• No results found

The   relative   binding   ratio   (RBR)   of   heterologous   DNA   was   expressed   as   the   percentage   of   the   homologous   binding   calculated   for   the   control   DNA   (RBR=   BRheteroduplex   /   BR   homoduplex   x   100).  

Finally,  for  each  experiment,  the  pooled  standard  deviation  was  calculated  as  √Σ  (X-­‐x)  2/n  (where  X  is  the   mean  of  the  RBR  values,  x  each  single  value,  and  n  the  number  of  single  values  obtained.  

   

5.  Sequencing  of  genes  and  phylogenetic  reconstructions    

5.1.  Sequencing  

Sequencing   of   the   complete   or   partial   genes   analyzed   in   this   work   was   made   in   technical   scientific   services  of  University  and  by  private  DNA  Sequencing  Services  (Genomex  in  France,  Secugen  in  Spain).  In   all  cases,  the  nucleotide  sequences  were  determined  using  the  Big  Dye  Terminator  Cycle  Sequencing  kit   (Applied   Biosystems)   according   to   manufacturer’s   recommendations   and   an   ABIPRISM   310   DNA   sequencer  (Applied  Biosystems).    

 

5.2.  Sequence  analyses  

Sequences   were   revised   and   corrected   with   Sequencher   v   4.7   (Gene   Codes   Corp.).   16S   rRNA   gene   alignments  were  produced  with  the  use  of  the  ARB  software  package  (Ludwig  et  al.,  2004)  (www.arb-­‐

home.de),   introducing   the   new   almost   complete   sequences   into   a   preexisting   alignment   available   of   about  208,000  single  sequences  (Pruesse  et  al.,  2007)  (www.arb-­‐silva.de).    

Housekeeping  gene  sequences  for  multilocus  sequence  analysis  (MLSA)  were  aligned  with  the  use  of  the   program  ClustalX  1.83,  and  the  alignments  were  improved  by  removing  hypervariable  positions  with  the   use   of   the   online   available   program   Gblocks   (http://molevol.ibmb.   csic.es/Gblocks_server.html)   using   the  conditions  previously  published  (Soria-­‐Carrasco  et  al.,  2007).    

 

5.3.  Phylogenetic  reconstructions  of  16S  rRNA  gene  and  concatenated  genes  

Phylogenetic  reconstructions  based  on  DNA  sequence  data  were  performed  using  the  neighbor  joining,   maximum  likelihood,  and  maximum  parsimony  algorithms  as  implemented  in  the  ARB  software  package   (Ludwig  et  al.,  2004),  or  online  (http://atgc.lirmm.fr/phyml/)  by  the  use  of  the  PHYML  program  package   (Guindon  &  Gascuel,  2003).  Topologies  and  branch  lengths  were  optimized  by  the  both  programs.  

Reference  sequences  of  16S  rRNA  gene  were  retrieved  from  the  SILVA  database  (Pruesse  et  al.,  2007).    

Materials  and  Methods    

 

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Type  strain  information  and  reference  alignment  was  extracted  from  the  All-­‐Species  Living  Tree  release   93  (Yarza  et  al.,  2008).  Alignments  were  performed  using  SINA  (Pruesse  et  al.,  2007)  implemented  in  the   ARB  package  (Ludwig  et  al.,  2004)  and  manually  improved  using  the  secondary  structure  ARB-­‐editor  by   following  the  reference  alignment  of  the  Living  Tree  Project.  Multiple  analyses  were  carry  out  to  find   topology   changes   due   to   the   effect   of   the   gene   composition   of   the   alignments   and   to   evaluate   tree   topology   stabilities,   as   previously   recommended   (Ludwig   &   Klenk,   2001).   Bootstrap   values   were   obtained  after  the  calculation  of  100  replicates,  as  implemented  in  the  PHYML  program  package.  

All  analyses  to  coding.protein  genes  were  performed  by  using  the  nucleotide  sequence  alignments,  since   their  translation  into  amino  acids  rendered  a  very  small  number  of  informative  positions.  Using  PHYML   program  package,  all  alignments  were  calculated  by  using  the  HKY  substitution  model  (Hasegawa  et  al.,   1985),  and  the  proportion  of  invariable  sites  and  the  transition/transversion  rates  were  estimated.  The   number   of   substitution   rate   categories   was   4.   Calculations   were   performed   by   using   a   BIONJ   starting   tree  (Guindon  &  Gascuel,  2003).      

