undernitrogenstarvaon, arthrospirasp ...hosting.umons.ac.be/aspnet/mdc2013/upload/153.pdf ·...
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To inves)gate the response to nitrogen starva)on, 3 biological replicates were analyzed at protein level through label-‐free mass spectrometry approach. Replicates were starved from nitrogen during 8 and 24 hours and then, we compared them to the control samples submiBed to an iden)cal treatment. To achieve this, proteins were separated on a RP column (L.: 25cm, ɸ: 75µm, par)cles: 3µm, outlet: 300nL/min, PepMap C18, Dionex) with an ACN gradient (4 to 35% v/v) and then analyzed in high resolu)on mass spectrometer (ABSciex 5600 Triple TOF®). 1604 proteins were iden)fied and quan)fied with a false discovery rate at pep)de level of 2.09% and 2.24% respec)vely for 8 and 24 hours of culture (Progenesis LC-‐MSTM (version 4.0)).
Under nitrogen starva.on, Arthrospira sp. PCC 8005 survived through alterna.ve pathways to the classical GS/GOGAT cycle: an integrated study
Frédéric Deschoenmaeker1, Bap.ste Leroy1, Hanene Badri 1, 2, Zhang C.-‐C.3 and Ruddy WaSez 1*
1 Department of Proteomic and Microbiology, Research Ins)tute for Biosciences, University of Mons, place du Parc 20, B-‐7000 Mons, Belgium, 2 Expert group for Molecular and Cellular Biology MCB, Belgian Nuclear Research Center SCK.CEN, B-‐2400 Mol, Belgium, 3 Laboratoire de Chimie Bactérienne, CNRS-‐UMR 7283, Aix-‐Marseille Université, 31 chemin Joseph Aiguier, 13402 Marseille
Cedex 20, France
>2.00
1.99 -‐ 1.70
1.69 -‐ 1.40
1.39 -‐ 1.25
1.25< x < 1
0.99 -‐ 0.70
0.69 -‐ 0.40
0.39 -‐ 0.20
< 0.19
N.D.
Cyanobacteria are photosynthe)c prokaryotes having a crucial importance in Earth’s nitrogen and carbon cycles as well as in solar light conversion into chemical energy. In addi)on, their major role in primary produc)on of biomass and oxygen has made them intensively studied, especially regarding their nitrogen metabolism. Nitrogen metabolism relies on the availability in the medium of either mineral sources such as ammonium, nitrate and nitrite ions or organic sources such as urea. Nitrogen assimila)on generally involves specific transporters such as ATP-‐binding casseBe type (ABC) or major facilitator transporters (MFS). In contrast to that, dinitrogen is consumed across its fixa)on in heterocysts. Ajer their specific transport, sources are metabolized by corresponding enzymes (such as nitrate (Nar) and nitrite (Nir) reductases) leading to ammonium produc)on.
10µm Legend: SEM morphology observation of Arthrospira sp. PCC 8005
(Deschoenmaeker F.)
Arthrospira sp. PCC 8005, belonging to the Oscilatoriale order, appears as non-‐heterocyst forming colonies named trichomes which are not able to fix dinitrogen. While some cyanobacteria are ojen concerned due to their toxin produc)on, our strain is edible and was consumed for centuries. Arthrospira sp. PCC 8005 was also selected by European Space Agency (ESA) to be one of the key members of an ar)ficial ecosystem called MELiSSA (MicroEcological Life Support System Alterna)ve).
Introduc.on
Then, glutamine synthetase (GS) and glutamine oxoglutarate aminotranferase (GOGAT) lead to glutamate produc)on. GS/GOGAT cycle involves 2-‐oxoglutarate consump)on leading to the regula)on of nitrogen metabolism according the carbon/nitrogen ra)o across key regulator ac)vity (such as NtcA and PII).
NtcA , … PII , …
2-OG
N2
NO3-‐
(/ NO2-‐)
NH4+
Urée
Nitrogenase N2
NO3-‐ NO2
-‐
NH4+
Urée
NH4+
Nar Nir Gln Glu
GS GOGAT
Ur CO2 P.
Cytoplasm
I.M.
O.M. Legend: Nitrogen metabolism overview through phylum of cyanobacteria
Conclusion & Outlook
The strain PCC 8005 of the genus Arthrospira showed abili)es to grow and survive during combined-‐nitrogen starva)on. Those ones seemed to be intrinsically related to catabolic pathways of alterna)ve sources of nitrogen such as arginin, cyanates or nitriles. In addi)on to previous men)oned abili)es, that cyanobacterium was able to survive ajer 30 days of nitrogen starva)on, poten)ally involving a kind of heterocyst differen)a)on and phenotypically corresponding to high-‐fluorescent cells in trichomes. In this context, phenotypic and molecular studies are required to well understand the long term mechanisms.
