Decreasing Mitochondrial RNA Polymerase Activity Reverses Biased Inheritance of Hypersuppressive mtDNA

Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that regions of rho+ mtDNA persisted after hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced hypersuppressive biased inheritance. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance relative to hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the respiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency.

COQ11 deletion mitigates respiratory deficiency caused by mutations in the gene encoding the coenzyme Q chaperone protein Coq10

Coenzyme Q (Qn) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1–coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6. The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because “fused” proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.

Adaptive regrowth in respiratory deficient strain of yeast Saccharomyces cerevisiae due to deletion of YKU70 gene

It is known that significant causes of malignant tumors are destabilization of the nuclear genome and mitochondrial dysfunction. Adaptive regrowth in yeast colonies (the appearance of cell subpopulations more adapted to unfavorable conditions under conditions of the death of the original culture) is used as a model of the initial stages of carcinogenesis. To study the features of the formation of adaptive regrowth, a reparationdefective and respiratory-deficient yeast strain of Saccharomyces cerevisiae was created. The thermosensitive mutation in the yku70 gene was used as an inducer of nuclear genome instability (at 37 оC it causes cell cycle arrest due to a reduction of the length of telomeric regions of chromosomes). Damage to the mitochondrial DNA of the ∆yku70 strain led to its respiratory deficiency (petite mutation). The isolated petite mutant ∆yku70 strain was cultured at optimal 28 оC and restrictive 37 оC temperatures, the state of the cell suspension was evaluated by light and fluorescence microscopy, to determine the viability of cells was used the analysis of microcolonies growth. Isolation of adaptive regrowth clones and analysis of their properties by the method of serial dilutions were conducted. To assess the genome stability of selected clones of adaptive regrowth, PCR analysis of the microsatellite sequences YOR267C, SC8132X, SCPTSY7 was conducted. When culturing the petite mutant of the strain ∆yku70 at a restrictive temperature of 37 оC for 7 days, the formation of viable subpopulations was detected, which can overcome the arrest of the cell cycle in the G2 / M phase. Further analysis of the isolated clones of adaptive regrowth showed that they differ in cell survival at restrictive temperature, resistance to UV radiation and the ability to form adaptive regrowth on colonies. In the analysis of microsatellite repeats in adaptive regrowth clones, no manifestations of instability of the studied sequences were detected.

Evaluation of fermentation potential of wild and UV-mutated yeasts screened from traditional murcha

Murcha (an amylolytic starter) from different parts of Eastern Nepal were screened for fermentative yeasts. The most potential one was UV-mutated (8W lamp at λ = 254 nm and an intensity of 44.21 Wm-2 for 5-50 s) to study the effect of mutation on growth and fermentation properties. Respiratory-deficient mutants (RDMs) that resulted from the mutation were identified by triphenyl tetrazolium chloride (TTC) overlay technique and replica-plated for further isolation. Cell growth, substrate utilization, and ethanol yield of the mutants were compared with normal cells by carrying out fermentation in high-test cane molasses broth of 30 °Bx. An exhaustive screening of the samples resulted in only two murcha viz., from Laxmimarga (LM) and Udayapur (UD), having the desirable fermentation properties. UV-mutation study of UD and LM yeasts (both identified as strains of Saccharomyces cerevisiae) showed 8-12% survival and ~ 22% RDMs yield of the survived cells. Out of the 8 randomly selected RDMs, only UDm4 (colony No. 4 from UD) showed fermentation properties worth further investigation. Comparison of UD, LM and UDm4 by fermenting molasses (high test) broth of 30°Bx showed the least growth of UDm4 but the highest alcohol yield (9% and 16% more compared to UD and LM, respectively). The present finding indicates that it is possible to improve fermentation properties of feral yeasts from murcha by relatively simple UV-mutation approach. Finding the right mutant (the selective screening part), however, may involve considerable time and effort.

Bactericidal Effect of Tomatidine-Tobramycin Combination against Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa Is Enhanced by Interspecific Small-Molecule Interactions

ABSTRACTThis study investigated the antibacterial activity of the plant alkaloid tomatidine (TO) againstStaphylococcus aureusgrown in the presence ofPseudomonas aeruginosa. Since theP. aeruginosaexoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) is known to cause a respiratory deficiency inS. aureusand respiratory-deficientS. aureusare known to be hypersensitive to TO, we assessed kill kinetics of TO (8 μg/ml) againstS. aureusin coculture withP. aeruginosa. Kill kinetics were also assessed usingP. aeruginosamutants deficient in the production of different exoproducts and quorum sensing-related compounds. After 24 h in coculture, TO increased the killing ofS. aureusby 3.4 log10CFU/ml in comparison to that observed in a coculture without TO. The effect of TO was abolished whenS. aureuswas in coculture with thelasRrhlR,pqsA,pqsL, orlasAmutant ofP. aeruginosa. The bactericidal effect of TO againstS. aureusin coculture with thepqsLmutant was restored by supplemental HQNO. In anS. aureusmonoculture, the combination of HQNO and TO was bacteriostatic, indicating that thepqsLmutant produced an additional factor required for the bactericidal effect. The bactericidal activity of TO was also observed against a tobramycin-resistant methicillin-resistantS. aureus(MRSA) in coculture withP. aeruginosa, and the addition of tobramycin significantly suppressed the growth of both microorganisms. TO shows a strong bactericidal effect againstS. aureuswhen cocultured withP. aeruginosa. The combination of TO and tobramycin may represent a new treatment approach for cystic fibrosis patients frequently cocolonized by MRSA andP. aeruginosa.