   

6.  Protein  extraction  and  quantification    

6.1.  Outer  membrane  protein  extraction  

Membrane  fractions  were  prepared  as  previously  described  (Bucarey  et  al.,  2006;  Lobos  &  Mora,  1991).  

Briefly,   bacteria   were   grown   under   optimal   conditions   (37ºC   with   shaking   at   125   rpm)   to   mid   exponential  phase.  Then,  2  or  4  ml  of  culture  were  taken,  chilled  on  ice,  pelleted  by  centrifugation  at   16.000  g   x   for   10   min   at   4°C.   Supernatant   was   discarded   and   pellet   was   resuspended   in   1   ml   of   lysis   buffer  (10  mM  Tris–HCl  pH  8),  sonicated  on  ice  for  100  seconds  (60%,  30  power,  10    pulses),  and  then   supplemented   with   30   µl   of   Pefabloc   (69.5mM,   Roche,   cat.   no   11429876001).   Whole   cells   and   debris   were   removed   by   low-­‐speed   centrifugation  (4500g,   5   min),   pellet   was   discarded   and   supernatant   was   recovered   in   a   new   tube.   Total   external   membrane   fractions   were   obtained  after   30   min   of   centrifugation  at  16.000  g  at  4°C.    Supernatant  was  discarded  and  pellet  resuspended  in  500  µl  of  fresh   solubilization  buffer  (10  mM  Tris–HCl  pH  8,  10  mM  MgCl2,  2%  triton  X-­‐100  or  Nonidet  40).  Mixture  was   incubated  for  30  min  at  37ºC,  shaking  occasionally.    

     

Materials  and  Methods    

 

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Inner  membrane  proteins  were  solubilized  with  2%  triton  X-­‐100  or  Nonidet  40,  and  the  outer  membrane   fraction  was  pelleted  by  centrifugation  at  16.000  g  for  30  min  at  4ºC,  and  solubilized  in  30  µl  of  100  mM   Tris–HCl  pH  8,  2%  SDS  buffer,  while  supernatants  were  recovered  in  a  new  tube  for  inner  membrane   protein   extraction   (see   bellow).   Outer   membrane   protein   extracts   were   boiled   during   5   min   and   maintained  at  -­‐20ºC  until  use.    

 

6.2.  Inner  membrane  protein  extraction  

Supernatants  recovered  from  outer  membrane  proteins  protocol  (Bucarey  et  al.,  2006;  Lobos  &  Mora,   1991),  containing  the  proteins  associated  with  the  inner  membrane  were  precipitated  by  the  addition  of   2   volumes   of   cold   acetone   and   centrifuged   at   16.000   g   for   10   min   at   4ºC.   Pellets   were   air   dried   and   resuspended  in  30  µl  of  Tris–HCl  100  mM,  pH  8  buffer,  2%  SDS,  then  boiled  during  5  min  and  maintained   at  -­‐20ºC  until  use.    

 

6.3.  Protein  quantification  

Based  on  the  method  described  by  Bradford  (Bradford,  1976),  outer  and  inner  proteins  were  quantified   by  using  Bio-­‐Rad  Protein  Assay  (cat  no.  500-­‐0006).  Dye  reagent  was  diluted  1:4  times  in  distilled  water   and   filtered   through   Whatman   filter   (celullose,   no.1,   grade   >11   µm)   to   remove   all   possible   reagent   particles.    

Five   dilutions   of   Bovine   Serum   Albumin   (BSA)   (0.5   mg   ml-­‐1,   Sigma)   were   prepared   to   elaborate   the   calibration  curve,  whose  concentrations  ranging  from  0  to  75  µg  ml-­‐1  (Table  6).  Samples  and  standards   were   prepared   in   0.15M   of   NaCl   (final   volume   of   200   µl).   After   the   addition   of   2ml   of   diluted   dye   reagent,   samples   were   vigorously   mixed   and   incubated   at   room   temperature   for   at   least   5   min.  

Standards  were  measured  in  duplicate  at  595  nm  in  a  Hitachi  U  2900  spectrophotometer.    

