Mitochondrial DNA Heteroplasmy in Candida glabrata after Mitochondrial Transformation

Abstract

ABSTRACTGenetic manipulation of mitochondrial DNA (mtDNA) is the most direct method for investigating mtDNA, but until now, this has been achieved only in the diploid yeastSaccharomyces cerevisiae. In this study, theATP6gene on mtDNA of the haploid yeastCandida glabrata(Torulopsis glabrata) was deleted by biolistic transformation of DNA fragments with a recodedARG8mmitochondrial genetic marker, flanked by homologous arms to theATP6gene. Transformants were identified by arginine prototrophy. However, in the transformants, the original mtDNA was not lost spontaneously, even under arginine selective pressure. Moreover, the mtDNA transformants selectively lost the transformed mtDNA under aerobic conditions. The mtDNA heteroplasmy in the transformants was characterized by PCR, quantitative PCR, and Southern blotting, showing that the heteroplasmy was relatively stable in the absence of arginine. Aerobic conditions facilitated the loss of the original mtDNA, and anaerobic conditions favored loss of the transformed mtDNA. Moreover, detailed investigations showed that increases in reactive oxygen species in mitochondria lackingATP6, along with their equal cell division, played important roles in determining the dynamics of heteroplasmy. Based on our analysis of mtDNA heteroplasmy inC. glabrata, we were able to generate homoplasmic Δatp6mtDNA strains.