in vitro synthetase reactions  protein preparation and 
  synthetase reactions employing maltose - binding protein - tagged substrate and 
  sagbcd were performed as described earlier 
  ( 3 ) . omission of the 
  substrate or the sagbcd synthetase resulted in no detectable hemolytic 
  activity ( data not shown ) . cytolytic activity assay  the in vitro and genetic 
  reconstitution assays described below were performed at least three times for 
  each substrate tested . because of lot - to - lot variation in commercial blood 
  sources and prep - to - prep variation in the specific activity of the synthetase 
  complex , we have elected to report activity semi - quantitatively . in 
  vitro hemolytic activity equal to wild type saga treated with sagbcd is 
  reported as three plus signs ( + + + ) . cytolytic activity that is 
  3070% of wild type saga is given as ( + + ) . detectable activity that 
  is less than 30% of saga is thus a single plus sign ( + ) , and nondetectable 
  activity is a single minus sign ( - ) . the cytolytic activity of mutant 
  substrates tested via genetic reconstitution was scored in an analogous 
  manner . all of the assays were internally normalized , and the base line was 
  adjusted using two positive controls ( triton x-100 and wild type saga treated 
  with sagbcd ) and two negative controls ( substrate and sagbcd alone ) . transformation and verification of s. pyogenes m1 saga 
  mutants  the saga allelic exchange mutant of s. 
  pyogenes m1 was made electrocompetent using a previously published 
  glycine / sucrose method ( 2 ) . maxiprepped pdcerm constructs ( 3 and 12 g ) were incubated with 
  electrocompetent s. pyogenes m1 saga and 
  electroporated using an eppendorf 2510 electroporator set to 1.5 kv . these 
  cells ( 50 and 150 l ) were then plated on todd - hewitt agar plates 
  supplemented with 2 g / ml erythromycin . clones harboring an appropriately sized 
  insert were initially screened for cytolytic activity by streaking bacteria on 
  blood agar plates ( hardy diagnostics ) . all of the clones were verified to be 
  s. pyogenes ( group a streptococcus ) by using the bbl 
  streptocard enzyme latex test ( bd diagnostics ) . because 
  of the possibility that nonphysiological concentrations could lead to 
  artifactual activity in vitro , the cytolytic activity of the peptide 
  substrates were also tested using genetic reconstitution . therefore , mutant substrates must also be 
  proteolytically processed and accepted by the sagghi export apparatus ( abc 
  transporters ) . because of toxicity in e. coli and transformation 
  difficulties , intrinsically lytic saga mutants were not tested by genetic 
  reconstitution . extracts containing bovine serum albumin - stabilized sls were 
  prepared in the following manner . overnight cultures ( 10 ml ) of s. 
  pyogenes m1 saga containing pdcerm - saga plasmids 
  were grown to a600 0.6 in todd hewitt broth 
  containing 2 g / ml erythromycin . the cultures were treated with bovine 
  serum albumin ( 10 mg / ml ) for 1 h at 37 c and then centrifuged ( 6,000 
   g , 10 min ) before passing the supernatant through a 0.2-m 
  acrodisc syringe filter ( pall corporation ) . these samples were centrifuged 
  again ( 6,000  g , 10 min ) , and the supernatants were assayed 
  for hemolytic activity by addition to defribinated sheep blood ( in v - bottom 
  microtiter plates at 1:25 and 1:50 dilutions ) . the blood was treated for 
  24 h before assessing hemolytic activity as previously reported 
  ( 3 ) . assessment of sls mutants in a murine skin infection model 
  the experiments were performed using models reported previously 
  ( 4 , 
  9 , 
  24 ) . s. pyogenes m1 
  saga complemented with saga - wt , saga - s39a , 
  and saga-7c / s were grown to log phase , harvested by centrifugation , 
  washed , resuspended in phosphate - buffered saline , and mixed 1:1 with cytodex 
  beads ( 1 mg / ml ; sigma ) . an inoculum of ( 1  10 
  colony - forming units in 100 l ) of s. pyogenes 
  saga complemented with either saga - s39a or the 
  saga-7c / s mutant was then injected subcutaneously into the right 
