Hhinsch Week 12

Outline

Article Info

• The journal club article I reviewed is: Pizzaro, Jewett, Nielson, Agosin. (2008) Growth Temperature Exerts Differential Physiological and Transcriptional Responses in Laboratory and Wine Strains of Saccharomyces Cerevisiae. "Applied and Environmental Microbiology", 74, 6358-6368, doi:10.1128/AEM.00602-08
• The full text can be found here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570279/pdf/0602-08.pdf

Definitions

1. Metabolome: the sum of all small molecular weight metabolites in a biological sample of interest. The metabolome of a given cell will vary greatly depending on its physiological or developmental state, its age, or its response to disease or drugs. [1]
2. Polyketide: any natural product synthesized via linear poly‐β‐ketones, which are themselves formed by repetitive head‐to‐tail addition of acetyl (or substituted acetyl) units indirectly derived from acetate (or a substituted acetate) by a mechanism similar to that for fatty‐acid biosynthesis but without the intermediate reductive steps. In many cases, acetyl‐CoA functions as the starter unit and malonyl‐CoA as the extending unit. Various molecules other than acetyl‐CoA may be used as starter, often with methylmalonyl‐CoA as the extending unit. The poly‐β‐ketones so formed may undergo modification by alkylation, cyclization, glycosylation, oxidation, or reduction. Polyketides include: coniine (of hemlock) and orsellinate (of lichens) – acetyl‐CoA; flavanoids and stilbenes – cinnamoyl‐CoA; tetracyclines – amide of malonyl‐CoA; urushiols (of poison‐ivy) – palmitoleoyl‐CoA; erythronolides – propionyl‐CoA and methylmalonyl‐CoA as extender. Polyketide synthases are large multidomain proteins that contain phosphopantheteine. [2]
3. Gas Chromatography/Mass Spectrometry: abbr.: GC/MS; an analytical technique that combines the separation process of gas chromatography with the highly selective detection technique of mass spectrometry.[3]
4. Metabolite: a chemical compound that is produced or consumed during metabolism. Polymeric biological molecules are excluded from this definition. Metabolites include low-molecular-weight compounds that are produced or converted by enzymes during metabolism or the precursors or breakdown products of biopolymers. [4]
5. Fermentation: an energy-yielding enzymatic breakdown of sugar molecules that takes place in bacteria and yeasts under anaerobic conditions.[5]
6. Gluconeogenesis: the formulation of glucose or other carbohydrates such as glycogen (glyconeogenesis) from noncarbohydrate precursors such as glycogenic amino acids, lactate, and Krebs TCA cycle intermediates. Gluconeogenesis occurs in the mammalian liver under conditions such as starvation or low carbohydrate intake. [6]
7. Diploid: referring to the situation or state in the life cycle where a cell or organism has two sets of chromosomes, one from the mother and one from the father. Diploidy results from the fusion of the haploid egg nucleus and a haploid sperm nucleus. [7]
8. Aneuploid: Describing a nucleus, cell, or organism in which one or more chromosomes have been added to or deleted from the complete set, so that the total number of chromosomes is not an exact multiple of the haploid number. [8]
9. Polyploid: Describing a nucleus that contains more than two sets of chromosomes (see diploid) or a cell or organism containing such nuclei. For example, triploid plants have three sets of chromosomes and tetraploid plants have four. Polyploidy is far more common in plants than in animals; many crops, in particular, are polyploid (bread wheat, for example, is hexaploid, i.e. 6n). It can be induced chemically with colchicine. [9]
10. Anaerobic Respiration: A type of respiration in which foodstuffs (usually carbohydrates) are partially oxidized, with the release of chemical energy, in a process not involving atmospheric oxygen. Since the substrate is never completely oxidized the energy yield of this type of respiration is lower than that of aerobic respiration. It occurs in some yeasts and bacteria and in muscle tissue when oxygen is absent (see oxygen debt). Obligate anaerobes are organisms that cannot use free oxygen for respiration; facultative anaerobes are normally aerobic but can respire anaerobically during periods of oxygen shortage. Alcoholic fermentation is a type of anaerobic respiration in which one of the end products is ethanol. [10]

