• No results found

Amplifications   with   archaeal   primers   were   carried   out   under   the   following   conditions:   20   cycles   (denaturation  at  94º  C  for  5  min,  annealing  at  75º  C  for  1  min  (with  a  touchdown  gradient  of  -­‐0.5ºC  per   cycle),  extension  at  72º  C  for  1  min),  preceded  by  steps  of  denaturation  (5  min    at  94º  C,    5  min  at  80º  C,   1  min  at  65ºC,  3  min  at  72ºC  and  4  min  at  94ºC)  and  followed  by  14  cycles  of  1  min    at  94º  C,    1  min  at   66º  C  and  3  min  at  72ºC.  In  addition,  the  reaction  was  completed  by  a  final  extension  of  10  min  at  72ºC   (Micaela  García  thesis,  2009).    

 

3.5.  Purification  of  PCR  products  

PCR   products   obtained   were   purified   with   Qiaquick   PCR   purification   kit   (Qiagen,   cat.no   28104)   and   MSB®  Spin  PCR  apace  kit  (Invitek,  cat.  nº  1020220200)  according  to  the  manufacturer’s  protocol.    

                                           

 

 

-­‐47-­‐  

 

                                             Table  5:    Characteristics  of  the  primers  used  in  this  study      

Name   Specificity   Sensea  and  

positionb   target   Tm  for  

PCR   Sequence  (5´-­‐3´)   Reference  

21F   Archaea   F:    7-­‐26   16S  rDNA   TTCCGGTTGATCCTGCCGGA   García-­‐Martínez  et  al.,  2000  

1492R   Arch-­‐Bact   R:  1492-­‐1509   16S  rDNA   55ºC  

TACGGYTACCTTGTTACG   Muyzer  et  al.,  1995  

344F   Archaea   F:  344-­‐363   16S  rDNA   ACGGGGYGCAGCAGGCGCGA   Raskin  et  al.,  1994  

915R   Archaea   R:  915-­‐935   16S  rDNA   66ºC  

GTGCTCCCCCGCCAATTCCT   Stalh  and  Amann,  1991  

915F   Archaea   F:915-­‐934   16S  rDNA   60ºCc   GTGCTCCCCCGCCAATTCC   Stalh  and  Amann,  1991  

Euryclus   Archaea   R:  24-­‐26   23S  rDNA   55ºC   TCGCAGCTTRSCACGYCCTTC   Benlloch  et  al.,  2001  

344-­‐CG   Archaea   F:  344-­‐363   16SrDNA   75-­‐65ºC   CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGC

CCCCGCCCGACGGGGYGCAGCAGGCGCGA   Casamayor  et  al,  2000  

GM5-­‐GC   Bacteria   F:  341-­‐357   16SrDNA   65-­‐55ºC   CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGC

CCCCGCCCGCCTACGGGAGGCAGCAG   Muyzer  et  al.,  1993  

GM3   Bacteria   F:  8–23   16S  rDNA   AGAGTTTGATCMTGGC   Muyzer  et  al.,  1995  

GM4   Bacteria   R:  1492-­‐1507   16S  rDNA   47  ºC  

TACCTTGTTACGACTT   Muyzer  et  al.,  1995  

RAPD1   Bacteria   unknown   16S  rDNA   55  ºC   TGCGAACTGTTGGGAAGGG   Sikorski  et  al.;  1999  

RAPD2   Bacteria   unknown   16S  rDNA   55  ºC   CGAGCTTCGCGTACCACCCC   Sikorski  et  al.;  1999  

RAPD3   Bacteria   unknown   16S  rDNA   55  ºC   CGCTGCGGTTGCGCGCCGCC   Sikorski  et  al.;  1999  

RAPD4   Bacteria   unknown   16S  rDNA   55  ºC   CTCAATGGCAGCGGCTATGG   Sikorski  et  al.;  1999  

 

 

-­‐48-­‐  

 

