Chemical-genomic profiling identifies genes that protect yeast from aluminium, gallium, and indium toxicity.

Aluminium, gallium, and indium are group 13 metals with similar chemical and physical properties. While aluminium is one of the most abundant elements in the Earth's crust, gallium and indium are present only in trace amounts. However, the increased use of the latter metals in novel technologies may result in increased human and environmental exposure. There is mounting evidence that these metals are toxic, but the underlying mechanisms remain poorly understood. Likewise, little is known about how cells protect themselves from these metals. Aluminium, gallium, and indium are relatively insoluble at neutral pH, and here we show that they precipitate in yeast culture medium at acidic pH as metal-phosphate species. Despite this, the dissolved metal concentrations are sufficient to induce toxicity in the yeast Saccharomyces cerevisiae. By chemical-genomic profiling of the S. cerevisiae gene deletion collection, we identified genes that maintain growth in the presence of the three metals. We found both shared and metal-specific genes that confer resistance. The shared gene products included functions related to calcium metabolism and Ire1/Hac1-mediated protection. Metal-specific gene products included functions in vesicle-mediated transport and autophagy for aluminium, protein folding and phospholipid metabolism for gallium, and chorismate metabolic processes for indium. Many of the identified yeast genes have human orthologues involved in disease processes. Thus, similar protective mechanisms may act in yeast and humans. The protective functions identified in this study provide a basis for further investigations into toxicity and resistance mechanisms in yeast, plants, and humans.

Genetically controlled mtDNA deletions prevent ROS damage by arresting oxidative phosphorylation.

Deletion of mitochondrial DNA in eukaryotes is currently attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that this process critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication through Rtg2 and Rtg3. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. This shows that oxidative stress-induced mitochondrial impairment may be under strict regulatory control. If the results extend to human cells, the results may prove to be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.

A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance.

The rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high-throughput. We then mix the resistance plasmid-carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals, and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within Escherichia coli populations, by screening the Keio deletion collection in high replication. We recover all seven known chromosomal gene mutants affecting conjugation as donors and identify many novel mutants, all of which diminish antibiotic resistance transmission. We validate nine of the novel genes' effects in liquid mating assays and complement one of the novel genes' effect on conjugation (rseA). The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improve the longevity of current and future antibiotics. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.IMPORTANCE The rapid transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within E. coli populations. We recover all previously known and many novel chromosomal gene mutants that affect conjugation efficiency. The new framework holds great potential for rapid screening of compounds that decrease transmission. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.

Study on montmorillonite/insulin/TiO2 hybrid nanocomposite as a new oral drug-delivery system.

This study was conducted in two main stages. In the first stage, drug-loaded montmorillonite nanocomposites were prepared by intercalation of insulin into the montmorillonite layers in acidic deionized (DI) water. In the second stage, to increase the release of insulin from the prepared nanocomposites they were coated with TiO2, an inorganic porous coating, by using titanium (IV) butoxide, as precursor. The prepared nanocomposites were characterized by FT-IR, XRD, FE-SEM, BET, DLS and Zeta potential analysis. After investigating the release behaviour of the nanocomposites by UV-Vis absorbance technique, the results revealed that incorporation of porous TiO2 coating increased the drug entrapment noticeably, and decreased the amount of drug release, so that nanocomposites without and with TiO2 coating released the drug after 60min and 22h in pH7.4, respectively. These results could be used in converting the insulin utilization from injection to oral.

Biofuels. Altered sterol composition renders yeast thermotolerant.

Ethanol production for use as a biofuel is mainly achieved through simultaneous saccharification and fermentation by yeast. Operating at ≥40°C would be beneficial in terms of increasing efficiency of the process and reducing costs, but yeast does not grow efficiently at those temperatures. We used adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at ≥40°C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol desaturase gene, and increased expression of genes involved in sterol biosynthesis. Additionally, large chromosome III rearrangements and mutations in genes associated with DNA damage and respiration were found, but contributed less to the thermotolerant phenotype.

Production of 2-butanol through meso-2,3-butanediol consumption in lactic acid bacteria.

2-Butanol has been an issue of industries in many areas, for example, biofuel production (as an advanced alternate fuel), fermented beverages, and food (as taste-altering component). Thus, its source of production, the biological pathway, and the enzymes involved are of high interest. In this study, 42 different isolates of lactic acid bacteria from nine different species were screened for their capability to consume meso-2,3-butanediol and produce 2-butanol. Lactobacillus brevis was the only species that showed any production of 2-butanol. Five of ten tested isolates of L. brevis were able to convert meso-2,3-butanediol to 2-butanol in a synthetic medium (SM2). However, none of them showed the same capability in a complex medium such as MRS indicating that the ability to produce 2-butanol is subject to some kind of repression mechanism. Furthermore, by evaluating the performance of the enzymes required to convert meso-2,3-butanediol to 2-butanol, that is, the secondary alcohol dehydrogenase and the diol dehydratase, it was shown that the latter needed the presence of a substrate to be expressed.

2-Butanol and butanone production in Saccharomyces cerevisiae through combination of a B12 dependent dehydratase and a secondary alcohol dehydrogenase using a TEV-based expression system.

2-Butanol and its chemical precursor butanone (methyl ethyl ketone--MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuteri), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.

Physiological adaptations of Saccharomyces cerevisiae evolved for improved butanol tolerance.

BACKGROUND: Butanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation. However, the end product toxicity is one of the main obstacles for developing the production process irrespective of the choice of production organism. The long-term goal of the present project is to produce 2-butanol in Saccharomyces cerevisiae. Therefore, unraveling the toxicity mechanisms of solvents such as butanol and understanding the mechanisms by which tolerant strains of S. cerevisiae adapt to them would be an important contribution to the development of a bio-based butanol production process. RESULTS: A butanol tolerant S. cerevisiae was achieved through a series of sequential batch cultures with gradual increase of 2-butanol concentration. The final mutant (JBA-mut) tolerates all different alcohols tested at higher concentrations compared to the wild type (JBA-wt). Proteomics analysis of the two strains grown under mild butanol-stress revealed 46 proteins changing their expression by more than 1.5-fold in JBA-mut, 34 of which were upregulated. Strikingly, 21 out of the 34 upregulated proteins were predicted constituents of mitochondria. Among the non-mitochondrial up-regulated proteins, the minor isoform of Glycerol-3-phosphatase (Gpp2) was the most notable, since it was the only tested protein whose overexpression was found to confer butanol tolerance. CONCLUSION: The study demonstrates several differences between the butanol tolerant mutant and the wild type. Upregulation of proteins involved in the mitochondrial ATP synthesizing machinery constituents and glycerol biosynthesis seem to be beneficial for a successful adaptation of yeast cells to butanol stress.