Characterization of Mycobacterium smegmatis mutant strains deficient in DNA repair and energy metabolism for increased mutagenesis

Abstract

Tuberculosis is an ancient menace caused by Mycobacterium tuberculosis (M. tb). Nearly, 1.4 million lives are claimed due to this disease annually. Upon infection, M. tb is engulfed by alveolar macrophages to form a granuloma as part of the host’s immune response to control the infection. The conditions within the granuloma are hostile which can cause DNA damage resulting in spontaneous mutations, drug resistance or leading to cell death. However, mycobacteria have several repair pathways including the base excision repair (BER) pathway which is important for repairing damaged single nucleotides for continued growth and maintenance of genome integrity. DNA glycosylases are the first enzymes in the BER pathway that recognize and excise damaged nucleotides. Previous work has shown that endonuclease III (nth) has antimutator properties and a synergistic relationship with the two endonuclease VIII (nei) homologs in maintaining mycobacterial genome stability under oxidative stress conditions. However, DNA repair deficiency may not be the only driver of mutagenesis in mycobacteria. Mycobacteria require oxygen for metabolism and therefore, under oxygen-depleted environments growth of M. tb will cease. M. tb can also alternate between energy pathways and up-regulate specific genes of the electron transport chain (ETC) under hypoxic conditions thus, enhancing the survival of the bacteria under unfavourable conditions. Therefore, both the BER pathway and the ETC confer M. tb with the ability to tolerate high oxidative stress environments generated within the host during pathogenesis. Hence, we hypothesized that deficiency of critical genes in the ETC pathway would exacerbate reactive oxygen species (ROS) levels resulting in increased levels of DNA damage that could be catastrophic in the absence of crucial DNA repair enzymes (from the BER pathway), leading to mutagenesis and eventually to cell death. In this study, we aimed to generate and characterize combinatorial mutants deficient in genes in both the BER and the ETC pathways in the non-pathogenic M. smegmatis strain to gain an understanding of the interplay between these two pathways in the maintenance of mycobacterial genome integrity. Previous findings have reported that mutants deficient in single genes in either of the pathways play a role in genome maintenance under unfavourable conditions. Based on this we hypothesized that combinatorial mutants deficient in genes from both the BER and ETC pathways when exposed to stress conditions, are more likely to show increased mutagenesis.