Proteins  samples  were  prepared  by  mixing  8  µl  of  the  extract  with  192  µl  of  0.15  M  NaCl  and  treated  in   the  same  way  as  standards.  A  linear  regression  absorbance/  concentration  was  calculated  and  the  exact   concentrations   of   the   unknown   samples   were   determined   by   interpolation   considering   the   dilution   factor   of   the   samples.   Five   microliters   of   loading   buffer   were   added   to   aliquots   containing   20   µg   of   protein   (30   µl   as   final   volume)   and,   as   indicated   above,   boiled   during   5   min   and   maintained   at   -­‐20ºC   until  use.  

     

Materials  and  Methods    

6.4.  Matrix  Assisted  Laser  Desorption/Ionization-­‐  time  of  flight  Mass  Spectrometry  (MALDI-­‐TOF  MS)   Cultures   (1.5   ml)   were   pelleted   at   16.000   g   for   3   min   and   resuspended   in   500μl   70   %   ethanol.   The  

Materials  and  Methods    

 

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6.4.1.  Mass  spectra  analyses  

All   mass   spectra   profiles   were   grouped   in   a   matrix   and   evaluated   by   Hierarchical   clustering   using   the   software   PRIMER-­‐E   ®   (Plymouth).   Based   on   the   presence   and   absent   of   signals,   a   dendrogram   of   the   mass   spectra   was   obtained   by   single   linkage   agglomerate   similarity  calculations.   In   addition,   the   data   were  analyzed  in  SIMCA-­‐P  11.5  (Umetrics,  Umea,  Sweden).  Supervised  partial  least  square  discriminative   analysis   (PLS-­‐DA)   applying   the   orthogonal   signal   correction   (OSC)   was   used   in   order   to   evaluate   the   dependence  of  signals  with  the  time  and  stress  conditions  (Sjöström  et  al.,  1986;  Stahle  &  Wold,  1987).    

   

7.  Lipopolysaccharide  (LPS)  analysis    

7.1.  LPS  extraction  

LPS   was   prepared   by   following   two   different   methods:   the   first   is   a   modification   of   the   method   described  by  Hitchcock  (Hitchcock  &  Brown,  1983),  in  which  exponential  cultures  of  M8  and  M31  strains   of  S.   ruber   were   harvested   by   centrifugation   and   pellets   were   resuspended   in   water   until   reaching   a  

DO420   nm=   0.4.     Suspensions   (1.5   ml)   were   centrifuged   at   16.000   g   for   10   minutes,   supernatants   were  

discarded  and  pellets  resuspended  in  50  µl  of  lysis  buffer  (2%  SDS,  4%  β-­‐mercaptoethanol,  10%  glycerol,   1M  Tris  pH  6.8,  0.02%  bromophenol-­‐blue)  and  boiled  for  5  min.  After  adding  5  µl  of  proteinase  K  (5  mg   ml-­‐1  in  water  solution,  Roche),  samples  were  incubated  for  1  hr  at  60  ºC.  Finally,  20  µl  of  solution  were   loaded  in  a  12%  polyacrylamide  gel  (see  below).  

In  the  second  method,  proposed  by  Busse  (Busse  et  al.,  1989),  2  ml  of  exponential  cultures  of  M8  and   M31   strains   were   harvested   by   centrifugation   at   16.000   g   for   10   min   at   4°C   and   bacterial   pellet   was   frozen  and  lyophilized.  Freeze-­‐dried  cultures  were  resuspended  in  700  µl  of  lysis  buffer  (0.5M  Tris  pH   6.8,  2%SDS,  10%  glycerol,  0.02%  bromophenol-­‐blue).  Samples  were  mixed  and  boiled  for  5  min.    When   samples  were  cold,  10  µl  of  proteinase  K  (5  mg  ml-­‐1  in  water  solution,  Roche)  were  added  and  incubated   for   1   h   at   65   ºC.   Finally   samples   were   boiled   for   5   min   to   inactivate   proteinase   K.   Samples   were   centrifuged   at   9.300   g   for   10   min,   and   20   µl   of   supernatant-­‐solution   were   loaded   in   a   12%  

polyacrylamide  gel  (see  below).    

       

Materials  and  Methods    

 

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7.2.  Detection  of  LPS  by  silver  staining  

To   check   the   LPS   composition,   extracts   were   subjected   to   the   polyacrylamide   gel   electrophoresis   (Laemmli  1970).  It  included  a  5%  stacking  gel  and  a  12%  separating  gel.  Tris-­‐glycine  buffer  (pH  8.3)  was   used   as   electrode   buffer.   The   electrophoresis   was   carried   out   as   indicated   in   seccion   8.2.  