Table  5:    Characteristics  of  the  primers  used  in  this  study  

Name   Specificity   Sensea  and  

positionb   target   Tm  for  

PCR   Sequence  (5´-­‐3´)   Reference  

RAPD5   Bacteria   unknown   16S  rDNA   55  ºC   GTTTCGCTCGATGCGCTACC   Sikorski  et  al.;  1999  

RAPD6   Bacteria   unknown   16S  rDNA   55  ºC   CGGCACACTG  TTCCTCGACG   Sikorski  et  al.;  1999  

BOX1AR   Bacteria   unknown   16S  rDNA   55  ºC   CTACGGCAAGGCGACGCT   Sikorski  et  al.;  1999  

ERIC1R   Bacteria   unknown   16S  rDNA   55  ºC   ATGTAAGCTCCTGGGGATTCAC   Sikorski  et  al.;  1999  

ERIC2   Bacteria   unknown   16S  rDNA   55  ºC   AAGTAAGTGACTGGGGTGAGCG   Sikorski  et  al.;  1999  

ef-­‐2_634F   Archaea   634-­‐652   ef-­‐2   TCC  GCG  CTB  TAY  AAS  TGG   Papke  et  al.,  2004  

ef-­‐2_1147R   Archaea   1126-­‐1147   ef-­‐2   50ºC  

GTG  GTC  GAT  GGW  YTC  GAA  HGG   Papke  et  al.,  2004   radA_prSJS252F   Archaea   252-­‐284   radA   ACS  GAR  KTS  TWC  GGS  GAR  TTC  GGS  KCS  

GGS  AA   Papke  et  al.,  2004  

radA_prSJS253R   Archaea   226-­‐253   radA   50ºC  

GTC  SGG  GTT  SGM  SAM  SAC  CTG  GTT  SGT   Papke  et  al.,  2004  

secY_356F   Archaea   356-­‐376   secY   TCT  ATC  AGG  GVB  YBC  AGA  AG   Papke  et  al.,  2004  

secY_914R   Archaea   896-­‐914   secY   50ºC  

CGA  ACG  AGK  ATM  AYB  GGC   Papke  et  al.,  2004  

atpB_409F   Archaea   F:  409-­‐436   atpB   GACATCGTCGGTGAGSCVATSAACCC   Papke  et  al.,  2004  

atpB_906R   Archaea   R  :  906-­‐927   atpB   50ºC  

GCCAGGTCVGTRTACATGTA   Papke  et  al.,  2004  

gyrB_43F   Archaea   F:  43-­‐59   gyrB   ATCGACGAGGCGCTT   This  work  

gyrB_1299R   Archaea   R:1299-­‐1318   gyrB   55ºC   CGGGTGTTTCTCGACGTT   This  work  

 

 

-­‐49-­‐  

 

Table  5:    Characteristics  of  the  primers  used  in  this  study   Name   Specificity   Sensea  and  

positionb   target   Tm  for  

PCR   Sequence  (5´-­‐3´)   Reference  

gyrB_339cF   Archaea   F:  339-­‐358   gyrB   54ºCc   TCGTCGAGCGGTTTCAGG   This  work  

gyrB_998cR   Archaea   R:  998-­‐1017   gyrB   48ºCc   GCCCTCGAAGTGATCATG   This  work  

eno_F   Bacteroidetes   F:100210-­‐228   eno   TCG  ACG  AGG  CTA  TCC  GAC   This  work  

eno_R   Bacteroidetes   R:998579-­‐597   eno   60  ºC  

GGC  GTG  CCG  CTA  TAC  CGC   This  work  

tuf_F   Bacteroidetes   F:1317810-­‐825   tuf   GAC  CAC  GGG  AAG  ACG   This  work  

tuf_R   Bacteroidetes   R:1318933-­‐948   tuf   48ºC  

GTT  ATC  CCC  CGG  CAT   This  work  

pyrG_F   Bacteroidetes   F:724839-­‐851   pyrG   CCG  TGC  AGA  CCA  AGT   This  work  

