This document summarizes a study that aimed to: 1) Elucidate the behavior of cyanobacteria under nitrate-nitrogen stress 2) Determine if the cyanobacterial cell wall specifically responds to nitrate stress 3) Develop tools to monitor cyanobacterial blooms The study found that nitrate depletion caused bleaching of cyanobacterial cells and slower growth. Analysis of cell wall proteins showed new proteins were synthesized under nitrate stress. The cell wall responded specifically to different nitrogen sources. The study concluded the cyanobacterial strain demonstrated a specific adaptation of the cell wall to nutrient stress.

1. Eco-physiological mechanims
involved in cyanobacterial bloom
Place: University of Amsterdam
Laboratorium of Microbiology
Participants: Prof. Dr. L. Mur
Dr. H.J.M. Matthijs
Dr. M. Briglia
Ir. J. Balke
Topic:

2. Eco-physiological mechanims
involved in cyanobacterial bloom
Why
Environmental factors:
Nutrient stress
(nitrate-N)
How
Cellular
Mechanisms:
Cell wall
response
(protein pattern)
Intervention
Topic:
Prevention
Cyanobacterial
bloom

3. 1. Elucidate cyanobacterial behaviour under
nitrate-N stress;
2. Determine whether the cyanobacterial cell
wall responds specifically to nitrate stress;
3. Develop molecular tools to monitor bloom-
warning signal/s (multiprobe array: identity +
activity).
AIMS
:
Nitrate-N stress:
Cellular behaviour
Proteomics of the cell wall
Study:

4. Because:
*N is necessary for growth (biological
macromolecules) and metabolism (energy
flow) of the cell;
*it is the most stable inorganic source of N;
Nitrate reduction:
NO2
- NH4
+ Gln
Glu
(∆G=~+500 mV)
NO3
-
Nitrate-N stress:
Cellular behaviour
Proteomics of the cell wall
Study:

5. * Batch culture system (rich and
depleted nitrate conditions);
* Continuous culture system
(nitrate inputs 0.5 and 0.05 mM,
d=0.015, NH4
+
input 0.05 mM);
* Cyanobacterium strain model
Synechocistys PCC 6803.
Experimental approach:
Nitrate-N stress:
Cellular behaviour
Proteomics of the cell wall
Study:

6. Effect of nitrate-N stress on the behaviour of
Synechocystis cells
 Under nitrate depletion Synechocystis
cells undergo to a quick loss of
pigments (bleaching);
 They keep dividing though at almost
undetectable level;
Results from batch study:

7. Behaviour of Synechocystis PCC 6803 under
nitrate-N stress
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0 2 4 6 8 10 12 14
Time (day)
Absorbance(750nm)
control
NH4+ limitation
NO3- limitation
light limitation
light limitation
Fig. 1
modulation phase
modulation phase

8. Effect of nitrate-N stress on the behaviour of
Synechocystis cells
 Low nitrate input slows down the growth;
 The type of nitrogen source influences the
growth of Synechocystis PCC 6803;
 The modulation phase of nitrate limited cells
is shorter than that one of ammonium limited
cells.
Results:

9.  Elucidate cyanobacterial behaviour under nitrate-N stress;
 Determine whether the cyanobacterial cell
wall responds specifically to nitrate-N stress;
 Develop molecular tools to monitor bloom-
warning signal/s (multiprobe array: identity +
activity).
AIMS:
Nitrate-N stress:
Cellular behaviour
Proteomics of the cell wall
Study:

10. Effect of nitrate-N stress on strain PCC 6803
cell wall: study of the protein pattern.
* Isolation of the cell wall
fraction (by flotation ultracentrifugation on
discontinuous sucrose density gradient);
* Analysis of the cell wall fraction (by
SDS-PAGE and polypeptide sequence
determination);
Experimental approach:

11. Experimental approach:
Isolation of the cell wall fraction
1. Cell disruption by shearing forces (bead beating);
2. Preparation of discontinuous sucrose density
gradient;
Steps:
10%
30%
45%
48%
55%
90%
Before
centrifuging
After
centrifuging
Citoplasmic
membrane
Cell wall