Lipopolysaccharides  extracts  (20  µl)  were  loaded  into  the  gel  and  the  electrophoresis  was  performed  at   70  V  during  1  hr  and  then  100  V,  until  the  dye  front  reached  the  bottom  of  the  gel  (about  2  h).  After   electrophoresis,  silver  staining  was  carried  out  according  to  Tsai  and  Frasch  (Tsai  &  Frasch,  1982).  The   gel  was  kept  overnight  in  a  fixative  solution  containing  40%  ethanol  and  5%  acetic  acid  in  a  clean  plastic   box.  Next,  the  fixative  solution  was  replaced  by  0.7%  periodic  acid  in  40%  ethanol  and  5%  acetic  acid  to   oxidize   the   LPS   during   5   min.   Subsequently,   three   washes   of   15   min   were   performed   using   double   distilled  water.  Finally,  the  gel  was  immersed  in  fresh  staining  reagent  (150  ml)  for  30  min.  The  staining   reagent  was  prepared  as  follows:  4  ml  of  concentrated  ammonium  and  28  ml  of  0.1  N  sodium  hydroxide   were   added   to   115   ml   of   double   distilled   water   and   5   ml   silver   nitrate   (1g   dissolved   in   5   ml   of   MiliQ   water).  The  concentrated  ammonium  was  added  drop  by  drop  with  magnetic  stirring.  Transient  brown   precipitate  was  formed  when  each  drop  of  silver  nitrate  solution  was  added,  but  it  disappeared  within   seconds.   After   all   silver   nitrate   solution   was   added,   the   staining   reagent   was   added   to   the   gel   and   incubated  under  agitation  at  room  temperature  during  30  min.    After  staining,  three  10  min  washes  in   water   were   performed.   Later,   the   gel   was   immersed   in   300   ml   of   formaldehyde   developer   solution   (containing  75  mg  of  citric  acid  and  0.75  ml  of  37%  formaldehyde)  and  incubated  with  slight  agitation  at   room  temperature  until  the  visualitzation  of  the  LPS  bands.  To  prevent  the  saturation  of  color  in  the  gel,   development  was  stopped  by  adding  1%  acetic  acid  solution  and  incubation  at  room  temperature  during   2  to  5  min.  Finally,  gels  were  dried  by  immersion  in  a  solution  containing  25%  ethanol  and  2%  glycerol   for  20  min.  

                 

Materials  and  Methods    

 

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8.  Electrophoresis    

8.1.  Agarose  gel  electrophoresis  

Nucleic  acids  and  amplification  products  were  resolved  in  1.5%  agarose  gels  (EEO,  Prodanisa)  or  1.5%  

agarose  MS  8  (Prodanisa)  in  1X  TAE  buffer  (40mM  Tris,  20  Mm  acetic  acid,  1  mM  EDTA)  at  5  V/cm.  Five   microliters   of   each   sample   was   mixed   with   10X   loading   buffer   (0.1%   bromophenol   blue,   0.1M   EDTA,   0.1%   SDS,   and   50%   glycerol).   Markers   1kb   (from   250   to   10.000   bp,   Promega),   100-­‐1000   bp   (mbl),   λ   DNA/Hind  III-­‐EcoRI  (from  564  to  21.226  bp,  REAL)  and  λ  DNA/PstI  (from  247  to  11501  bp,  Fermentas)   were  used  as  molecular  weight  references.  

Gels   were   stained   during   10   min   in   fluorescent   dye   ethidium   bromide   solution   (1µg   ml-­‐1),   washed   in   distilled  water  for  20  min  and  visualized  in  an  UV  transiluminator  (Syngene)  at  312  nm.  Pictures  of  gels   were   taken   by   G-­‐BOX   system   (Syngene)   or   Gel   printer   plus   system   (TDI)   and   printed   in   Sony   digital   graphic  printer  UP-­‐D897.  

