EXPERIMENT RECORD N° 9145
YEAST-B - Yeast In No Gravity - Part 2 (aka YING-B)
  1. 2009 • ISS Increments 19-20
Life Sciences:
  • Cell and Molecular Biology
BIOLAB
Jason Hatton
jason.hatton@esa.int
R. Willaert (1), F. Delvaux (2), B. Devreese (3), J. Nielsen (4), G. Van Minnebruggen (5), L. Wyns (1)
(1)  
Lab Structural Biology Brussels
Vrije Universiteit Brussel
Pleinlaan 2
1050 Brussel
BELGIUM
Tel:  
+32(0)2.6291846
Fax:  
+32(0)2.6291963
e-mail:  
Ronnie.Willaert@vib-vub.be
(2)  
Katholieke Universiteit Leuven
BELGIUM
(3)  
Universiteit Gent
BELGIUM
(4)  
Systems Biology
Department of Chemical and Biological Engineering
Chalmers University of Technology
Kemivägen 10
41296 Gothenburg
SWEDEN
(5)  
VIB Micro Array Facility
BELGIUM
Yeast-B was the second in a series of three experiments (the first of which was the YING-A experiment).

AIM

Determine the effect of microgravity on expression and functionality of Flo proteins from Saccharomyces cerevisae

Specific Goals

1. Determine influence on microgravity on yeast flocculation (cell-cell interaction)
2. Influence of microgravity on yeast biofilm formation
3. Influence of microgravity on invasive growth of yeast
4. Influence of microgravity on Flo protein associated processes

This project studied the influence of microgravity on Flo proteins of the yeast Saccharomyces cerevisiae, which are involved in cell surface interactions on solid substrate and cell-cell interactions in liquid media.

The final goal was to obtain information on the importance of gravity on the formation of organized cell structures (flocculation, biofilm, invasion) and the entire “Flo processes” itself.

Comparison of experiments performed under normal gravity and microgravity were studied at morphology, physiology and molecular (transcriptome, proteome, protein structures, etc) levels. Yeast cells were cultured on solid and in liquid media (batch and continuous cultures). Different strains of the same species (classical, low or strong flocculating cells, overexpressing strains for certain proteins) were utilised.

Yeast use adhesion molecules for interactions between cells, as well as with surfaces. In Saccaromyces cerevisiae the Flo family of cell wall glycoproteins (adhesines) play important and distinct roles which are illustrated here. Flocculation is the process of cell-cell adhesion (eg. clumping, with sedimentation of clumps). This is regulated by the FLO1, 5, 9 and 10 genes. Invasive growth is the process where the yeast cells penetrate within a substrate (eg. agar), FLO11 regulates this process. Yeast cells can also grow on surfaces as a biofilm and again the FLO11 protein regulates this process.

The experiment consists of two separate components:
YING-B1 with liquid cultures only - the samples were accomodated in boxes 2,3,4,5

YING-B2 with solid surface culture only - the samples were accomodated in triangle 1,2
see attachment: YING hardware

These experiment sub-units will be performed in separate experiment hardware and have different scientific requirements.

YING B-1
Biological Samples:
- Saccharomyces cerevisiae BY4742 wt (wild-type strain);
- Saccharomyces cerevisiae BY4742::FLO8
- S. cerevisiae BY4742 FLO1 overexpressing;
- S. cerevisiae BY4742 FLO5 overexpressing;
- S. cerevisiae BY4742 FLO9 overexpressing;
- S. cerevisiae BY4742 FLO10 overexpressing;
- S. cerevisiae BY4742 FLO11 overexpressing;
- S. cerevisiae flocculating industrial brewer´s strain;

Number of samples per condition / g-level:

26 samples in total, of which 18 in microgravity and 8 in 1g reference centrifuge.

General Experiment Procedure:

– Experiment prepared at launch site & handed over for launch integration
– Upload in passive stowage
– Incubation of the experiment
– Cell cultures & culture medium are mixed at activation – CO2 produced by cell culture during growth should be removed as far as possible (see below for performance requirements)
– Filtration of cells from medium.
– Fixative added to cells, medium unfixed
– Following fixation samples should be stored refrigerated
– Samples downloaded in passive temperature control
– Ground transportation until hand-over

Requirements on telemetry / data downlink / storage:
• No telemetry downlinked
• Temperature data: 10 minute time resolution, 0.5°C; data to be returned with samples. Measurements from hand-over to hand-over.
• Time of experiment activities to be recorded; facility data to be downlinked after completion of mission (within a few weeks of end of mission preferable)
• Passive ionizing radiation measurements (High LET & Low LET radiation dosimeters)
No requirements on commands uplink
No imagery requirements

