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

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Time (h)

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HglK  (pep.:0;  4)  

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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.