12. Experimental approach:
Analysis of the cell wall protein pattern
1) SDS-PAGE of the cell wall protein pattern of PCC
6803 cells submitted to rich (+) and depleted (-)
nitrate treatment.
+ + +- - -
66,2 Kb
45 Kb
2.2ųg 1.8ųg 1.4ųg 1.2ųg 0.7ųg 0.6ųg
31 Kb
21,5 Kb
97,4 Kb
116,2 Kb
200 Kb

13. Experimental approach:
Analysis of the cell wall protein pattern
2) SDS-PAGE of the cell wall protein pattern of PCC 6803 cells
submitted to sufficient (+), limeted (-) nitrate and ammonium
(NH) treatment.
200 Kb
97,4 Kb
116,2 Kb
66,2 Kb
45 Kb
31 Kb
21,5 Kb
+N -N -NH
7ųl 10ųl 10ųl 10ųl7ųl 7ųl15ųl 15ųl 15ųl

14. Effect of nitrate-N stress on strain PCC 6803
cell wall: study of the protein pattern.
* Depletion of nitrate-N induces synthesis of
new polypetides in the cell wall of strain
PCC 6803 as shown by SDS-PAGE
analysis;
* Induction of the synthesis of these proteins
occurs already at low nitrate-N concentration
(0,05 mM);
* Low concentration (0,05 mM) of ammonium-
N induces also synthesis of new protein.
Results:

15. 1. Elucidate cyanobacterial behaviour under
nitrate-N stress;
2. Determine whether the cyanobacterial cell
wall responds specifically to nitrate stress;
3. Develop molecular tools to monitor bloom-
warning signal/s (multiprobe array: identity +
activity).
AIMS:
Nitrate-N stress:
Cellular behaviour
Proteomics of the cell wall
Study:

16. Conclusions:
Molecular ecophysiology of strain PCC
6803/cyanobacteria under nitrate-N stress.
1) Indeed strain PCC 6803/cyanobacteria
respond specifically to the stress of
different Nitrogen source.
2) In strain PCC 6803/cyanobacteria
nutrient stress (N) induces a specific
adaptation of the cell wall rather than a
non-specific increase of its permeability.

17. CBS-Project:
Co-existence of bacteria and fungi in
soils.
A) Contribution of bacterial and fungal
activities to the degradation of organic
matter;
B) Antagonistic activities between
bacteria and fungi;
C) Interaction and dynamics between
fungal and bacterial population
structures;

18. Original Paper: Applied Microbiology …, 2002
Bioavailable nitrate detection in water by an immobilized luminescent
cyanobacterial reporter strain.
F. Mbeunkui, C. Richaud, A.-L. Etienne, R. Schmid and T. Bachmann
Cyanobacteria are a major group of photosynthetic bacteria that can accumulate
in surface water as so-called "blooms" in response to environmental factors
such as temperature, light and certain nutrients such as N, P, and Fe.
Some species of cyanobacteria produce toxins, causing a considerable danger for
human and livestock health.
As a consequence, monitoring of bloom formation and toxin production of drinking
water supplies has become a major concern.
To enable prediction and monitoring of cyanobacterial blooms, tools to detect nutrient
bioavailability in water would be advantageous.
A whole-cell biosensor was developed for monitoring nitrate (NO3-)
bioavailability in aquatic ecosystems using the recombinant bioluminescent
cyanobacterial strain Synechocystis PCC 6803 harboring an insertion of a
luxAB-kmr fusion with nblA1 in its chromosomal DNA, leading to PnblA::luxAB-kmr.
This reporter strain was designated N1LuxKm. Cells were immobilized in microtiter plates
and showed a dose-dependent response to nitrate deprivation. The resultant CyanoSensor
could detect nitrate in the 4-100 µM concentration Range after a sample incubation time of
10 h under continuous illumination (50 µE m-2 s-1).