Science deliverables
• Record of time of experiment steps (automatically recorded)
• Temperature profile
• Housekeeping data from facility, including temperature, centrifuge start /stop (can be downlinked after completion of mission)
• RNAlater fixed Saccharomyces cerevisiae samples subject to experimental protocol
• Filtered culture medium
• Passive radiation detectors (low LET & high LET range) exposed to flight environment in close proximity to experiment samples

YING B-2

Solid surface culture, with CO2 removal. Yeast cultures are allowed to grow on the agar surface before fixation. Cell cultures will be observable for photography during the active phase of the experiment. See figure.

Biological Samples:
- Saccharomyces cerevisiae yeast strains;
o Sigma 1278b MAT a/α wt (wild-type)
o Sigma 1278b MAT a/α flo11Δ/flo11Δ
o Sigma 1278b MATa wt
o Sigma 1278b MATa flo11Δ
o BY4742 MATα wt
o BY4742::FLO8 MATα

Number of samples per condition / g-level:

24 samples in microgravity.

General Experiment Procedure:

- Experiment prepared at launch site & handed over for launch.
- Upload in passive stowage, It is desirable to have as stable a temperature as possible within the specified range.
- Incubation of the experiment in ISS cabin environment. It is desirable to have as stable a temperature as possible within the specified range.
- Two sessions of photography of cultures in-flight.
- CO2 produced by cell culture during growth, this should be removed as far as possible.
- Addition of fixative to culture, displacing air above culture.
- Following fixation samples should be stored refrigerated.
- Samples downloaded in passive temperature control.
- Ground transportation until hand-over.

Requirements on telemetry / data downlink / storage:
• Temperature data: 10 minute time resolution, 0.5°C; data to be returned with samples. Measurements from hand-over to hand-over.
• Passive ionizing radiation measurement (High LET & Low LET radiation dosimeters)
• Time of experiment activities to be recorded; facility data to be downlinked after completion of mission (within a few weeks of end of mission preferable)

Imagery requirements:

Photography:
 
-  If possible, there shall be two photo-sessions during the active phase of the experiment. This is an “optional” requirement that would benefit to the scientific outcome of the experiment.
 
- Two sessions of photography in-flight, preferably FD4 & FD8 (+/-1 day).
- Field of view shall include the entire culture plate, with at least half of the samples visible.
- At least half of each culture vessel must be visible.
- Photograph must be focussed on culture surface i.e. yeast colony, the size of each yeast colony should be visible.
- Resolution of photos should be in millimetre range, reflections are to be minimised.
- Two photo-sessions for each container is the optimal situation. Two photo-sessions for two containers is the second-best option. Two sessions for only one container is still better than no photo-sessions at all.
- In order for the colonies to be observable for photography, the culture chamber shall not allow condensation to build-up.

EXECUTION OF EXPERIMENT
The experiment was carried to the ISS on the Soyuz 20S mission, which launched on 30 September 2009. The experiment used two configurations, one in liquid culture and one with solid substrate. The 26 experiment containers were processed between 2 and 7 October 2009, with only 2 of 26 cultivation chambers failing. As part of the sample processing and fixation, automated filtration and fixation of the cultivation chamber was successfully commanded from BIOLAB. After termination of the processing the experiment containers were stored at +4 degrees Celsius in the Thermal Control Unit of Biolab and returned with flight 18S.

Science deliverables
• Record of time of experiment steps (automatically recorded)
• Temperature profile
• Photo’s (detailed resolution requirements to be defined.
• RNAlater fixed Saccharomyces cerevisiae samples on solid substrate subject to experimental protocol
• Passive radiation detectors (low LET & high LET range) exposed to flight environment in close proximity to experiment samples