pyrG_R   Bacteroidetes   R:726518-­‐533   pyrG   50ºC  

CAT  GTC  GAC  CGA  CGC   This  work  

gap_F   Bacteroidetes   F:1512707-­‐722   gap   GCT  TGG  AAT  TAA  TGG   This  work  

gap_R   Bacteroidetes   R:1511687-­‐702   gap   48ºC  

GCC  GCT  CCA  CGA  GAT   This  work  

glyA_F   Bacteroidetes   F:986903-­‐918   glyA   CGC  TCC  GCA  ACC  AAG   This  work  

glyA_R   Bacteroidetes   R:988178   glyA  

48ºC  

CGT  ACA  GCG  GGT  GCT   This  work  

rpsE_F   Bacteroidetes   F:1328848-­‐863   rpsE   GGC  GGA  TCG  AAA  AGA   This  work  

rpsE_R   Bacteroidetes   R:1329375-­‐360   rpsE  

48ºC  

CCC  GTT  GAA  CAC  CTT   This  work  

 

 

-­‐50-­‐  

 

Table  5:    Characteristics  of  the  primers  used  in  this  study   Name   Specificity   Sensea  and  

positionb   target   Tm  for  

PCR   Sequence  (5´-­‐3´)   Reference  

rpsC_F   Bacteroidetes   F:1323483-­‐495   rpsC   CCC  GTG  GGC  CAG  AAA   This  work  

rpsC_R   Bacteroidetes   R:1324153-­‐168   rpsC   48ºC  

GCT  GGG  GCG  ACT  CCT   This  work  

groEL_F   Bacteroidetes   F:314922-­‐937   groEL   ACG  AAG  GAC  GGC  GTC   This  work  

groEL_R   Bacteroidetes   R:  316619-­‐634   groEL   50ºC   GAC  CTT  CGT  CGG  GTC   This  work  

uvrA_F   Bacteroidetes   F:604024-­‐039   uvrA   AAC  CCG  CGC  TCG  ACG   This  work  

uvrA_R   Bacteroidetes   R:  601362-­‐377   uvrA   54-­‐56ºC  

CTC  CTC  GGG  CGT  GCC   This  work  

pgk_F   Bacteroidetes   F:  1511623-­‐638   pgk   GCT  TGG  AAT  TAA  TGG   This  work  

pgk_R   Bacteroidetes   R:  1510412-­‐427   pgk  

48ºC  

GCC  GCT  CCA  CGA  GAT   This  work  

thrS_F   Bacteroidetes   F:  3458674-­‐689   thrS   GCA  TCG  ACA  TCA  CCC   This  work  

thrS_R   Bacteroidetes   R:3460872-­‐887   thrS   50ºC  

GCG  TCG  GCT  CGA  ACT   This  work  

recA_F   Bacteroidetes   F:1908864-­‐878   recA   CG  CTC  GAC  KAC  GCS   This  work  

recA_R   Bacteroidetes   R:1907794-­‐809   recA   52ºC  

GTT  YTC  RCG  YCC  CTG   This  work    

                                                                       a      F,  forward;  R,  reverse  

                                                                       b  Referred  to  Escherichia  coli  (for  bacterial  primers),  Halobacterium  halobium  (for  archaeal  primers)  16S  or  23S  rRNA  nucleotide  position  and  S.  ruber  M31T  genome  (for  Bacteroidetes).    

                                                                       c    for  sequencing  

 

Materials  and  Methods    

 

-­‐51-­‐  

 

4.  Whole-­‐DNA  analyses    

4.1.  Determination  of  G+C  mol  percentage  

The  G+C  content  of  the  strains  that  were  isolated  from  nostrils  was  analyzed  by  hydrolyszing  the  DNA  to   its  nucleosides  and  quantifiying  them  by  high-­‐performance  liquid  chromatography  (HPLC).  To  determine   the   base   composition   of   the   DNAs,   a   previously   reported   reversed-­‐phase   HPLC   method   (Tamaoka   &  

Komagata,  1984;  Ziemke  et  al.,  1998)  was  followed  with  some  modifications  (Urdiain  et  al.,  2008).    