Glutamine
Carbamoyl-‐phoshate
CarA (pep.: 4; 1)
Citrulline
Ornithine Argl (pep.: 2; 2)
Argininosuccinate
ArgG (pep.: 2; 3)
Aspartate
Glutamate
GS (pep.: 18; 24)
Aat2 (pep.:4; 3)
Nitrile
ammonium
NCH (pep.: 1; 1)
Cyanate
CynS (pep.: 3; 4)
Formamide FmdA
(pep.: 5; 6)
Arginine
Fumarate ArgH
(pep.: 1; 1)
Glutamate
Formate
Carboxylate
Glutamyl-‐tRNA
tRNAglu
Agma)ne
CO2
H2O
Urea Putrescine
CO2
Speb-‐like (pep.: 13; 8)
CO2
Glutamate-‐1-‐semialdehyde
5-‐Amino-‐levulinate
Porphobilinogen
Hydroxymethylbilane Uroporphyrinogen I
Coproporphyrinogen I
Urinoporphyrin I
Coproporphyrin I
Uroporphyrinogen III
Coproporphyrinogen III
Protoporphyrinogen IX
Protoporphyrin IX Cytochrome anabolic pathway
Protoporphyrin IX -‐
Mg
Mg-‐ Protoporphyrinogen IX 13-‐monomethylester
131 -‐hydroxi-‐Mg-‐protoporphyrin IX 13-‐monomethylester
Divinyl proto-‐chlorophyllide
Chlorophyll anabolic pathway
Spontaneous
131 -‐oxo-‐Mg-‐protoporphyrin IX 13-‐monomethylester
HemA (pep.: 1; 1)
HemL (pep.: 10; 8)
HemB (pep.: 2; 4)
HemC (pep.: 6; 6)
HemE (pep.: 3; 2)
HemF (pep.: 5; 7)
ChlH (pep.: 1, 2)
AcsF (pep.: 3; 3)
AcsF (pep.: 3; 3)
HemE (pep.: 3; 2)
CO2
Fold change scale
The strain PCC 8005 of Arthrospira sp. PCC 8005 was cul)vated in the presence and in the absence of combined-‐nitrogen sources (n=4). The bacterial growth was monitored through the O.D.750 measurements over )me.
Besides bacterial cultures under nitrate-‐depleted condi)ons, sedimenta)on and the well-‐known bleaching phenomena were no)ced.
During first several hours of culture under combined-‐nitrogen depleted condi)ons, a growth phenomenon was first observed while a survival phenomenon was no)ced over 50 hours.
1. Growth under nitrogen-‐starva.on condi.ons
3. Bleaching phenomenon
The obvious sedimenta)on was inves)gated through O.D.750nm measurements (n=4). While no sedimenta)on was observed in control medium, this phenomenon significantly increased under nitrogen-‐starva)on condi)ons.
2. Sedimenta.on phenomenon
To inves)gate the bleaching, a differen)al extrac)on of photosynthe)c pigments was achieved (n=4). Allophycocyanin (λ650nm) and phycocyanin (λ620nm) content significantly decreased under depleted condi)ons (both p-‐value < 1x10-‐³). While chlorophyll a (λ665) content was not showed a significant varia)on (p-‐value=0.526) over )me, its content significantly increased in control condi)ons (p-‐value at 72H: 0.032 ).
+ N -‐ N + N -‐ N
Acknowledgements
Ajer a nitrogen starva)on over 30 days, Arthrospira sp. PCC 8005 was incubated under nitrate-‐replete condi)ons and the growth was monitored according to the O.D.750 measurements (n=4). Results showed a growth before 100 hours sugges)ng a long term survival ability.
In the same )me, fluorescence of trichomes was observed under confocal microscopy (n=4). Ajer a long term nitrogen starva)on, only few isolated cells in trichomes exhibited a high fluorescence level. The replete condi)ons induced an increase of these cells which characterized all cells ajer 96 hours of culture.
While the genus Arthrospira is a non-‐heterocystous bacterium, mass spectrometry suggested that abundance of HetR and HglK proteins significantly increased ajer 24 hours of culture in absence of any combined-‐nitrogen sources.
