bacillus thuringiensis cells. Bam35 morphology and genome organization resemble those of PRD1, a lytic phage infecting gram-negative bacteria. Bam35 and PRDI have an outer protein coat surrounding a membrane that encloses the viral DNA. We used electrochemical methods to investigate physiological changes of the lysogenic and nonlysogenic hosts during Bam35 DNA entry and host cell lysis. During viral DNA entry, there was an early temporal decrease of membrane voltage associated with [K.sup.+] efflux that took place when either lysogenic or nonlysogenic hosts were infected. Approximately 40 min postinfection, a second strong [K.sup.+] efflux was registered that was proposed to be associated with the insertion of holin molecules into the plasma membrane. This phenomenon occurred only when nonlysogenic cells were infected. Lysogenic hosts rarely were observed entering the lytic cycle as demonstrated by thin-section electron microscopy.

The icosabedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane. It is the only representative of the Corticoviridae family. How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marine Pseudoalteromonas host has been an intriguing question. Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated. During the infection process, the host cell outer membrane becomes transiently permeable to lipophillc gramicidin D molecules proposing fusion with the viral membrane. One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration. Entry of the virion also causes a limited depolarization of the cytoplasmic membrane. These phenomena differ considerably from those observed in the entry process of bacteriophage PRDI, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.

Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.

Bacteriophage PM2 is the only described member of the Corticoviridae family. It is an icosahedral dsDNA virus with a membrane residing underneath the protein coat. PM2 infects some gram-negative Pseudoalteromonas spp. In the present study, we mapped the viral promoters and showed that the PM2 genome consists of three operons. Four new virus genes were assigned based on their function in transcription. Proteins P15 and P16 are shown to repress early transcription, and proteins P13 and P14 are shown to activate late transcription events. The early regulatory region, containing genes for proteins P15 and P16, as well as the newly identified early promoter region in PM2, has significant sequence similarity with the Pseudoalteromonas pAS28 plasmid. P14, the transcription activator for the structural genes, has a zinc finger motif homologous to archaeal and eukaryotic TFIIS-type regulatory factors.

The nucleocapsid shell subunit (P8) of bacteriophage phi6, an enveloped dsRNA bacteriophage of Pseudomonas syringae, and its assembly are discussed. The regions of P8 involved in subunit/subunit interactions in the intact shell undergo rapid exchanges, likely because of local unfolding events with low activation barriers.

Results show that purified phage protein P2 is monomeric, has a receptor binding constant of 0.20 nM, interferes with binding of pRD1 bacteriophage to the receptor and ensures proper DNA insertion. A hypothesis for the manner in which DNA is ejected from the bacteriophage is presented.

Viral evolution as revealed by human adenovirus coat protein structures and bacteriophage PRD1 is discussed. The unusual PRD1 has a membrane beneath the outer icosahedral protein coat. Each of three interlocking subunits in the major coat protein has two 8-stranded viral jelly rolls that are normal for the viral capsid. The P3 molecule, which is the major coat protein, is much like hexon, the equivalent in human adenovirus. The striking similarities strongly suggest evolutionary relationship.

IncP-group plasmids are broad-host-range plasmids carrying antibiotic resistance determinants. The mating pair formation (Mpf) complex is a receptor for IncP-specific phages such as PRD1. A phage receptor saturation technique was used to quantify the Mpf complexes on the cell surface. Mpf complex was shown to increase cell envelope permeability to lipophilic compounds and ATP.

The double-stranded DNA bacterial virus PRD1 has an internal membrane and a protein-primed replication mechanism. The effects of PRD1 adsorption and DNA entry on the magnitudes of host cell Delta Psi and K+ gradients were studied using ion-selective electrodes and a potentiometric monitoring system. PRD1 DNA cell penetration was characterized by lower intracellular K+ concentration, raised OM permeability to lipophilic compounds and raise membrane voltage.

The binding of bacteriophage PRD1 to cell surface receptors coded by the IncP-type plasmid RP1 was investigated. The adsorption and electron microscopic results indicate that the surfaces of Salmonella typhimurium(RP1) and Escherichia coli(RP1) cells contain approximately 50 and 20 PRD1 binding sites, respectively. It was also shown that PRD1 resistance is due to Tn5 insertion into RP1 and that all of the PRD1-resistant transposon mutants are transfer deficient.

The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection. [ABSTRACT FROM AUTHOR]

P3 has been imaged with X-ray crystallography to reveal a trimeric molecule with strikingly similar characteristics to hexon, the major coat protein of adenovirus. The structure of native P3 has now been extended to 1.65 Å resolution (Rwork = 19.0% and Rfree = 20.8%). The new high-resolution model shows that P3 forms crystals through hydrophobic patches solvated by 2-methyl-2,4-pentanediol molecules. It reveals details of how the molecule's high stability may be achieved through ordered solvent in addition to intra- and intersubunit interactions. Of particular importance is a 'puddle' at the top of the molecule containing a four-layer deep hydration shell that cross-links a complex structural feature formed by 'trimerization loops'. These loops also link subunits by extending over a neighbor to reach the third subunit in the trimer. As each subunit has two eight-stranded viral jelly rolls, the trimer has a pseudo-hexagonal shape to allow close packing in its 240 hexavalent capsid positions. Flexible regions in P3 facilitate these interactions within the capsid and with the underlying membrane. A selenomethionine P3 derivative, with which the structure was solved, has been refined to 2.2 Å resolution (Rwork = 20.1% and Rfree = 22.8%). The derivatized molecule is essentially unchanged, although synchrotron radiation has the curious effect of causing it to rotate about its threefold axis. P3 is a second example of a trimeric 'double-barrel' protein that forms a stable building block with optimal shape for constructing a large icosahedral viral capsid. A major difference is that hexon has long variable loops that distinguish different adenovirus species. The short loops in P3 and the severe constraints of its various interactions explain why the PRD1 family has highly conserved coat proteins. [ABSTRACT FROM AUTHOR]

Focuses on the purification and characterization of the assembly factor P17 of the lipid-containing bacteriophage PRD1. Information on assembly factors; Details on the high heat stability of assembly factor P17; Analysis of the structure of the assembly factor.

Studies the functional organization of the bacteriophage PRD1 genome. Replication through a protein-primed mechanism; Chloramphenicol acetyltransferase; Identification of promoter regions controlling PRD1 transcription.