Defining the mechanism of NAD-mediated mutagenesis
Bacteria require the ability to rapidly evolve and adapt in order to overcome the physiological stresses that they often face. Genetic variation and natural selection drive adaptive mutation, however the mechanism behind adaptive mutation is controversial. A novel discovery indicated adaptive mutation, through hypermutation, occurs in Mesorhizobium loti. Mesorhizobium loti is an α-proteobacterium and is a nitrogen-fixing microsymbiont of several Lotus species. The investigations for this study were carried out using three M. loti strains: M. loti R7A, M. loti R7ANS and M. loti R7Adct. M. loti R7A contains a mobile symbiosis island, ICEMlSymR7A, M. loti R7ANS is a non-symbiotic derivative of R7A that lacks ICEMlSymR7A, and M. loti R7Adct contains a deletion of the otherwise-functional C4-dicarboxylate transport system that is encoded on ICEMlSymR7A. These strains contain a non-functional (cryptic) C4-dicarboxylate transport (dct) system on their chromosome and thus they are unable to grow on media with succinate as a sole carbon source. However when cultured on such media, a sub-set of the strains give rise to Dct+ colonies over time that contain mutations that activate the cryptic dct genes. This ability to mutate was found to be due to nadQABC genes located on the mobile symbiosis island, ICEMlSymR7A. It was found that in the presence of the nadQABC locus, or of a large excess of nicotinic acid (NA), the Dct- non-symbiotic mesorhizobia and strain R7Adct give rise to Dct+ mutants over time. Strains lacking the nadQABC genes and grown in the presence of low concentrations of NA never give rise to Dct+ mutants. Furthermore the mutants seem to arise only after growth has stopped. Mutagenesis under non-growing conditions is known as stress-induced mutagenesis (SIM) or stationary-phase mutagenesis.The purpose of this study was to investigate the hypothesis that the Dct+ mutants arise as a result of oxidative DNA damage. We propose that hypermutation should result in accumulation of mutations in the genome due to oxidative stress caused by an imbalance in NAD homeostasis and that potentially deleterious mutations will be repaired.In this study, a series of single gene insertion duplication mutants (IDM) and a markerless deletion mutant were constructed for genes involved in DNA repair and in oxidative stress tolerance. It was hypothesised that these mutants would show an increase in both deleterious and beneficial mutations. The genes that were targeted encode proteins involved in the GO repair system (MutM, MutT, MutY) and Mismatch Repair (MMR) system (MutS) as well as a monofunctional catalase (KatE) and a bifunctional catalase/peroxidase (KatG). Mutants in each of the genes were constructed in M. loti strains R7A, R7ANS and R7AΔdct by either insertional duplication mutagenesis or in-frame deletion and mutants confirmed via PCR and Southern hybridisation techniques. The mutants in the R7ANS and R7A∆dct backgrounds were assayed for mutator phenotypes in the presence or absence of an excess of NA. The results demonstrated a considerable increase in mutation rate for the katG, mutS, mutT and mutY mutants, while the katE and mutM mutants showed wild-type mutation rates. Overall, these results provide compelling evidence for a mechanism whereby under conditions of stress, elevated levels of ROS and NADH lead to HO• formation via the Fenton reaction. This damage leads to wide-spread mutation resulting in both deleterious and beneficial mutations. The deleterious mutations are constantly repaired, and when a beneficial mutation occurs, the conditions of stress are alleviated, removing the source of SIM and restoring mutation rates to their original state.
Advisor: Ronson, Clive; Sullivan, John
Degree Name: Master of Science
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: Adaptive mutation; Stress-induced mutagenesis; Hypermutation; Oxidative stress; NAD; Mutagenesis; Mesorhizobium loti; Stationary-phase mutagenesis; Catalase; MMR; GO system; KatG; MutT; MutM; MutY; MutS
Research Type: Thesis