1. Research program: Systems Biology of the Living Cell 1
Subprogram: Molecular Biology & Microbial Food Safety

2. Swammerdam Institute for Life Sciences
Molecular Biology and Microbial Food Safety theme: Stress response in bio(medical) systems research approach Systems Biology
Environmental stress induced response on physiology (S. cerevisiae and C. albicans)
Drug induced mitochondrial dysfunction (C. elegans)
Quantitative systems analysis of stress induced biochemical and physiological cellular responses (bioenergetics of temperature, pH & antimicrobial stress)
Microbial stress: sporulation and antibiotic resistance.
Swammerdam Institute for Life Sciences

3. Swammerdam Institute for Life Sciences
The role of mitochondria in temperature stress: Dynamic control of yeast respiratory efficiency
30oC
37oC
38oC
38oC→30oC
Why? To prevent irreversible damage? ROS?
Swammerdam Institute for Life Sciences
Postmus et al., Microbiology 2011

4. Systems biology  answering the why
Aim:
Systems biology of ROS and ageing: Gertien Smits, Hans v/d Spek
Integrated approach C. elegans & S. cerevisiae
Integration of quantitative biochemistry, physiology, molecular biology and cell biology in quantitative, time resolved models of stress and age induced ROS production, ROS damage induction, and cellular responses
Collaborators: Chris de Koster, Natal van Riel, Takenori Yamamoto, Ron Wanders, Peter Reiss, Frank Baas
Ageing: developmental drift or ROS induced damage
New researcher in the group: McGillarvy fellow Yelena Budovskaya (+PhD)
Swammerdam Institute for Life Sciences
Budovskaya et al., Cell

5. Swammerdam Institute for Life Sciences
Molecular Biology and Microbial Food Safety theme: Stress response in bio(medical) systems research approach Systems Biology
Environmental stress induced response on physiology (S. cerevisiae and C. albicans)
Drug induced mitochondrial dysfunction (C. elegans)
“Omics” approaches to understand adaptation
to pH, temperature and antimicrobial stress
(and to prevent it):
Frans Klis, Gertien Smits
Stanley Brul, Benno ter Kuile with collaborations:
De Koster, Manders, Rep.
RUMC, VWA, AMC
Microbial stress: sporulation and antibiotic resistance.
Swammerdam Institute for Life Sciences

6. Molecular Biology and Microbial Food Safety theme: Stress response in biomedical systems the role of the intracellular pH
Orij et al., BBA 2011

7. Regulation of yeast lipid biosynthesis by pHcyt Young,…. Orij…
Regulation of yeast lipid biosynthesis by pHcyt Young,….Orij…..Smits and Loewen Science (329)
Growth
No Growth
Young et al.,Science 2010

8. Intracellular pH: a second messenger for cellular decision making?
pHc
growth rate
ln (OD600 cor)
pH 5.0
pH 4.5
pH 4.0
pH 3.5
pH 3.0
pH 2.5
pH 2.0
0.04
0.08
0.12
0.16
0.2
6.8 7
7.2
7.4 pH
cyt
6.9
7.0
7.1
7.3
fraction
wild type
mutants
Mutants
Primary screen
Rescreening
Total found
% Identified
Table 1 Screen statistics
low
high 19 8 57 99 76
107 84 93 5 10 15 *
*** **
cellular nitrogen compound
metabolic process
mitochondrion organization
regulation of intracellular pH
tRNA aminoacylation
structural constituent of ribosome
hydrogen ion transmembrane
transporter activity
aminoacyl -
tRNA ligase activity
mitochondrial inner membrane
nucleoplasm
mitochondrial large ribosomal subunit
proton
transporting V
type ATPase complex
transporting two
sector
ATPase complex, catalytic domain
component
function
process
fold enrichment
t (hours)
Swammerdam Institute for Life Sciences
Orij et al., submitted

9. Intracellular pH: a second messenger for cellular decision making?
Swammerdam Institute for Life Sciences
Orij et al., submitted

10. The role of the wall proteome of Candida albicans in mucosal and systemic infections
Before washing After washing
pH 7 invasive
pH 7
pH 4
pH 7
pH 4
pH 4
Sosinska et al. Microbiology 2010; Sorgo et al. Euk. Cell 2011; Klis et al. Future Microbiology, 2011

11. Swammerdam Institute for Life Sciences
Quantitation of the adaptations in the wall proteome of Candida albicans in biomats grown at pH 7 and 4
1. pH-dependent isoforms
Phr1 vs. Phr2
- Sod5/Sod4
- Als1, Als3 vs. Als4
. Hypha-specific WPs such as Sod5, Phr1, Hyr1, Als3
Goals
● Determine wall protein levels during infection-related stress conditions using
MS-based quantitation (FT-MS)
● Identify wall protein-based vaccine targets and potential diagnostic proteins
Collaborations
● Chris de Koster (MS), Eric Manders, Martijn Rep (Fusarium oxysporum)
● Mihai Netea, Univ Nijmegen (vaccine targets, diagnostics)
Swammerdam Institute for Life Sciences
Heilmann et al., Microbiology 2011