 

8.2.  Polyacrylamide  gel  electrophoresis  (SDS-­‐PAGE)  

Sodium   dodecyl   sulfate   polyacrylamide   electrophoresis   was   performed   by   the   method   of   Laemmli   (Laemmli,  1970),  with  5%  (w/v)  of  30%  acrylamide  mix  (acrylamide/bis  29:1  (Biorad))  in  the  stacking  gel   and   12%   or   12.5%   of   30%   acrylamide   mix   (Biorad)   in   the   resolving   gel.   Compositions   of   stacking   and   resolving  gels  are  shown  in  table  7.  Only  outer  membrane  proteins  were  separated  in  12.5%  resolving   gel   that   also   contained   NaCl   for   a   better   visualization   as   recommended   by   Lobos   and   Mora   (Lobos   &  

Mora,  1991).  Extract  proteins  samples  (20  µg  in  a  maximum  volume  of  30  µl)    were  mixed  with  5  µl  of   loading   buffer   (65.5   mM   Tris-­‐HCl   pH   6.75,   20%   glycerol,   4%   SDS,   0.05%   β-­‐mercaptoethanol,   0.002%  

bromophenol-­‐blue),  and  boiled  during  5  min  before  loading  onto  the  gel.  

Electrophoresis  was  performed  in  a  Protean  dual-­‐vertical-­‐slab-­‐gel  apparatus  (Bio-­‐Rad)  with  1X  running   Tris-­‐Glycine  buffer  (3.4%  Tris,  14%  glycine,  and  1%  SDS)  at  a  constant  voltage  of  70V  for  the  stacking  gel   (1  hour)  and  100  V  for  the  resolving  gel  during  4  h.  Gels  were  rinsed  in  distilled  water  for  few  minutes,   fixed  for  20-­‐30  min  in  the  fixing  solution  (10%  trichloroacetic  acid,  50%  isopropanol  alcohol)  and  stained   over   night   with   Coomasie   blue   solution   (BioRad).   Gels   were   unstained   in   10%   acetic   acid,   15%  

isopropanol  solution  or  in  distilled  water.  Protein-­‐patterns  of  samples  were  analyzed  two  times  and  in   some  cases  four  times.  

   

Materials  and  Methods    

8.3.  Denaturing  Gradient  Gel  Electrophoresis  (DGGE)  

One   milimeter   thick   6%   (w/v)   polyacrilamide   gels   (40%   acrylamide/bis   37.5   :1   (Biorad))   with   40-­‐80%  

Materials  and  Methods    

 

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9.  Physiological  and  biochemical  tests    

Salt  requirement  for  growth  was  determined  by  growing  the  isolates  in  liquid  SW  medium  with  different   concentrations  of  NaCl  (5,  10,  15,  20,  25  and  30%).  Similarly,  the  requirement  for  Mg+2  was  tested  in   medium   containing   MgCl2  at   the   following   concentrations:   0,   5,   10,   50,   170   and   400   mM.   The   experiments  were  performed  in  duplicate  in  96-­‐well  microtiter  plates.  Two  hundred  microliters  of  each   medium  were  inoculated  with  5  µl  of  a  well  grown  suspension  of  each  strain  (OD600nm  =  1.0).  Growth  was   monitored   spectrophotometrically   at   600   nm   after   the   incubation   period   (7–14   days   at   37ºC   without   shaking).  The  pH  range  for  growth  was  determined  in  liquid  25%  SW  medium  adjusted  to  the  desired  pH   values  (5.0,  5.2,  5.8,  6.2,  7.2,  7.4,  8.2  and  8.5).  The  microtiter  plates  were  inoculated  and  incubated  for  4   weeks  as  described  above.  Catalase  production  was  performed  by  suspending  single  colonies  in  3%  (v/v)   hydrogen   peroxide;   production   of   gas   was   checked   for   up   to   5  min   post-­‐hydrogen   peroxide   addition,   while  cytochrome  oxidase  production  was  evaluated  by  moisting  a  piece  of  filter  paper  with  N,N,N',N'-­‐

tetramethyl-­‐p-­‐phenylenediamine   solution   (1%)   and   spreading   a   colony   on   the   moisten   filter   paper   (Smibert   &   Krieg,   1994;   Stan-­‐Lotter   et   al.,   2002).   The   analytical   systems   API   ZYM   and   API   20NE   (BioMerieux)   were   used   for   the   analysis   of   additional   enzyme   activities   and   for   assimilation   tests   respectively  (Stan-­‐Lotter  et  al.,  2002).  

