El cuerpo como símbolo

The   experimental   design   of   the   REE   allowed   the   identification   of   the   genetic   changes  that  were  responsible  for  the  rise  of  different  phenotypes  throughout  the   evolutionary   history   of   the   12   parallel   lineages   depending   on   the   environment   (static  or  shaken).  Thus  it  was  possible  to  reproduce  the  entire  mutational  history   of  the  switcher  genotypes.  As  already  mentioned,  the  switchers  in  Line  1  and  Line   6  appeared  after  eight  preceding  mutations  (see  Chapter  1,  section  1.5).  The  ninth   mutation   in   both   lineages   gave   rise   to   the   switchers   referred   to   as   1w4   (Line   1)   and  6w4  (Line  6).  Both  switchers  are  essentially  identical.  The  fact  that  two  out  of   12  replicate  lineages  evolved  this  novel  phenotype  independently  indicates  a  high   degree  of  parallel  phenotypic  evolution.  However  when  comparing  the  underlying   genotypes   (Tab.   3.1)   it   becomes   apparent   that   the   causal   switcher   mutations   in   both  lineages  are  quite  different.  The  switcher  mutation  in  Line  1  occurred  in  the  

carB  gene  whereas  in  Line  6  it  occurred  in  the  rpoD  gene  (Tab.  3.1;  Gallie,  2009).      

Table   3.1:   Mutational   history   of   the   switcher   type   in   Line   1   and   Line   6.  After   nine  rounds  of  selection  (either  in  a  static  or  a  shaken  environment)  a  switcher  type   (SW)  occurred  in  two  out  of  12  lineages  under  static  conditions.  Listed  are  the  eight   preceding  mutations  and  the  switcher  mutations  in  Line  1  and  Line  6.  

Selection  

round   Environment  

Line  1     Line  6  

Name   Mutational     Name   Mutational                 history       history   1   static   1w0   mwsR   6w0   wspF  

2   shaken   1s1   mwsR   6s1   wspF  

3   static   1w1   awsX   6w1   awsX  

4   shaken   1s2   awsR   6s2   awsR  

5   static   1w2   wspF   6w2   wspF  

6   shaken   1s3   wssA   6s3   wssB  

7   static   1w3   mwsR   6w3   nlpD  

8   shaken   1s4   mwsR   6s4   nlpD   9   static   1w4  (SW)   carB   6w4  (SW)   rpoD  

Although  the  mutational  histories  of  both  lineages  diverge  from  each  other  there   are  some  noticeable  parallels  between  them.  Line  1  and  Line  6  have  genetic  loci  in  

common  that  were  affected  by  a  mutation,  such  as  mwsR,  aws,  wspF  and  wss,  but   the   order   of   the   mutation   occurrences   shows   some   variation   (Tab.   3.1).   It   is   thought   that   in   most   cases   the   genetic   loci   have   a   modular   nature   in   which   one   mutation   activates   the   WS   phenotype   (static   environment)   and   a   subsequent   mutation  in  the  same  locus  causes  the  phenotype  to  reverse  to  a  SM  ancestral  type   (shaken   environment;   see   section   1.4.2).   Interestingly,   four   genes   were   almost   identical  between  Line  1  and  Line  6.  Here  mutations  occurred  in  awsX  followed  by  

awsR,  and  afterwards  in  wspF  followed  by  wssA  in  Line  1,  or  wssB  in  Line  6  during   the  same  selection  round  (Tab.  3.1).    

Of  note  is  the  non-­‐modular  character  of  the  wspF  mutation  followed  by  a  mutation   in  wssA   (Line   1)   or   wssB   (Line   6;   Tab.   3.1).   Here   the   mutations   occurred   in   different   genetic   loci,  wspF  and  wssA/wssB.   It   is   noteworthy   that   both   are   linked   with  each  other  via  a  signal  transduction  pathway  (Bantinaki  et  al.,  2007).  The  wsp  