Outline

Abstract

• The scientists studied two different strains of Saccharomyces cerevisiae.
  • A wine strain and a lab strain.
    • The lab strains are primarily studied for mapping of eukaryotic cells and possible understanding of diseases.
    • The wine strains are primarily used for making wine that has organoleptic properties.
• A two-factor experiment was done to examine the responses of a laboratory strain and an industrial wine strain at a temperature of 15 and 30 degrees Celsius.
  • The studies were done in nitrogen-limited, anaerobic, steady-state chemostat cultures.
• Results showed that the biomass of each strain was effected greatly.
  • Laboratory strains were found to yield higher fermentation rates.
  • Industrial wine strains were found to yield higher biomass production at the lower temperatures.
• DNA Microarrays and targeted metabolome analysis were used.
  • Experimenteries identified 1,007 temperature-dependent genes and 473 strain-dependent genes.
  • Study helps give information that scientists can use to determine how growth temperature affects differential physiological and transcriptional responses.

Introduction

• Saccharomyces cerevisiae is an important organism.
  • Is used in bakeries, distilleries, breweries, or wineries.
  • It is also the first sequenced eukaryote.
  • Saccharomyces cerevisiae has also been used as a producer of pharmaceuticals.
• S. cerevisiae S288c is the labratory strain the experimenters used.
  • This has been popular for almost a century.
  • Used for genetics studies.
  • Laboratory yeasts can't produce ethanol so they get stuck in the fermentation process.
• Microarrays have helped determine what effects possible transcriptional regulation among cells.
  • Over the past ten years, microarrays have given scientists the ability to study what causal impact different physiological conditions have on the transcription of genes.
• Temperature effects the wine-fermentation.
  • Temperatures that are not great for fermentation are consistently used in white wine fermentation for desired sensory effects.
  • Physiological studies of yeast have shown that protein translation rates, cell membrane fluidity, RNA secondary structure stability, enzymatic activity, protein folding rates, and heat shock protein regulation are significantly affected.
• Used the two-factor design for to show difference in responses between the lab strain and the wine strain of yeast.
  • Used continual chemostat cultures which is different from earlier studies with wine yeast.
  • Used DNA microarrays and a sensitive gas chromatography-mass spectrometry (GC-MS) method for quantification of intra- and extracellular metabolites which helped expose a causal relationship between physiological changes and transcription.

Materials and Methods

• S. cerevisiae wild-type laboratory strain CEN.PK113-7D (Mata) was used as the lab yeast strain.
• The commercial wine strain S. cerevisiae var. bayanus EC1118 was used as the wine yeast.
  • Both of these strains were grown in 2 liter chemostats at both 15 and 30 degrees Celsius.
  • The biomass of the two strains was determined by filtering the culture through a pre-weighed 0.45-nm nitrocellulose filter.
  • The remaining biomass was then dried in a microwave.
  • Culture samples (10 ml) used for determination of glucose, ethanol, glycerol, acetate, pyruvate, and succinate concentrations were immediately filtered through a 0.22-nm-pore-size cellulose acetate filter and then stored at -80 degrees Celsius in order to analyze later in the experiment.
• Most of the cultures were stored at very cool temperatures in order to maintain consistency throughout the course of the experiment.
• Washing and staining of arrays was done using lab equipment: GeneChip fluidics station 400 and scanning with an Affymetrix GeneChip Scanner 3000.
  • A lot of specific genetic equipment was needed to perform this study.
  • SGD was also used to find the significantly effected gene ontology within the upregulated and downregulated temperature-dependent and strain-dependent genes.
  • The 5,814 unique S. cerevisiae open reading frames were extracted from the 10,765 transcript features on the Yeast Genome 2.0 arrays.