  flank of 4-week - old male hairless crl : skh1(hrhr)br mice ( n = 
  8/group ) . s. pyogenes saga complemented with 
  saga - wt was injected into the left flank of the same animal for 
  identical comparison . the animals were monitored daily for development of 
  necrotic ulcers . at 4 days post - infection , biopsies were performed on injection sites for histopathologic assessment 
  ( hematoxylin / eosin staining ) after measuring the size of tissue ulcers . generation of [ s]met - sagbcd  radioactive 
  synthetase was prepared using maxiprepped pet28-maltose - binding protein - sagbcd 
  and in vitro transcription / translation under t7 promoter control . rabbit reticulocyte extract ( promega ) gave superior yield and purity to wheat 
  germ and s30 extracts . a typical 50-l reaction was set up as follows : 
  13 g of plasmid dna was added to a master mix containing 2 l of 
  transcription / translation buffer , 25 l of rabbit reticulocyte extract , 1 
  l of rnasin , 1 l of minus met amino acid mix , 1 l of t7 rna 
  polymerase , 10 ci of [ s]met ( perkinelmer life sciences ) , and 
  dnase / rnase - free water . a small aliquot ( 0.5 l ) of the radiolabeled 
  product was separated by sds - page , dried , and visualized by autoradiography 
  using a kodak biomax low energy isotope intensifying screen ( 20 h , -80 
  c ) . peptide array immobilized peptides were synthesized on 
  cellulose membranes using a multipep autospot synthesis robot following the 
  manufacturer 's instructions ( intavis ag ) . irradiation with 254 nm ( uv ) light 
  gave an indication of the relative amount of peptide per spot . [ s]met - sagb , -c , and -d were allowed to bind to the array for 15 
  h at 4 c in tris - buffered saline / tween 20 ( 0.1% v / v ) supplemented with 
  2.5% nonfat milk and 2.5% bovine serum albumin ( both w / w ) . the array was then 
  extensively washed with tris - buffered saline / tween ( 4  10 min , 23 
  c ) before exposing film with a kodak biomax low energy isotope 
  intensifying screen ( 20 h , -80 c ) . under these conditions , sagb and sagd did not bind tightly to the array , as indicated by a weak radioactive signal , 
  even after longer exposure times ( 4 days ) . using sagbcd together led to a 
  substantial amount of  radioactive precipitation  on areas of the 
  cellulose membrane that did not contain peptide . the saga leader peptide provides substrate recognition the 
  precursor peptide from c. botulinum , closa , shares significant amino 
  acid sequence similarity with saga ( 62% ) and was converted to a cytolysin by 
  recombinant sagbcd ( 3 ) . the 
  microcin b17 precursor , mcba , is only moderately similar ( 32% ) but was also 
  accepted as a substrate by sagbcd . therefore , we hypothesized that sagbcd 
  would accept numerous noncognate substrates 
  ( fig . to 
  demonstrate that the permissive behavior of sagbcd on closa was not limited to 
  in vitro biochemical studies with purified proteins , genetic 
  complementation studies were performed using a saga deletion mutant 
  produced in an m1 serotype strain of s. pyogenes 
  ( saga ) . by transforming this strain with a plasmid encoding 
  the desired peptide substrate , -hemolysis on blood agar was restored when either the 
  wild type saga or closa gene was provided to the s. 
  pyogenes saga mutant bacteria . to increase sensitivity , the sls is extracted from the 
  s. pyogenes lipoteichoic acid layer using bovine serum albumin as a 
  carrier / stabilizer . this sample is mixed with sheep blood , and the amount of 
  hemoglobin released from lysed erythrocytes is quantified by absorbance . using 
  this assay , we confirmed that closa was converted to a cytolysin by endogenous 
  sagbcd but calculated the level of activity to be reduced by  
  relative to that of saga ( fig . interestingly , the related peptide substrates from s. aureus 
  ( stapha ) and l. monocytogenes ( lista ) did not exhibit detectable 
  hemolytic activity with purified recombinant proteins or in genetically 
  complemented s. pyogenes saga 
  ( fig . ( 18 ) have 
  recently shown that the sag - like genetic cluster from l. 
