Calcium ion-dependent entry of the membrane-containing bacteriophage PM2 into its Pseudoalteromonas host.

Marine bacteriophage PM2 infects gram-negative Pseudoalteromonas species and is currently the only assigned member of the Corticoviridae family. The icosahedral protein shell covers an internal protein-rich phage membrane that encloses the highly supercoiled dsDNA genome. In this study we investigated PM2 entry into the host. Our results indicate that PM2 adsorption to the host is dependent on the intracellular ATP concentration, while genome penetration through the cytoplasmic membrane depends on the presence of millimolar concentrations of calcium ions in the medium. In the absence of Ca(2+) the infection is arrested at the entry stage but can be rescued by the addition of Ca(2+). Interestingly, PM2 entry induces abrupt cell lysis if the host outer membrane is not stabilized by divalent cations. Experimental data described in this study in combination with results obtained previously allowed us to propose a sequential model describing the entry of bacteriophage PM2 into the host cells.

A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophage.

In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2.

Penetration of membrane-containing double-stranded-DNA bacteriophage PM2 into Pseudoalteromonas hosts.

The icosahedral 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 lipophilic 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 PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.