Strips   were   inoculated   with   a   cell   culture   grown   in   4   M   NaCl   and   brought   to   a   suspension   density   recommended  by  the  manufacturers,  incubated  for  up  to  24  h  (API  ZYM)  or  3  weeks  (API  20NE).  Tests   were  performed  by  triplicate  for  each  strain.  

   

10.  Metabolomics    

10.1.  Metabolite  extracts  preparation  

A  total  of  3ml  of  cell  suspension  grown  on  liquid  media  were  collected  by  centrifugation  (16,000  g,  2  min   at   4   ºC).   Cell-­‐free   supernatant   (2   ml)   was   stored   for   further   chromatographic   extraction.   Supernatant   was  acidified  by  the  addition  of  50  ml  of  98–100%  formic  acid  (Merck  KGaA).  Pelleted  biomass  was  then   resuspended  in  1ml  of  bidistilled  water,  and  sonicated  to  obtain  a  clear  lysate  extract.  This  lysate  was   then  acidified  by  the  addition  of  50  ml  of  98–100%  formic  acid.  After  the  acidification,  the  clear  lysate   formed  insoluble  aggregates  that  could  be  separated  from  the  soluble  fraction  by  centrifugation.    

 

Materials  and  Methods    

 

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The  clear  supernatant  was  stored  for  further  fractionation,  and  the  insoluble  pellet  was  resuspended  in   500   ml   of   methanol.   Sample   preparation   resulted   in   three   complementary   fractions:   the   extracellular   (E),  cellular  soluble  (CS)  and  cellular  insoluble  (CI)  fractions.  

 

10.2.  Solid-­‐phase  extraction  

Both  acidified  extracellular  and  cellular  soluble  fractions  were  solid  phase  extracted  using  Bond  Elut  C18   columns  (Varian  Inc).  This  chromatography  enables  the  isolation  of  the  organic  molecules  on  the  basis  of   their  nonspecific  interaction  and  retention  to  the  C18  material.  Columns  were  activated  by  washing  the   column   with   2ml   of   methanol,   followed   by   2ml   MilliQ   water   and   finally   2ml   1%   formic   acid   in   MilliQ   water.    Fractions  were  mixed  with  3  ml  of  1%  formic  acid  and  placed  within  the  columns  to  bind  organics   compounds.  This  purification  procedure  removes  the  high-­‐salt  charge  of  the  media  and  extracts,  which   may   interfere   during   the   electrospray   procedure   by   ion   suppression   (Li   et   al.,   2006).   Columns   were   finally   washed   with   2ml   of   1%   formic   acid   and   the   retained   fraction   was   recovered   with   2ml   of   methanol.  

 

10.3.  ICR-­‐FT/MS  procedure  

Broad  band  mass  spectra  were  acquired  on  a  Bruker  (Bremen,  Germany)  APEX  Qe  ICR-­‐FT/MS  with  12  T   superconducting   magnet   and   an   Apollo   I   electrospray   (ESI)   source,   whereas   high-­‐resolution   spectra   were  acquired  with  an  Apollo  II  ESI  source  in  positive  and  negative  modes.  The  samples  were  infused  in   methanol  with  a  microelectrospray  source  at  a  flow  rate  of  120  ml  h-­‐1  with  a  nebulizer  gas  pressure  of   20  p.s.i.  and  a  drying  gas  pressure  of  15  p.s.i.  (200  ºC).  Spectra  were  externally  calibrated  on  clusters  of   arginine  (10  mg  L-­‐1  in  methanol),  and  calibration  errors  in  the  relevant  mass  ranges  were  always  below   100  p.p.b.,  which  is  the  pre-­‐requisite  for  an  adequate  elementary  composition  assignment.  

Relative  standard  deviation  in  the  intensity  values  of  the  peaks  was  routinely  lower  than  5%  in  these   analysis  conditions.  The  spectra  were  acquired  with  a  time  domain  of  1  megaword  (where  1  data  word   corresponds  to  32  bits)  with  a  mass  range  of  150–  2000m/z.  The  spectra  were  zero  filled  to  a  processing   size  of  2  megawords.  A  sine  apodization  (technical  term  for  changing  the  shape  of  an  electrical  signal)   was  performed  before  Fourier  transformation  of  the  time  domain  transient.  The  ion  accumulation  time   in   the   ion   source   was   set   to   0.2   s   and   1024   scans   were   accumulated   for   one   spectrum.   ICR-­‐FT/MS   spectra   were   exported   to   peak   lists   at   a   signal-­‐to-­‐noise   ratio   (S/N)=1.   From   these   lists,   possible   elemental   formulas   were   calculated   for   each   peak   in   batch   mode   by   a   software   tool   written   in   Helmholtz  Zentrum  München  (Formulae®).    

