4. Discussion
The results obtained allow us to conclude that lysogenization by the
phage φ24B of diverse E. coli strains producing O antigens was
not due to an unusual ability of this virus to penetrate the O antigen
shield, but was mediated by spontaneous formation of bacterial rough
mutants or of mutants with significantly compromised O antigen
biosynthesis. It is not clear why the lysogenization was not effective
for some strains. The activity of antiviral systems, such as
restriction-modification, avoiding the lysogenization at stages after
the viral DNA penetration into the cell46, cannot be
excluded. Also, the effect may be due to point mutations present in BamA
protein or lower frequency of rough mutants in particular strains.
In the conditions of our experiment, the high concentration of
bacteriophage used allowed almost all the cells potentially susceptible
to the phage to be infected. However, in vivo the populations ofE. coli are very unlikely to face such a massive viral attack.
The fraction of rough mutants in natural habitats is hard to estimate,
but we can speculate that it should be lower than in in vitroconditions because such mutants have compromised protection not only
from the phage attack but also from immune system agents such as serum
bactericidal activity47-49 and from other
environmental factors32 and therefore should be
counter-selected. Moreover, if stx-phage lysogens were formed by
infection of such rough mutants, their expected fitness and/or virulence
would be significantly lower than that of the parental strains. These
strains, noteworthily, were highly sensitive to SBA of the horse serum
to which the parental O-antigen producing strains were completely
resistant. Therefore the lysogens for the φ24B phage are expected to
have reduced virulence. Thus, the factor of non-specific protection of
the bacterial cells by the O antigen should not be neglected during the
evaluation of the potential significance of stx-converting phage
transmission in nature (as it currently is neglected in many
studies25, 26).
We also should note that the lysogenization by φ24B:cat appears to be a
simple and efficient procedure for selection for mutants with
compromised or completely abolished O antigen synthesis. This procedure
may be particularly valuable for the researchers working with field
isolates of E. coli for which the genomic sequences are not yet
available and/or in which other rapid techniques such as recombination
with PCR fragments for genes knockout50 are frequently
less effective than in laboratory E. coli.