Eco-physiological mechanims involved in cyanobacterial

bloom Place: University of Amsterdam

Laboratorium of Microbiology

Participants: Prof. Dr. L. MurDr. H.J.M. MatthijsDr. M. BrigliaIr. J. Balke

Topic:

Eco-physiological mechanims involved in cyanobacterial

bloom

WhyEnvironmental factors:Nutrient stress

(nitrate-N)

HowCellular Mechanisms:Cell wall response

(protein pattern)

Intervention

Topic:

Prevention

Cyanobacterial bloom

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:

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+ GlnGlu

(G=~+500 mV)NO3

-

Nitrate-N stress:

Cellular behaviour

Proteomics of the cell wall

Study:

* 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:

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:

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)

Abso

rban

ce (7

50 nm

)

control

NH4+ limitation

NO3- limitation

light limitation

light limitation

Fig. 1

modulation phase

modulation phase

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:

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:

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:

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%

Beforecentrifuging

Aftercentrifuging

Citoplasmic membrane

Cell wall

Experimental approach:

Analysis of the cell wall protein pattern1) 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 Kb116,2 Kb

200 Kb

Experimental approach:

Analysis of the cell wall protein pattern2) 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 Kb116,2 Kb66,2 Kb

45 Kb

31 Kb 21,5 Kb

+N -N -NH7ųl

10ųl 10ųl 10ųl7ųl 7ųl15ųl 15ųl 15ųl

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:

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:

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.

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;

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).