Results

• To compare the difference between the lab yeast and the wine yeast the experimenters used a systems approach.
  • This systems approach correlated physiological, transcriptional, and metabolomic responses.
• Physiological differences under all conditions were studied first.
  • There were significant changes in the biomass.
  • The lab and yeast cultures were found to have 30% and 50% decreases in biomass which is consistent with previous results.
• One of the most notable effects was that of the adaptation of to growth at low temperature was the effect on nitrogen metabolism.
  • Both strains produced a lower biomass field at 30% and 50%.
  • At 30% and 50% the wine yeast produced more biomass than that of the lab yeast.
  • This suggests that the wine yeast is suited to grow better with limited nitrogen.
  • The experiments found there to be increases in the protein and RNA contents and decreases in the storage carbohydrate contents.
    • This was found at the lower temperature.
  • There weren't any amino acids detected in the extracellular medium.
  • This is all in consistent with previous results.
• The yield of ethanol on glucose was another significant physiological finding.
  • The reduction of temperature caused a significant yield of ethanol for both strains.
    • Under anaerobic conditions, the fermentation capacity is related to ethanol production.
      • This wasn't significant at the lower temperature.
• There wasn't a change in levels of ethanol yields on glucose between strains which suggests that this energetic process is strain independent.
• Statistical Analysis of Microarray Data was used in order to find out which genes significantly changed expression.
  • A four way analysis was performed. This analysis showed differences in the expressions of the genes at both temperatures.
    • This showed differences between temperatures and between strains.

Conclusions

• This was the first study to utilize a two-factor design to control for and study the underlying mechanisms for temperature adaptation in wine yeasts compared to laboratory yeasts by using chemostat cultures.
• Growth at the low temperatures altered the biomass compositions of the strains which yielded more nitrogen rich macromolecules.
  • This in turn yielded less biomass.
  • At the lower temperature, lower sugar uptakes were observed.
  • These two factors reduced the efficiency of fermentation.
• The lab yeast and the wine yeast responded differently to growth at the lower temperature.
  • The study suggests that the lab yeast uses carbon sources better.
    • This could be determined by the increase in fermentation rates of the lab yeast.
  • The study suggests that the wine yeast was better adapted for growth in nitrogen limited environments.
    • This happened at both temperatures because of the alterations in nitrogen metabolism.

Journal Club Presentation Powerpoint Slides

Media:MaryHaydenWeek12JournalClubPresentation.pptx ‎

References

1. King, R., Mulligan, P., & Stansfield, W.(2013). A Dictionary of Genetics. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444.
2. (2006). polyketide. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J.(Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-15977.
3. (2006). gas chromatography/mass spectrometry. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J.(Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-7653.
4. King, R., Mulligan, P., & Stansfield, W.(2013). metabolite. In A Dictionary of Genetics. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-4071.
5. King, R., Mulligan, P., & Stansfield, W.(2013). fermentation. In A Dictionary of Genetics. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-2328.
6. King, R., Mulligan, P., & Stansfield, W.(2013). gluconeogenesis. In A Dictionary of Genetics. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-2707.
7. King, R., Mulligan, P., & Stansfield, W.(2013). diploid. In A Dictionary of Genetics. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-1796.
8. (2016). aneuploid. In Hine, R., & Martin, E.(Eds.), A Dictionary of Biology. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780198714378.001.0001/acref-9780198714378-e-230.
9. (2016). polyploid. In Hine, R., & Martin, E.(Eds.), A Dictionary of Biology. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780198714378.001.0001/acref-9780198714378-e-3532.
10. (2016). anaerobic respiration. In Hine, R., & Martin, E.(Eds.), A Dictionary of Biology. : Oxford University Press. Retrieved 19 Nov. 2017, from http://electra.lmu.edu:2218/view/10.1093/acref/9780198714378.001.0001/acref-9780198714378-e-217.
11. Pizarro, F. J., Jewett, M. C., Nielsen, J., & Agosin, E. (2008). Growth Temperature Exerts Differential Physiological and Transcriptional Responses in Laboratory and Wine Strains of Saccharomyces cerevisiae . Applied and Environmental Microbiology, 74(20), 6358–6368. http://doi.org/10.1128/AEM.00602-08
12. LMU BioDB 2017. (2017). Week 12. Retrieved November 19, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_12

Acknowledgments

1. Mary and I worked over text and in person in order to complete the deliverables of this assignment.
2. The whole group worked over text in order to complete the deliverables of the team's page.
3. Dr. Dioniso and Dr. Dahlquist helped explain the deliverables of the assignment in class.
4. While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.Hhinsch (talk) 18:09, 20 November 2017 (PST)

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