J L Van Etten

RNA polymerase activity associated with bacteriophage φ6 nucleocapsid

Summary The Pseudomonas phaseolicola bacteriophage φ6 nucleocapsid incorporated ATP, UTP, CTP and GTP into ssRNA products which sedimented at the same rate as heat-denatured small and medium dsRNA components. Hybridization of the separated ssRNA products with denatured dsRNA revealed that the small ssRNA hybridized only to small dsRNA and the medium ssRNA to the medium dsRNA. Synthesis of the large ssRNA was not observed. The RNA polymerase reaction proceeded through a replicative intermediate-like RNA probably via a semiconservative mechanism.

Mutual exclusion occurs in a Chlorella-like green alga inoculated with two viruses.

Progeny viruses resulting from dual inoculations with different and near-isogenic viruses of a Chlorella-like green alga were distinguished by immunoblotting. Plaques arising from single cells inoculated with two viruses usually contained only one of the viruses. Thus the viruses mutually exclude one another. In some combinations the ratio of viruses (as infective centres) recovered differed significantly from the input ratio.

Resistance to bacteriophage φ6 by Pseudomonas phaseolicola

Summary Resistance to bacteriophage φ6 by Pseudomonas phaseolicola HB10Y occurred at a frequency of about 1 in 120000 cells. Statistical analysis by the fluctuation and re-spreading tests indicated that the majority of φ6r bacteria resulted from random mutation rather than from contact with the phage. However, exposing the bacteria to heated φ6 (50 °C for 1 min) or φ6 nucleocapsid (virus minus the lipid envelope) prior to testing for resistance increased the frequency two- to threefold; φ6 dsRNA, unheated φ6, or heated φ91 did not have this effect. Nine resistant variants were selected for further characterization. When DNA isolated from the variants was subjected to agarose gel electrophoresis, seven of the nine strains had two plasmid DNA bands typical of HB10Y; the remaining two strains had three plasmid bands. φ6 attached to the pili of six of the nine variants; four of the variants actually had higher adsorption rate constants than HB10Y. Culture supernatants from the φ6r strains did not inactivate φ6. One of the resistant strains, D5, produced large amounts of infectious φ6 particles during its exponential phase of growth without a significant effect on its growth rate. Treatment of D5 with φ6-antiserum resulted in the loss of φ6 production; however, unlike typical carrier-state cells, D5 retained its resistance to φ6. The results indicate that resistance to φ6, although occurring at a relatively high frequency, is not due to one common event, such as loss of a plasmid and/or attachment sites.

Ultrastructure of bacteriophage phi 6: arrangement of the double-stranded RNA and envelope.

Pseudomonas phaseolicola cells synchronously infected with phi 6 were fixed and embedded by several procedures. The diameter of phi 6 measured 75 nm and its nucleocapsid 60 nm. Virus nucleocapsids were icosahedral and surrounded by a double membrane. In planar sections the nucleic acid appeared as a hexagonal ring and in cross section as two angular structures.