About  1-­‐3  µg  of  DNA  were  diluted  in  a  10µl  volume  of  water.  Each  sample  was  boiled  for  about  5  min   and  immediately  chilled  on  ice.  To  the  denature  DNA  mixture,  10  µl  nuclease  S1  (Roche)  at  4  units  µl-­‐1  in   hydrolysis  buffer  (33mM  sodium  acetate,  50mM  NaCl,  and  0.033mM  ZnSO4,  pH  4.5)  were  added,  and   the  solution  was  incubated  for  1h  at  37ºC.  Following  the  DNA  hydrolysis,  12  µl  alkaline  phosphatase  at  2   units  µl-­‐1  (Ammersham)  in  dephosphorylation  buffer  (0.1M  Tris–HCl,  pH8.1)  were  added  to  the  mixture   and   incubated   for   2h   at   37ºC.   Standards   were   prepared   from   commercial   deoxynu-­‐   cleotides   (dATP,   dCTP,  dGTP,  dCTP,  PCR  grade;  Roche).  Equimolar  AT  and  GC  mixtures  at  a  final  concentration  of  1  nmol   µl-­‐1   for   each   nucleotide   were   used   to   prepare   different   standard   G+C   mole   percentages   (20–70%).  

Standards  were  treated  as  the  DNA  samples,  but  omitting  the  addition  of  the  nuclease  S1  enzyme.  The   final  hydrolysis  mixtures  of  60  µl  were  deproteinized  by  the  addition  of  an  equal  volume  of  chloroform:  

isoamylalcohol   (24:1).   After   centrifugation   (5   min   at   16,000   g)   the   supernatants   were   collected   and   stored  frozen  until  their  injection  in  the  HPLC.  Nucleoside  mixtures  were  separated  by  reversed-­‐phase   chromatography   using   a   C-­‐18   column   (Aventis)   in   a   HPLC   system   (Watersdetector,   PDA2996),   using   a   mobile  phase  of  0.1M  (NH4)  H2PO4  pH  4  with  5%  acetonitrile  (Panreac),  at  a  flow  rate  of  1ml  min-­‐1.  The   chromatography  was  completed  in  8–10min.  

 

4.2.  DNA-­‐DNA  hybridizations  (DDH)  

DDH  experiments  were  carried  out  following  a  microtiter  plate  non-­‐radioactive  method.  

 

4.2.1.  Labelling  and  separation  of  DNA  

Reference  Halococcus  strains  DNAs  were  double-­‐labelled  using  DIG-­‐11-­‐dUTP  (Roche,  cat.  no.  1093088)   and  biotin-­‐16-­‐dUTP  (Roche,  cat.  no.  1  093070)  by  using  the  nick-­‐translation  kit  (Roche,  cat.  no.  976776).  

The  optimum  ratio  of  the  nucleotide  mixture  was  0.75  µl  DIG-­‐11-­‐dUTP:  0.25  µl  biotin-­‐16-­‐dUTP  (vol:  vol).  

DNA  (2  µg)  was  labelled  according  to  the  manufacturer’s  recommendations  (90  min).  Labelled  DNA  was   precipitated   with   890   µl   ethanol,   45µl   of   3M   sodium   acetate   pH   7,   and   380   µl   sterile   Milli   Q   water,   mixed  by  inverting  and  frozen  at  -­‐20ºC  for  30  min.    

