A Study of the penetration of host cells by bacteriophages

The object of the work described in this thesis was to investigate the attachment, and penetration of the host cell walls and membranes by the minute bacteriophage ΦX 174. In an attempt to simplify the investigation, the interaction between viral particles and cell walls isolated from the bacterial host, Escherichia coli strain C, was examined. Conditions for maximum, irreversible adsorption to cell walls were shown to be in buffered solutions at pH 6–9, at 35–45°, in the presence of calcium or magnesium ions. It was established that these conditions gave efficient attachment to intact bacteria.
When phage particles labelled with 32P-phosphate became attached to isolated bacterial cell walls, up to 30% of the viral 32P was converted to a form accessible to the enzyme, pancreatic deoxyribonuclease (DNase). This indicated that some of the phage deoxyribonucleic acid (DNA) was released on attachment of virus to cell walls. At the same time, some of the adsorbed ΦX 174 virus were converted to particles which could be eluted from the cell walls by a solution of sodium borate, containing ethylene diamine tetra acetate (EDTA). The particles were resolved into two components on fractionation by sedimentation through sucrose density gradients. One component consisted of particles with a sedimentation coefficient of 45-50S (50S particles). When viral preparations, doubly labelled with 32P- and 35S- were used, comparison of the value of the 35S/32P ratio in the original phage and in the 50S particles, showed that the latter contained the same quantity of DNA as the original virus. In contrast to intact virus, however, the 50S particles were infectious towards bacterial spheroplasts, but not towards whole bacteria. Examination in the electron microscope of 50S particles, isolated by sedimentation through sucrose gradients, failed to reveal any gross contamination with cell wall or other material, which might have caused anomalous sedimentation behaviour. It was concluded that a conformational change in the arrangement of the viral protein sub-units occurred on attachment to the cell walls and was responsible for the changes in infectivity and sedimentation coefficient. Similar investigations on the second component, showed that it had a sedimentation coefficient of 70, was not infectious towards whole bacteria or spheroplasts, and h&d; a reduced DNA complement. The particles appeared to be identical with the "top component" previously identified by others in preparations of ΦX 174 (e.g. Sinsheirner R.L., (1959) J. Mol. Biol. 1:37).
Attachment of ΦX 174 to intact, starved bacteria suspended in buffer also resulted in the formation of 50S and 70S particles, which were eluted from the bacteria by sodium borate and EDTA. After prolonged incubation (30 minutes) of virus with either cell walls, or intact bacteria, only the 70S component could be detected in eluates made with borate-EDTA solution.
When the viral material eluted from cell walls was treated with DNase there was no loss in infectivity detectable in the bacterial spheroplast assay. It was concluded that all of the infectivity resided in the 50S component, and none in the viral DNA released on attachment of the phage particles.
The results show that changes occur in ΦX 174 particles when these attach to isolated cell walls or to non-metabolising bacteria. The possible role of the 50S component in the natural infectious process could not be determined from the experiments described here. Its properties suggest that it contains a hitherto unrecognised particle which can be formed by conformational changes in whole intact ΦX 174.