Microgravity is “sensed” by yeast cells as a stress condition and several mitogen‐activated protein kinases (MAPK) signaling pathways are activated, including the cell wall integrity (CWI)/protein kinase C (PKC), the high osmolarity glycerol (HOG) and the target of rapamycin (TOR) pathways. One of the indicators of morphological changes is an increase at random bud scar profile. Microgravity influences on the growth rate of yeast cells have been observed. The colony growth rate of the agar invasive S. cerevisiae Σ1278b strain was reduced as well as its agar invasiveness. Post‐flight growth experiments of a brewer’s top yeast strain showed an increase in G2/M and a decrease in Sub-G1 cell population; an increased viability, a decreased lipid peroxidation level, increased glycogen content, and changes in carbohydrate metabolic enzyme activities were also observed. Using the S. cerevisiae BY4741 deletion collection, genes that provide a survival advantage in space, were identified in a batch growth experiment; no difference in growth rate was observed. Freeze-dried strains showed significant changes in the cell wall thickness. Spaceflight unique gene expression changes were observed in stress response element (STRE) genes with transcription regulation involving Sfp1 (which is involved in the TOR pathway) and Msn4. Some of the components of the ribosome biogenesis (which is under the control of Sfp1) as well as components of the proteasome were down regulated in microgravity. Recent results indicate that microgravity imposes a “microgravity” stress on the cells, which has the characteristics of an osmotic stress. Cellular energy is directed towards protective measures such as cell wall biosynthesis (cell wall integrity pathway activation) and the production of compounds (glycerol, trehalose) that increase the osmotolerancy (HOG pathway).
[1]  
T.B. Reynolds, G.R. Fink, (2001), "Baker's yeast, a model for fungal biofilm formation", Science 291: 878-881.
[2]  
K. Verstrepen, G. Derdelinckx, H. Verachtert, F.R. Delvaux, (2003), "Yeast flocculation: what brewers should know", Appl. Microbiol. Biotechnol. 61: 197-203.
[3]  
J. Nielsen, (2003), "It is all about metabolic fluxes", J. Bacteriol. 185: 7031-7035..
[4]  
K.J. Verstrepen, T.B. Reynolds, G.R. Fink, (2004), "Origins of variation in the fungal cell surface", Nat. Rev. Microbiol. 2: 533-540.
[5]  
S.E. Van Mulders, E. Christianen, S.M.G. Saerens, L. Daenen, P.J. Verbelen, R. Willaert, K.J. Verstrepen, F.R. Delvaux, (2008), "Phenotypic diversity of Flo protein family-mediated adhesion in Saccharomyces cerevisiae", FEMS Yeast Research, pp. 178-190.
[6]  
R. Willaert, (2009), "Engineering aspects of cell immobilization.", Encyclopedia of industrial biotechnology, John Wiley & Sons, New Jersey, in press..
[7]  
S.E. Van Mulders, E. Christianen, S.M. Saerens, L. Daenen, P.J. Verbelen, R. Willaert, K.J. Verstrepen, F.R. Delvaux, (2009), "Phenotypic diversity of Flo protein family-mediated adhesion in Saccharomyces cerevisiae", FEMS Yeast Res. 9(2): 178-190.
[8]  
K. Goossens, R. Willaert, (2010), "Flocculation protein structure and cell-cell adhesion mechanism in Saccharomyces cerevisiae", Biotechnology Letters, 32, pp. 1571-1585.
[9]  
S.E. Van Mulders, M. Ghequire, L. Daenen, P.J. Verbelen, K.J. Verstrepen, F.R. Delvaux, (2010), "Flocculation gene variability in industrial brewer's yeast strains", Applied Microbiology and Biotechnology, 88, pp. 1321-1331.
[10]  
S.E. Van Mulders, (2010), "Phenotypic diversity of Flo protein family-mediated adhesion", PhD Thesis, KU. Leuven, Leuven, Belgium.
[11]  
K. Goossens, C. Stassen, I. Stals, D. Donohue, B. Devreese, H. De Greve, R. Willaert, (2011), "The N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae binds specifically to mannose carbohydrates", Eukaryotic Cell, 10, pp. 110-117.
[12]  
S.E. Van Mulders, C. Stassen, L. Daenen, B. Devreese, V. Siewers, R.D. van Eijsden, J. Nielsen, F.R. Delvaux, R. Willaert, (2011), "The influence of microgravity on invasive growth in Saccharomyces cerevisiae", Astrobiology, 11, 1, DOI: 10.1089/ast.2010.0518, pp. 45-55.
[13]  
C. Stassen, (2012), "The influence of microgravity on the model organism Sacharomyces cerevisiae: a proteomic study", PhD Thesis, Ghent University, Belgium.
[14]  
R.G. Willaert, (2013), "The growth behavior of the model eukaryotic yeast Saccharomyces cerevisiae in microgravity", Current Biotechnology, 2, 3, pp. 226-234.
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Experiments concepts

YING B-2 Working concept

YING B-1 and YING B-2
 
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