Materials  and  Methods    

 

-­‐52-­‐  

 

Then,   labeled   DNA   was   centrifuged   for   15   min   at   16.000   g,   supernatant   was   discharged   and   the   dry   pellet  was  resuspended  in  200  µl  sterile  Milli  Q  water.    Two  microlitres  of  the  suspension  were  diluted  in   800  µl  PBS,  and  400  µl  of  the  dilution  were  mixed  with  400  µl  PBS.  Finally,  200  µl  of  each  of  the  two   dilutions   were   used   to   measure   the   labbeling   efficiency   following   the   microtitre   detection   protocol   mentioned   below.   DDH   experiments   were   performed   using   a   modification   of   the   Lind   and   Ursing   method   (Rocap   et   al.,   2003).   Unlabelled   DNAs   from   nostrils   isolated   strains   were   first   diluted   to   a   concentration   of   0.3   µg   ml-­‐1.   Target   DNAs   were   simultaneously   prepared   with   the   same   spectrophotometer   in   order   to   standardize   working   concentrations,   and   avoid   biases   due   to   concentration   measurement   techniques.   Any   single   DDH   set   contained   at   least   one   homologous   reaction,  in  addition  to  all  the  different  mixtures  to  be  assayed.  Fifteen  micrograms  of  unlabelled  DNA   were  mixed  in  a  0.2  µl  reaction  cap  with  100–150  ng  of  labeled  DNA  (between  10  and  15  µl  depending   on   the   labelling   efficiency),   and   filled   to   72   µl   with   sterile   milliQ   water.   The   mixed   solution   was   denatured  by  boiling  for  5  min  and  immediately  chilled  on  ice.  After  a  short  spin  of  the  DNA  mixture,  28   µl  of  1M  phosphate  buffer  (PB;  decreasing  the  pH  of  a  1M  Na2HPO4  solution  with  1M  NaH2PO4  to  pH  6.9)   were  added  and  mixed  by  vortexing.  The  100  µl  hybridization  mixtures  were  covered  with  50  µl  of  light   mineral  oil  (Sigma)  in  order  to  avoid  evaporation  and  volume  changes  during  incubation.    

Finally,   all   the   solutions   were   incubated   for   16h   at   30ºC   (non-­‐restrictive),   below   the   melting   point   temperature   (Tm)   of   the   homologous   DNA   in   0.28M   PB   (Rosselló-­‐Móra,   2006).   The   Tm   of   each   homologous   DNA   was   calculated   from   its   G+C   mole   content   [Tm=(G+C+182.2)/   2.44],   in   this   case   considering  a  G+C  content  of  61%  mol  content  for  all  strains  and  resulting  in  69ºC.  Single-­‐  and  double-­‐

stranded   DNA   were   eluted   on   hydroxyapatite   (HA)   following   the   scaled   batch   procedure   previously   reported  (Ziemke  et  al.,  1998).    

 

Prior   to   chain   separation,   HA   (DNA   grade   Bio-­‐Gel   HTP,   Bio-­‐Rad)   was   equilibrated   with   0.14M   PB   as   follows:  1g  HA  was  mixed  with  10ml  PB  and  vigorously  shaken.  Aliquots  of  200  µl  (two  for  each  single   mixture  with  about  20mg  HA  dry  weight)  were  transferred  to  1.5ml  microtubes  and  centrifuged  for  1   min  at  13,000g.  Clear  supernatant  was  removed,  and  the  wet  HA  was  stored  for  chain  separation.  Each   DDH  mixture  was  diluted  with  sterile  Milli  Q  water  to  a  final  volume  of  0.2ml,  with  a  final  PB  ion  strength   of  0.14M.  Two  50  µl  aliquots  of  each  single  DDH  mixture  were  transferred  to  two  microtubes  containing   equilibrated  HA,  respectively.  Transfer  was  carried  out  carefully  in  order  to  avoid  introducing  mineral  oil   that   had   been   previously   wiped   from   the   pipette   tip   with   a   clean   tissue.   The   DDH   solution   was   well   mixed  with  the  HA  and  incubated  for  15  min  at  35  ºC  below  the  association  temperature  (64.8  ºC).    

