Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation

Abstract

Genetic manipulation of mitochondrial DNA (mtDNA) is the most direct method for investigating mtDNA, but until now, this has been achieved only in the diploid yeast Saccharomyces cerevisiae. In this study, the ATP6 gene on mtDNA of the haploid yeast Candida glabrata (Torulopsis glabrata) was deleted by biolistic transformation of DNA fragments with a recoded ARG8(m) mitochondrial genetic marker, flanked by homologous arms to the ATP6 gene. 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 lacking ATP6, along with their equal cell division, played important roles in determining the dynamics of heteroplasmy. Based on our analysis of mtDNA heteroplasmy in C. glabrata, we were able to generate homoplasmic Deltaatp6 mtDNA strains.

Fig. 1.

Biolistic transformation and characteristics of mtDNA transformants. (A) Transformants on bombarded plates. A YPD culture of 20 ml (30°C, 200 rpm, 24 h) of C. glabrata Δura3 Δarg8 cells was concentrated, washed with 1 mol·liter⁻¹ sorbitol, spread on MM plates, and transformed with 5 μl of 2-μg·μl⁻¹ pUC19-atp6::ARG8m using a particle gun. Plates were incubated at 30°C for 72 h until transformants appeared. (B) mtDNA transformants on MM and YPG plates. Overnight cultures (10 μl) of C. glabrata Δura3 Δarg8 (CK) and mtDNA transformants were spread on YPD and YPG and cultured at 30°C for 72 h. mtDNA transformants yielded colonies similar to those of CK. mtDNA transformants also grew on YPG, although more weakly. (C) mtDNA transformants lose the ability to grow on MM. After aerobic growth in YPD, some colonies lost the ability to grow on MM while retaining the ability to grow on SM. The ratio of MM to SM colonies was 32.5% based on the analysis of a total of 500 colonies.

Fig. 2.

Heteroplasmy. (A) Primers for confirming ATP6 or ARG8^m mtDNA. (B) Probes and restriction enzyme sites for Southern blot analysis. (C) Electrophoresis of a typical colony using the primers shown in panel A. ATP6 or ARG8^m was amplified using P1/P2, giving PCR products of 1,507 bp for the original mtDNA or 2,023 bp for Δatp6::ARG8^m mtDNA (Table 1). Primers P3 and P4 were used for further validation. (D) Southern blot. mtDNA from C. glabrata Δura3 Δarg8, mtDNA primary transformants, and a homoplasmic Δatp6::ARG8m deletion strain were digested with SwaI or TaqI. DNA fragments were separated in 1% agarose and transferred to nitrocellulose. Membranes were hybridized with digoxigenin-labeled ATP6 and ARG8^m probes. The relevant SwaI and TaqI restriction sites and positions of the probes (bar labeled with an asterisk) are shown in panel B.

Fig. 3.

Mitochondrial biogenesis under anaerobiosis. To visualize mitochondria, cells were stained with MitoTracker Green FM. Discrete green staining creates fluorescence from mitochondria. Phase-contrast (A) and fluorescence (B) images of Δatp6 heteroplasmic cells from 0 h, 4 h, 8 h, and 12 h of anaerobic growth in MM are shown.

Fig. 4.

Effect of anaerobic growth on the mitochondrial copy number. mtDNA copy number is expressed as the relative value for cells during aerobic growth at the indicated time point. The approximate value of the mtDNA copy number shown here decreased with time during anaerobic growth. mtDNA copy number is a highly approximate value of the number of mitochondria and is not exact because of mtDNA concatemers (52) and mitochondria without mtDNA (48). Error bars indicate standard deviations.

Fig. 5.

Elimination of mtDNA heteroplasmy. The relative ratio of mtDNA(ATP6) and mtDNA(Δatp6::ARG8^m) could be determined by qPCR of ATP6 and ARG8^m because the two genes were unique in the two kinds of mtDNA molecules. Error bars indicate standard deviations.

Fig. 6.

ROS production in different kinds of yeast cells. ▵, C. glabrata with only mtDNA(ATP6); □, C. glabrata with both mtDNA(ATP6) and mtDNA(Δatp6::ARG8^m); ▪, C. glabrata with only mtDNA(Δatp6::ARG8^m). Error bars indicate standard deviations.

Fig. 7.

Process of mtDNA transformation and heteroplasmy dynamics. The original mtDNA contained all essential genes for oxidative phosphorylation, which supplied the cell with more ATP and facilitated growth. The transformed mtDNA lacked the ATP6 gene, which is essential for oxidative phosphorylation, but contained the ARG8^m gene, which is essential for cell growth on medium without arginine.