Materials  and  Methods    

 

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The  generated  formulas  were  validated  by  setting  sensible  chemical  constraints  (nitrogen  rule,  atomic   oxygen   to   carbon   ratio   O/C   ≤   1,   element   counts:   hydrogen   H≤   (2+C2),   carbon   C≤   100,   oxygen   O≤80,   nitrogen   N≤   5   and   sulfur   S≤   1)   and   only   the   masses   in   conjunction   with   their   automated   generated   theoretical  isotope  pattern  (existence  of  the  13C  isotope)  were  taken  into  consideration  (Hertkorn  et  al.,   2007).   The   obtained   reduced   peak   lists   were   compared   in   m/z   at   5   p.p.m.   and   the   corresponding   intensity  matrices  were  generated  for  further  statistical  analysis.  

 

10.4.  Statistical  analysis  

The  data  were  imported  and  analyzed  in  SIMCA-­‐P  11.5  (Umetrics,  Umea,  Sweden)  and  SAS  version  9.1   (SAS   Institute   Inc.,   Cary,   NC,   USA).   Different   multivariate   techniques   from   unsupervised   principal   component  analysis  (PCA)  to  supervised  partial  least  square  discriminative  analysis  (PLS-­‐DA)  were  used   in   order   to   reduce   the   different   datasets   and   extrapolate   informative   masses   from   the   different   experimental  conditions  (Sjöström  et  al.,  1986;  Stahle  &  Wold,  1987)  (Kemsley,  1996;  Vong  et  al.,  1988).  

PCA   is   a   mathematical   procedure   that   uses   an   orthogonal   transformation   to   convert   a   set   of   observations   of   possibly   correlated   variables   into   a   set   of   values   of   uncorrelated   variables   called   principal   components.   Partial   least   squares   (PLS)   projections   to   latent   structures   are   a   regression   extension  of  PCA  (Wold  et  al.,  1987).  It  uses  the  X  variables  (matrix  of  masses)  as  predictors,  and  dummy   variables  (belonging  or  not  belonging  to  a  given  class  coded  as  1/0;  that  is,  origin  of  isolation,  stage  of   growth,  and  stress  condition)  as  response  variables  (Y  variables).  Besides,  the  PLS-­‐DA  and  PLS  (applying   the  orthogonal  signal  correction  (OSC))  modelling  could  be  used  to  determine  the  relative  concentration   of   the   metabolites   of   interest.   PLS-­‐DA   are   the   modelling   often   used   in   metabolomic   field   for   classification  of  the  samples  (Barker  et  al.  2003;  Bylesjö  et  al.  2006;  Trygg  and  Wold  et  al.  2002a,  b).  

In   all   cases,   three   modalities   (extracellular,   cellular   soluble   and   cellular   insoluble)   were   calculated   independently  but  in  each  analyzed  dataset  only  a  fraction  was  chosen  as  the  descriptive  power  of  the   model.  The  descriptive  power  can  be  defined  by  several  terms,  most  directly  the  fraction  of  the  sum  of   squares  (SS)  of  all  the  Y  explained  by  the  current  component  (R2Y(cum))  and  Q2(cum).  R2Y  provides  an   estimate   of   how   well   the   model   fits   the   Y   data,   and   Q2   provides   an   estimate   of   how   well   the   model   predicts  the  Y  data.  Pareto  scaling  of  the  intensity  values  with  a  logarithmic  transformation  of  the  data   was   chosen   in   some   cases   to   consider   all   masses   equally,   including   those   with   medium-­‐   and   low-­‐

intensity  values  (van  den  Berg  et  al.,  2006).    

   

Materials  and  Methods    

 

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Score  scatter  plots  and  loading  plots  were  generated.  The  score  scatter  plots  present  a  view  of  how  well   the   classes   are   separated   on   the   basis   of   their   x   variables.   In   the   loading   plots,   the   different   masses   characteristic   for   each   of   the   classes   are   differentiated.   From   these   analyses,   a   list   of   discriminative   masses   (m/z)   for   the   different   geographical   area,   growth   stage   or   stress   conditions   were   chosen   according  to  their  correlation  coefficient  value.    Those  having  the  highest  coefficients  were  considered   to  be  relevant  (that  is,  variables  (m/z)  with  a  correlation  value  higher  than  |0.002|).    

