The Physiological Functions Of ATP And Related Mechanisms In Torulopsis Glabrata

In this dissertation, a multi-vitamin auxotroph (i. e., thiamine, biotin, pyrodoxin and nicotinic acid) Torulopsis glabrata strain, CCTCC M202019 was used as a model to demonstrate the effect of the energy metabolism on the physiological processes in eukaryotic microorganisms. Based on the well understanding of the energy metabolism, the mechanisms in the role of ATP metabolism in the intracellular micro-environment and related physiological processes were investigated. The main results were described as follows:(1) A reusable method, which systematically integrated the fusion PCR, high-efficiency electroporation transformation, nystatin enrichment and limited media screening, for construction of non-marker large fragment deletion Torulopsis glabrata auxotroph strains was established. By using the method, three T. glabrata auxotroph strains were obtained, i. e., a uracil auxotroph strain (Δura3), an arginine auxotroph strain (Δarg8) and a uracil arginine double auxtroph strain (Δura3Δarg8). The stability of 2μm bearing vectors was assayed in the T. glabrataΔura3 strain. The result proved that the 2μm bearing vector was highly stable and could express target green fluorescent protein in T. glabrataΔura3 strain.(2) ATP8, ATP6 and ATP9 are three genes that encode three key subunits of F₀F₁-ATPase, which are essential for the respiration, and locate on the mitochondrial genome (mtDNA). To knockout of the three genes, a homologous knockout box with a recoded ARG8 gene ARG8m, were transformed into the T. glabrataΔura3Δarg8 strain by biolistic transformation. The mtDNA transformants were primarily screened by the back-mutation of arginine auxotroph. However, it was found out that both the wild-type mtDNA and transformed mtDNA co-existed in the transformant cells and the ratio of the two kinds of mtDNA varied under different culture conditions and growth periods. The phenomenon was designated as Single Cell Mitochondrial Genome Polymorphism (SCMGP). It was found that the instability of mtDNA, mitochondrial fusion/fission and selective loss of mtDNA played crucial roles in the SCMGP. Three mtDNA homoplasmic cells without ATP8, ATP6 and ATP9 were obtained based on the characterization of the SCMGP and named as ATP8, ATP6 and ATP9, respectively.(3) Deficiency in ATP6 could increase the loss ratio of mtDNA to 42% and 63% on minimum medium and argine supplement medium after 24 generations, respectively. Through the assay of intracellular ATP level, production of reactive oxygen species (ROS), intracellular ATP level, the pH in mitochonchondrial intermembrane space (MIMS) and the transcriptional and enzyme activity assay of aconitase, it was found out that the H+ accumulation in MIMS was the dominant factor for the mtDNA instability of T. glabrata cells deficient in ATP6. In normal cells, the H+ accumulation in MIMS could be released by the F₀F₁-ATPase accompanied with the ATP synthesis. However, in cells deficient in ATP6, which encodes the ion channel subunit a, the H+ could not be released through the F₀F₁-ATPase. The accumulation of H+ in MIMS further improved theΔΨm and ROS, interrupted the translocation of mitochondrial matrix located proteins, and thus systematically caused the mtDNA instability in T. glabrata cells deficient in ATP6. The expression of two NADH oxidation associated genes, AOX1 from Histoplasma capsulatum and noxE from Lactobacillus lactis, could significantly improve the stability of mtDNA in ATP6. The result further proved that the conclusion. The two strains were names as AOX and NOX, respectively.(4) The effects of ATP metabolism on the central carbon metabolism were systematically investigated using strains ATP8, ATP6, ATP9 and NOX. The deficiency in the F₀F₁-ATPase significantly decreased the intracellular level, inhibited the cell growth and released the inhibitory effect of ATP during the initial 28 h. However, the relased inhibitory effect of ATP was mostly compsensated by decreased ability to deal with the tolerance to acidic stress and osmotic stress. The deficiency of F₀F₁-ATPase resulted in increased intracellular ROS level. The overexpression of noxE could recover the intracellular ROS level caused by the ATP6 deficiency and improved the intracellular micro-environment, thus enhanced the cell growth and pyruvate accumulation. The metabolic networks analysis, transcriptional and enzyme activities analysis of central metabolism key enzymes revealed that the deterioration of intracellular micro-environment caused by the deficiency in F₀F₁-ATPase affected the central carbon metabolism on different levels thus prohibited the further increase of pyruvate production. The phenomenon also suggested the importance of sub-cellular compartmentation in the protection of biomass synthesis and energy metabolism in the mitochondrial matrix.(5) In eukaryotic microorganisms, the pH in different subcellular compartmentations is kept in a suitable range thus the enzymes in these compartmentations could play their normal functions. Eukaryotic microorganisms realized the pH homeostasis in different cell compartmentation through a series of ATPase, which consumed additional ATP besides the normal cell growth. To determine the effect of ATP in the tolerance to lower pH, the intracellular micro-environment under enhanced ATP supply conditions was investigated by the citrate addition. The result showed that the enhanced the ATP supply could facilitate the ATP-dependent H+-transportation processes, thus kept higher pH gradients among extracellular environment, cytoplasm and vacuole. The quantitative relationship between the intracellular ATP content and the pH gradients showed that the increased intracellular ATP level could significantly promoted the pH homeostasis processes. Compared with the pH homeostasis process in Saccharomyces cerevisiae, it was found out that the lower pH homeostasis ability in T. glabrata CCTCC M202019 should be a positive factor for the pyruvate accumulation.