Development of mucobacteriophage L5 as a marker for mutation induction in mycobacteria

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.