A new approach to testing the efficacy of drinking water disinfectants.

New disinfection procedures are being developed and proposed for use in drinking-water production. Authorising their use requires an effective test strategy that can simulate conditions in practice. For this purpose, we developed a test rig working in a flow-through mode similar to the disinfection procedures in waterworks, but under tightly defined conditions, including very short contact times. To quantify the influence of DOC, temperature and pH on the efficacy of two standard disinfectants, chlorine and chlorine dioxide, simulated use tests were systematically performed. This test rig enabled quantitative comparison of the reduction of four test organisms, two viruses and two bacteria, in response to disinfection. Chlorine was substantially more effective against Enterococcus faecium than chlorine dioxide whereas the latter was more effective against the bacteriophage MS2, especially at pH values of >7.5 at which chlorine efficacies already decline. Contrary to expectation, bacteria were not generally reduced more quickly than viruses. Overall, the results confirm a high efficacy of chlorine and chlorine dioxide, validating them as standard disinfectants for assessing the efficacy of new disinfectants. Furthermore, these data demonstrate that the test rig is an appropriate tool for testing new disinfectants as well as disinfection procedures.

Mechanism of membranous tunnelling nanotube formation in viral genome delivery.

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.

Optimal preparation and purification of PRD1-like bacteriophages for use in environmental fate and transport studies.

Bacteriophages are bacterial viruses with unique characteristics that make them excellent surrogates for mammalian pathogenic viruses in environmental studies. Simple and reliable methodologies for isolation, detection, characterization and enumeration of somatic and F-specific bacteriophage are available in the literature. Limited information or methods are available for producing high-titer purified phage suspensions for studying microbial transport and survival in natural and engineered environments. This deficiency arises because most research on the production of high-titer phage suspensions was completed over half a century ago and more recent advances on these methods have not been compiled in a single publication. We present a review of the available methods and new data on the propagation, concentration and purification of two bacteriophage host systems (somatic PRD1/Salmonella thyphimurium and F-specific PR772/Escherichia coli) that are commonly utilized in laboratory and field-scale assessments of subsurface microbial transport and survival. The focus of the present study is to recommend the approach(es) that will ensure maximum bacteriophage yields while optimizing suspension purification (i.e. avoiding modification of surface charge of the phage capsids and/or inadvertent introduction of dissolved organic matter to the study system).

Drastically lowering the titer of waterborne bacteriophage PRD1 by exposure to immobilized hydrophobic polycations.

Decrease in the titer of bacteriophage PRD1 (a model of animal adenoviruses) in aqueous solutions caused by the presence of systematically chemically derivatized surfaces was kinetically investigated. The greatest loss of infectivity--up to a 4-log reduction in the titer--was observed with immobilized hydrophobic polyethylenimine-based and dendrimer-based polycations.

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.

The lytic enzyme of bacteriophage PRD1 is associated with the viral membrane.

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble beta-1,4-N-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.