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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.

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.

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.

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.