The translocation of genetic material from the viral capsid to the cell is an essential part of the viral infection process. Whether the energetics of this process is driven by the energy stored within the confined nucleic acid or cellular processes pull the genome into the cell has been the subject of discussion. However, in vitro studies of genome ejection have been limited to a few head-tailed bacteriophages with a double-stranded DNA genome. Here we describe a DNA release system that operates in an archaeal virus. This virus infects an archaeon Haloarcula hispanica that was isolated from a hypersaline environment. The DNA-ejection velocity of His1, determined by single-molecule experiments, is comparable to that of bacterial viruses. We found that the ejection process is modulated by the external osmotic pressure (polyethylene glycol (PEG)) and by increased ion (Mg(2+) and Na(+)) concentration. The observed ejection was unidirectional, randomly paused, and incomplete, which suggests that cellular processes are required to complete the DNA transfer.

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 ؉ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K ؉ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only ϳ5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.For most bacteriophages, host cell lysis requires, at a minimum, two proteins: an endolysin and a holin. Endolysins are small enzymes that degrade cell wall peptidoglycan. These enzymes fall into four groups depending on their activity, which is directed against the three different covalent linkages that maintain the integrity of the cell wall: (i) glycosylase and (ii) transglycosylase activities targeting the glycosidic linkages, and (iii) amidase and (iv) endopeptidase activities targeting the oligopeptide cross-linkages (39). Most endolysins characterized to date have no signal sequence and therefore accumulate in the cytosol during infection. Holins are small hydrophobic integral membrane proteins that permeabilize the cytoplasmic membrane (CM) and allow the endolysins to attack the peptidoglycan (20,38,39). In addition, holins may work as activators of the endolysins (39). Holins are grouped into two classes based on their primary structure. Class I holins, such as bacteriophage S protein, generally have more than 95 residues and form three transmembrane helices. Class II holins are smaller (65 to 95 residues) and form two transmembrane helices (18, 39).Holin-dependent lysis systems are highly regulated. The precise temporal regulation likely is dependent on the energy state of the CM, because adding a metabolic poison (e.g