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Leviviruses,
which are single-stranded RNA bacteriophages, are known to be produced
by only three genera of bacteria: Escherichia, Pseudomonas,
and Caulobacter. Although studies of these viruses in
E. coli have been of considerable importance to the elucidation
of the molecular details of translation and of the use of overlapping
sequences as viral genes, understanding of the steps leading to
viral genome-ribosome association is limited to two observations:
Leviviruses can attach to pili (which may or may not be the virion
receptor sites), and viral RNA is transiently exposed during virus
penetration of the cell.
The summer project was one component of
a research program directed toward elucidation of the early steps
in Levivirus infection in C. crescentus. During the project,
clones of strain CB2 that survived phage infection were isolated
and characterized with respect to their susceptibility to the RNA
phage ØCb5, as well as to two DNA phages ØCd1 and ØCb13. As is often observed with RNA phage
infections, survivors of ØCb5 infection in batch broth cultures retained
full phage susceptibility. In contrast, two of 25 clones that survived
ØCd1 infection in batch broth culture proved
resistant to plaque formation by both ØCd1 (the selecting phage) and ØCb5 (the RNA phage) and to be unable to
adsorb either of those phages. They were interpreted as spontaneous
phage-resistant mutants whose resistance could be attributed to
loss of a phage attachment site common to the infection path of
both phages.
Methods developed and refined during the
experiments with the first mutants were subsequently employed for
the characterization of 1,000 clones of CB2 isolated as survivors
of perpetual ØCb5 infection established and sustained
in chemostat cultures. The initial screening detected more than
100 bacterial clones with altered phage susceptibilities. However,
in contrast to the phage-resistant mutants obtained by batch-culture
infection, these clones exhibited a diversity of phenotypes that
included, in addition to resistance to both phages: resistance to
the RNA phage only; to both ØCb5 (the selecting phage) and ØCd1, but with acquired susceptibility to
ØCb13, to which the parent strain is not
susceptible; and hypersusceptibility to all three phages. The perpetual
infection therefore yielded a collection of strains with various
modifications whose further study should assist in elucidation of
steps of RNA phage attachment to and penetration of the C. crescentus
cell surface.
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