12. Spores from the genus Bacillus are extremely stress resistant (heat!)
Bacterial spore formers are of prime concern to microbial food stability:
Spores from the genus Bacillus are extremely stress resistant (heat!)
This allows surviving spores to germinate and grow causing food spoilage
Brul et al., Food Microbiology 2011

13. Johan van Beilen (PhDst)
Weak Organic Acid Stress: Membrane & Intracellular pH
Johan van Beilen (PhDst)
In vivo pH meter: pHulorin
Elucidating the function of RodZ in weak organic acid resistance in B. subtilis.
Genetic Metabolic Diseases (AMC):
Prof. Ron Wanders
Ter Beek and Brul, Curr. Opin Biotech. 2010

14. Problem of spore germination & outgrowth:
Heterogeneity!
Single-cell live imaging of heterogeneous germination and outgrowth of Bacillus subtilis (cells &) spores
Germination
Time-resolved single cell analysis
Time-resolved single spore
germination & outgrowth analysis
Heat treatment
Centre for Advanced Microscopy:
Dr. Erik Manders
Rachna Pandey (ERASMUS MUNDUS PhDst)

15. (ERASMUS MUNDUS, PhDst)
The spore coat proteome of Bacilli
Wishwas Abhyankar
(ERASMUS MUNDUS, PhDst)
TSB MOPS MOPS MOPS
dilutions pre-culture Sporulation
medium medium
96 hours
Spore coat isolation
&
SDS extraction
Trypsin
digestion
Desalting
LC-ESI-MS/MS
MASCOT analysis
Harvesting of spores
Reduction
alkylation
of disulfide bridges
Mass Spectrometry of Biomacromolecules: Prof. Chris de Koster
Abhyankar et al., Proteomics 2011

16. Targeted Inhibition of Bacterial Spores (TIBS)
Goal: To find targets that prevent spore germination & outgrowth under conditions of (mild) preservation
Alex Ter Beek (PD)
We need to understand the mechanistic basis of spore germination & outgrowth (in control & stress conditions)
heat
weak organic acids (sorbic-, lactic acid)
Expression studies of
B. subtilis spore outgrowth
under (mild) stress conditions 5 10 15 20 25 30 50 40 60 70 80 90
100
110
120
130
Time (minutes)
Microarray Department:
Dr. Timo Breit
Ter Beek et al., Food Microbiology 2011

17. Swammerdam Institute for Life Sciences
Adaptations to pH & antimicrobial stress; challenges & collaborations within SILS
Goals Biomedical yeasts
● Determine Candida albicans wall protein levels during infection-related stress
conditions (important pH perturbation) using MS-based quantitation (FT-MS)
● Identify wall protein-based vaccine targets and potential diagnostic proteins
Collaborations (apart from yeast groups UvA, VU, FINSysB EU)
● Chris de Koster (MS), Eric Manders, Martijn Rep (Fusarium oxysporum)
● Mihai Netea, Radboud Univ. Med. Centre (vaccine targets, diagnostics)
Goals bacterial (food)spoilage organisms
● Determine the role of the intracellular pH in regulating cell growth
● Identify targets to interfere with spore germination & growth of Bacilli
● Identify the genetic basis (mutations) underlying stress adaptation
Collaborations (apart from the European Spore Conference core)
● Chris de Koster (MS), Eric Manders, (Live Imaging), AMC (genome seq.)
● TNO (screening antimicrobial compounds), FES partners for spore purification
1. pH-dependent isoforms
Phr1 vs. Phr2
- Sod5/Sod4
- Als1, Als3 vs. Als4
. Hypha-specific WPs such as Sod5, Phr1, Hyr1, Als3
Swammerdam Institute for Life Sciences

18. Swammerdam Institute for Life Sciences
Molecular Biology and Microbial Food Safety; research focus of our group members
Systems Biology of Stress & Ageing
Gertien Smits (staff), Hans van der Spek (staff), Yelena Budovskaya (staff McGillarvy), R. de Boer (PhD), Rueben Smith (PhD), Marco Lezzerini (PhD McGillarvy), Belinda Koenders (technical support 0,5), Marcel Scholte (supp. 0,4)
Medical yeasts
Gertien Smits (staff), Frans Klis (staff), Stanley Brul (staff), Alice Sorgo (PhD), Clemens Heilmann (PhD), Azmat Ullah (PhD)
Food spoilage bacteria
Stanley Brul (staff), Benno Ter Kuile (VWA & staff), Jan Smelt (seior scientist) Merijn Schuurmans (post-doc*), Nadine Händel (PhD VWA), Alex Ter Beek (post-doc), Johan van Beilen (PhD), Wishwas Abhyankar (PhD), Rachna Pandey (PhD), NN4 (PhD FES).
1. pH-dependent isoforms
Phr1 vs. Phr2
- Sod5/Sod4
- Als1, Als3 vs. Als4
. Hypha-specific WPs such as Sod5, Phr1, Hyr1, Als3
General Support
Marian de Jong and Muus de Haan (both ~1 day a week. Rest teaching & SILS)
Swammerdam Institute for Life Sciences