Interpretation   of   the   regression   coefficients   provides   information   pertaining   to   the   metabolic   explanation   of   class   differences   (Holmes   and   Antti,   2002)   based   on   the   fact   that   each   coefficient   is   related   to   a   specific   elemental   composition.   Thus,   masses   associated   with   the   highest   correlation   coefficient   were   represented   in   the   van   Krevelen   projection   in   order   to   visualize   their   differences   in   chemical  composition  (H/C  versus  O/C)  (Holmes  &  Antti,  2002;  Wu  et  al.,  2004).    

 

10.5.  Metabolites  identification  

Lists   of   discriminative   masses   were   evaluated   and   assigned   with   the   use   of   bioinformatics   tools   as   MassTRIX   (www.masstrix.org)   (Suhre   &   Schmitt-­‐Kopplin,   2008)   and   the   Japanese   metabolome   (www.metabolome.jp)   databases.   The   masses   that   were   found   to   be   discriminative   for   each   of   the   different   stress   responses   were   crosschecked   using   the   MassTRIX   database   and   the   annotation   was   done  using  the  published  genome  of  the  M31  strain  as  a  reference  (Mongodin  et  al.  2005).  Additionally   MassTRIX  gave  valuable  information  through  the  identification  of  certain  metabolites;  in  the  meanwhile   it  was  possible  to  characterize  the  experiments  in  relation  with  its  biological  context.    

   

11.  Microscopy  techniques    

Cell   numbers   and   morphology   of   the   organisms   were   observed   under   an   optic   microscope   Zeiss   Axio   Imager  A1.  

 

11.1.  Sample  fixation    

Culture   samples   (900   µl)   were   fixed   at   4ºC   during   16   h,   with   37%   formaldehyde   (Sigma)   in   order   to   obtain  a  final  concentration  of  4%  formaldehyde  (Antón  et  al.,  1999).  Samples  for  fluorescence  in  situ   hybridization  analyses  were  pelleted  at  16.000  g  for  10  min  and  resuspended  in  cold  ice  50%  PBS  4X-­‐  

ethanol  solution  and  storaged  at  -­‐20ºC  until  use.  

Materials  and  Methods    

 

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Before  staining,  all  samples  were  diluted  (10-­‐1  and  10-­‐2)  in  sterile  4X  PBS,  and  100  µl  of  suspensions  were   filtered  thought  GTTP  Isopore  filters  of  0.22  µm  pore  size  and  16  mm  diameter  (Milipore).  Filters  were   stored  at  -­‐20ºC  until  use.  

 

11.2.  4’-­‐6-­‐diamidino-­‐2-­‐phenylindole  stain  (DAPI)  

In  order  to  determine  the  number  of  cells  present  in  each  culture,  a  piece  of  filter  was  stained  with  25-­‐

30  µl  of  4'-­‐6-­‐diamidino-­‐2-­‐phenylindole  solution  (1  µg  ml-­‐1)(Porter  &  Feig,  1980)  during  1.5  min  at  room   temperature.   Then,   washed   with   sterile   MiliQ   water   and   absolute   ethanol   and   dried   at   room   temperature   and   darkness.   Finally,   filter   was   mounted   with   a   drop   of   Citiflour   AF1   (Citifluor   ltd)   and   covered  in  a  microscope  slide.  Cells  were  quantified  using  a  fluorescence  microscope  (Axio  imager.A1,  

30  µl  of  4'-­‐6-­‐diamidino-­‐2-­‐phenylindole  solution  (1  µg  ml-­‐1)(Porter  &  Feig,  1980)  during  1.5  min  at  room   temperature.   Then,   washed   with   sterile   MiliQ   water   and   absolute   ethanol   and   dried   at   room   temperature   and   darkness.   Finally,   filter   was   mounted   with   a   drop   of   Citiflour   AF1   (Citifluor   ltd)   and   covered  in  a  microscope  slide.  Cells  were  quantified  using  a  fluorescence  microscope  (Axio  imager.A1,