0h 4h 8h
24h 48h 96h
Nitrogen starva8on
HetR (pep.:1; 3)
Absence of nitrogen-‐combined sources in the medium significantly (p-‐value<0.05) induced altera)ons of several biological func)ons such as amino acid metabolism as well as photosynthesis. Several catabolic pathways could be s)mulated to establish a glutamate-‐glutamine pool probably allowed surviving over )me.
hBp://www.chm.bris.ac.uk/motm/oec/motm.htm
Moreover, the obtained results suggested a decrease of photosynthe)c ac)vi)es by decrease of proteins such as :
4. Long term nitrogen starvation
Luque, I., E. Flores, and A. Herrero, Molecular mechanism for the opera8on of nitrogen control in cyanobacteria. Embo J, 1994. 13(23): p. 5794. Flores, E. and A. Herrero, Chapter 16: Assimilatory Nitrogen Metabolism and Its Regula8on, ed. D.A. Bryant. 1994: Klüwer Academic Publishers. Aichi, M. and T. Omata, Involvement of NtcB, a LysR family transcrip8on factor, in nitrite ac8va8on of the nitrate assimila8on operon in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol, 1997. 179(15): p. 4671-‐5. Berman-‐Frank, I., P. Lundgren, and P. Falkowski, Nitrogen fixa8on and photosynthe8c oxygen evolu8on in cyanobacteria. Res Microbiol, 2003. 154(3): p. 157-‐64. Zhang, C.C., Heterocyst differen8a8on and paPern forma8on in cyanobacteria: a chorus of signals. Mol Microbiol, 2006. 59(2): p. 367-‐75. Collier, J.L., B. Brahamsha, and B. Palenik, The marine cyanobacterium Synechococcus sp. WH7805 requires urease (urea amidohydrolase, EC 3.5.1.5) to u8lize urea as a nitrogen source: molecular-‐gene8c and biochemical analysis of the enzyme. Microbiology, 1999. 145 ( Pt 2): p. 447-‐59. Herrero, A., A.M. Muro-‐Pastor, and E. Flores, Nitrogen control in cyanobacteria. J Bacteriol, 2001. 183(2): p. 411-‐25. Janssen, P.J., Genome sequence of the edible cyanobacterium Arthrospira sp. PCC 8005. J Bacteriol, 2010. 192(9): p. 2465-‐6.
Phenotypic analysis results
0 25 50 75 1000.0
0.2
0.4
0.6
0.8
1.0 + Nitrate - Nitrate
Time (h)
O.D
. at 7
50nm
0 10 20 30 40 50 60 70 800
5
10
15
20
25
30+ Nitrate - Nitrate
Time (h)
O.D
. at 6
65nm
/O.D
. at 7
50nm
0 10 20 30 40 50 60 70 800
5
10
15
20
25
30
Time (h)
O.D
. at 6
20nm
/O.D
. at 7
50nm
0 10 20 30 40 50 60 70 800
5
10
15
20
25
30
Time (h)
O.D
. at 6
50nm
/O.D
. at 7
50nm
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
+ Nitrate
– Nitrate
Time (h)
Perc
enta
ge o
f sed
imen
tatio
n (%
)
Proteomic analysis results
HglK (pep.:0; 4)
0 50 100 150 200 250 300 350 4000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Time (h)
O.D
. at 7
50nm
3,0.104
2,5.104
2,0.104
1,5.104
1,0.104
0,5.104
0
Aver
age
abun
danc
e
Time (h) 8 24
PII – N PII + N
Enzymes involved in the porphyrin, cytochrom and chlorophyll anabolic pathway showed lower abundance sugges)ng a reduced ac)vity of this one.
PII (pep.: 2; 2) protein showed a significant (p-‐value<0.05) higher abundance ajer 24 hours of starva)on, sugges)ng a classical regula)on of nitrogen metabolism.
Allophycocyanin β (pep.: 32; 21), NAD(P)H-‐quinone oxidoreductase (pep.: S.U.I: 2; 4 & S.U.K: 3; 2),
Enzymes opera)ng the assembly of PS I (pep.: Ycf3: 1; 2 & Ycf4: 1; 1), Enzyme opera)ng the assembly of thylakoïds Thf1 (pep.: 4; 3), Etc.
I thank De Meur Q. for his art supervising. This research is supported by European Space Agency (Prodex « Melgen-‐3 » project).
Deschoenmaeker F. is a F.R.I.A. PhD student.
*
N.S.