signal  transduction  pathway  model  was  described  in  the  introduction  (see  Chapter   1,  Fig.  1.5).  A  mutation  in  wspF  almost  certainly  abolishes  its  function  as  a  negative   regulator   of   WspR.   As   a   result   WspR   is   activated   and   produces   c-­‐di-­‐GMP   constitutively   (Goymer   et   al.,   2006;   Malone   et   al.,   2007).   Since   no   deactivating   mutation  was  detected  in  wspR,  either  in  Line  1  or  in  Line  6  later  on  (Tab.  3.1),  it  is   likely  that  all  genotypes  that  occurred  after  the  wspF  mutation  have  an  increased   level   of   c-­‐di-­‐GMP.   It   has   been   shown   that   increased   c-­‐di-­‐GMP   synthesis   has   phenotypic  consequences  for  the  genotypes  after  the  wspF  mutation,  such  as  the   production   of   a   cellulosic   polymer.   It   is   thought   that   WssA   is   responsible   for   the   localization  of  the  cellulose-­‐producing  unit  within  the  cell  and  WssB  is  part  of  the   cellulose  synthase  complex  (Spiers  et  al.,  2002).  Phenotypic  analyses  have  shown   that  mutations  in  wssA  or  wssB  decrease  the  amount  of  cellulose  production,  but   the  polymer  is  still  produced  by  the  cell  (Gallie,  2009).  It  is  likely  that  increased  c-­‐ di-­‐GMP  concentration  might  influence  the  capsulation  process  with  an  effect  on  the   switcher  phenotypes  in  Line  1  and  Line  6,  which  is  similar  to  observations  in  other   organisms,   such   as  Vibrio  parahaemolyticus  (Boles   &   McCarter,   2002;   Guvener   &   McCarter,  2003).  

3.1.4 Replaying  the  tape  of  life  in  P.  fluorescens    

During   the   REE   the   same   phenotypic   innovation,   a   stochastically   switching   phenotype,  emerged  in  two  out  of  12  replicate  lineages  of  P.  fluorescens.  It  has  been   shown   that   switching   between   different   phenotypic   stages   is   a   winning   strategy   under   the   selective   regime   experienced   by   the   bacteria   (Beaumont  et   al.,   2009;   Libby   &   Rainey,   2011;   Rainey   et   al.,   2011).   The   fact   that   two   lineages   independently  evolved  a  switcher  shows  that  the  evolution  of  this  key  innovation   can  be  achieved  repeatedly.  It  is  not  clear  whether  this  is  the  result  of  history  or   deterministic   processes.   In   both   switcher   lineages   the   new   phenotype   occurred   after   eight   prior   mutations   and   although   the   causative   switcher   mutation   itself   emerged  in  different  genes  it  is  known  that  the  mutational  histories  overlap  partly   between   the   lines,   indicating   a   possible   impact   of   mutational   history   in   the   evolution  of  the  switcher  (Beaumont  et  al.,  2009).  

To  investigate  these  hypotheses  I  used  the  opportunity  to  go  back  in  time,  revive   frozen  bacterial  cultures  from  a  ‘frozen  fossil  record’,  and  ‘replay’  evolution  from  a   particular   timepoint.   The   aim   of   the   experiment   was   to   compare   four   independently  evolved  lineages  of  P.  fluorescens  (Line  1,  Line  3,  Line  6  and  Line  12)   in  their  ability  to  produce  a  switcher  genotype.  The  four  lineages  evolved  during   the  course  of  the  long-­‐term  experiment  REE  (see  Chapter  1,  section  1.4.4)  and  each   lineage  had  eight  mutations  that  occurred  over  time,  but  the  mutations  differed  in   their  order  and  partly  in  the  genes  that  were  affected  by  a  mutation  (Tab.  3.2).  The   immediate  ancestors  of  the  switcher  in  Line  1  (1s4)  and  Line  6  (6s4)  are  already   known   for   their   ability   to   evolve   this   novel   type   (Beaumont  et   al.,   2009;   Gallie,   2009).   Replaying   evolution   in   two   additional   lineages   that   are   not   known   for   switcher   evolution   from   the   same   starting   position   as   the   immediate   ancestor   of   the   switcher   may   clarify   to   what   degree   switching   can   repeatedly   evolve.   Line   3   and  Line  12  were  chosen  as  additional  lineages  because  their  mutational  histories   were  except  for  one  mutation  in  Line  12  completely  known  at  the  time  when  the   experiment   started.   Knowledge   of   the   individual   mutational   histories   of   each   lineage  may  shed  some  light  on  the  significance  of  the  mutations  and  their  order   for  switcher  evolution.