Abstract:
Due to the paucity of sensitive mutation markers available for studying mycobacterial
species it was decided to explore the suitability of mycobacteriophage L5 as an
analogous mutation detection system to phage Lambda in E. coli. The system relies on
the detection of an increased production of clear plaque mutants (CPM) arising from
turbid plaques, in response to DNA damage. A number of L5 phage experimental tools
were developed and optimized, including a lysogen-based CPM confirmation assay.
The mutant induction system was applied to wild type M. smegmatis mc2155 and its
recA mutant, dinP mutant as well as an M. smegmatis(L5) lysogen. The lysogen system
proved to be insensitive with respect to mutant induction since elevated CPM
frequencies could not be detected. Interestingly, the wild type M. smegmatis mc2155
system demonstrated slightly elevated CPM frequencies in response to transfection of
untreated L5 on UV irradiated host cells. This result suggests that a host SOS mutagenic
system is able to act on normal, undamaged DNA bases. The involvement of the SOS
response in untargeted mutagenesis was confirmed by the abrogation of increased CPM
frequency, in an M. smegmatis recA mutant. This data supports suggestions that RecA is
responsible for the control of the SOS response. The M. smegmatis dinP mutant system
showed a decrease in CPM frequency which supports evidence that this gene does have
mutator polymerase activity, as is in seen E. coli dinP homologues.