Materials  and  Methods    

 

-­‐53-­‐  

 

During   incubation,   double-­‐stranded   DNA   was   bound   to   HA   but,   to   optimize   the   procedure,   the   suspensions  were  mixed  repeatedly,  since  the  HA  tended  to  sediment  out  very  quickly.  After  incubation,   450  µl  of  PB  (0.14M,  0.2%SDS)  were  added  to  each  tube,  mixed  thoroughly  and  incubated  with  repeated   mixing  for  5min  at  the  same  temperature.  Single  stranded  DNA  remained  unbound  in  the  supernatant,   and  was  collected  after  centrifugation  (2  min  at  13,000g)  and  stored  in  a  clean  tube.  The  HA  pellet  was   once  more  washed  with  500  µl  PB  (0.14M,  0.2%  SDS)  at  the  same  temperature  and  for  the  same  time.  

After   centrifugation,   the   500   ml   of   supernatant   were   added   to   that   previously   recovered.   The   total   quantity  of  supernatant  harbouring  single  stranded  DNA  (SS)  was  1  ml.    

The  HA  pellet  containing  bound  double-­‐stranded  DNA  was  well  mixed  with  200  µl  0.4M  PB,  and  kept  at   room  temperature  for  1–2  min.  Supernatant  was  collected  after  centrifugation  (2  min  at  13,000  g),  and   the  pellet  was  washed  again  with  200  µl  0.4M  PB.  The  final  volume  of  double-­‐stranded  DNA  (DS)  was   400  µl.  These  samples  were  denatured  by  boiling,  and  they  were  ice  chilled  prior  to  their  detection  on   microtitre  plates.  

 

4.2.2.  Detection  of  eluted  DNA  

Each  single  DDH  mixture  was  eluted  in  two  independent  HA  treatments.  Single  stranded  DNA  (1  ml)  was   amended  with  10  µl  of  bovine  serum  albumin  (BSA,  10%  in  1X  PBS),  and  double-­‐stranded  DNA  (0.4  ml)   was   amended   with   4   µl   of   10%   BSA.   From   each   single   elute,   200   µl   were   transferred   to   a   well   of   a   streptavidin  coated  microtitre  plate  (Roche),  and  incubated  for  2h  at  room  temperature  with  vigorous   shaking.  Unbound  DNA  was  then  discarded  and  the  wells  were  thoroughly  washed  at  least  three  times   with  1X  PBS.  In  each  well,  200  µl  of  the  antibody  mixture  (anti-­‐digoxygenin  AP,  Roche;  1:5000  in  1X  PBS,   1%  BSA)  were  added  and  incubated  for  1h  at  room  temperature  with  vigorous  shaking.  Wells  were  then   thoroughly  washed  at  least  three  times  with  1X  PBS.  

Finally,   250   µl   of   developing   solution   (7.5Mm   Na2CO3,   15.5mM   NaHCO3,   1mM   MgCl2   x   6H2O,   pH   9.6)   with   1   mg   ml-­‐1   p-­‐nitrophenyl   phosphate   (Sigma)   were   added   to   each   well.   The   microtitre   plate   was   incubated  at  37ºC,  without  agitation,  and  colour  development  was  measured  at  405nm  (Tijssen,  1985)   in  an  absorbance  plate  reader  (Sunrise,  Tecan).  

 

4.2.3.  Treatment  of  hybridization  data    

Homologous   and   heterologous   reassociations   were   processed   simultaneously.   The   degree   of   reassociation   (binding   ratio)   was   expressed   as   the   percentage   of   labeled   DNA   released   with   0.4M   PB   (DS)  compared  to  the  total  labeled  DNA  released  (DS+SS)  (BR=  DS  x  2  /  (SS  x  5  +  DS  x  2)  x  100).  

Materials  and  Methods    

 

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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  

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