  monocytogenes synthesizes an sls - like cytolytic factor . therefore , the 
  lack of activity in our assays must originate at the level of substrate 
  recognition or enzymatic tolerance . previous work from the walsh laboratory 
  has shown that the n - terminal leader peptide of mcba is required for substrate 
  recognition ( 14 ) . the most 
  important residues implicated in this process are mcba - f8 and -l12 , which are 
  proposed to lie on the same face of an -helix 
  ( 15 ) . alignment with other 
  substrates shows that a similar motif ( fxxxb , where x is any 
  amino acid and b is a branched chain amino acid ) is found in the leader 
  peptides of saga and closa but is lacking in stapha and lista 
  ( fig . this 
  finding prompted the construction of a double alanine mutant of the 
  fxxxb motif ( saga - fia ) and chimeric substrates comprised of the saga 
  leader peptide fused to the stapha and lista c terminus 
  ( fig . if the saga 
  leader peptide contains adequate substrate recognition information , then 
  activity should be restored upon treating the chimera with purified sagbcd . furthermore , if the fxxxb motif is required for substrate 
  recognition , then cytolytic activity should be reduced for similarly treated 
  saga - fia . a full restoration of in vitro cytolytic activity was 
  observed for both the saga - lista and saga - stapha chimera . in contrast , genetic 
  complementation revealed that only saga - stapha retained activity , indicating 
  that either the endogenous rules of cytolytic conversion are more restrictive 
  or the saga - lista substrate is not efficiently exported . enzymatic promiscuity 
  could theoretically be amplified in vitro by the presence of large 
  amounts of purified sagbcd . this is supported by the observation that saga - fia 
  has detectable activity using purified enzymes but not under genetic 
  reconstitution conditions ( fig . unnatural saga analogs were prepared to shed light on the positional 
  requirements of heterocycle formation . we first designed an artificial 
  substrate , sagx , in an attempt to assess the minimum features for the 
  formation of a cytolysin . four criteria were used to design this potential 
  substrate : ( i ) the saga leader peptide to provide recognition , ( ii ) a stretch 
  of contiguous heterocyclizable residues adjacent to the leader peptide 
  cleavage site , ( iii ) 30% glycine evenly distributed through the c - terminal 
  half , and ( iv ) a serine flanked by two glycine residues . akin to saga - lista , 
  this artificial substrate gave activity equal to saga in the in vitro 
  assay but no activity upon complementation of the saga mutant 
  of s. pyogenes . the region of saga between the cysteine - rich region 
  and the last heterocyclizable residue ( fsia ... gsyt ) 
  was further probed by designing two additional unnatural substrates . saga 
  inverse simply inverted residues 3350 of saga , whereas in saga scramble 
  this region was reordered in a randomized fashion . despite having the highest 
  protoxin similarity to wild type saga , the saga inverse substrate did not 
  yield lytic activity in either assay . saga scramble was active in 
  vitro but did not complement hemolytic activity in s. pyogenes 
  saga . these data show that the leader peptide , especially the 
  fxxxb motif , is important for substrate acceptance and that 
  heterocyclizable positions must be available at precise positions to allow 
  creation of an sls - like cytolytic factor . additionally , these data indicate 
  that even though sagbcd accepts artificial substrates , there is a limit to the 
  enzymatic promiscuity . these limits are more pronounced during genetic 
  complementation experiments most likely because substrate recognition and 
  enzymatic tolerance are bypassed to an extent when using purified sagbcd . an 
  alternative explanation is that the unnatural substrates are secreted from 
  s. pyogenes with differing efficiencies . analysis of saga leader peptide binding requirements  because 
  the above activity assays measure both substrate binding and heterocycle 
  formation in a simultaneous and indirect fashion , peptide arrays were 
  synthesized to directly evaluate binding . based on a perceived importance of 
  the fxxxb motif in sagbcd substrate acceptance , the first array 
  consisted of a panel of leader peptides that contain the fxxxb motif . next to each wild type ( wt ) sequence , the fxxxb double alanine 
  mutation was synthesized for saga , closa , mcba , and a substrate from 
  pyrococcus furiosus , designated paga 
  ( fig . equal 
  peptide loading was confirmed by irradiating the array with uv ( 254 nm ) light . 
  to assess binding , [ s]met - labeled sagbcd was prepared by in 
  vitro transcription / translation ( supplemental fig . s1 ) and allowed to 
  interact with the peptide array as described under  experimental 
  procedures . initially , the sagbcd complex was tested for binding in 
  approximately a 1:1:1 ratio , as judged by the number of methionines and the 
  intensity of exposure . unfortunately , this led to a substantial amount of 
  precipitation that could not be removed from the cellulose membrane and 
  rendered the binding information unreliable . sagb and sagd did not bind tightly to 
  the leader peptide array and were washed off before exposing the film ( data 
  not shown ) . sagc , however , bound with high affinity to the array . for the wt 
  sequences , sagc binding was highest to saga , followed by closa and paga 
  ( fig . importantly , 
  binding to each fxxxb mutant was reduced in comparison with the 
  corresponding wt leader peptide . a , saga is converted into an active cytolysin , pro - streptolysin - s 
  ( pro - sls ) , by the actions of sagbcd ( a trimeric oxazole / thiazole 
  synthetase ) . heterocycles are schematically represented as shaded 
  pentagons . a marginally conserved motif in the saga leader peptide , 
  fxxxb ( where b is a branched chain amino acid ) , is highlighted in 
  red . individual reactions catalyzed by sagc ( cyclodehydratase ) and 
  sagb ( fmn - dehydrogenase ) b , representative amino acid 
  sequences and cytolytic activity of saga - like substrates . shown in 
  red the putative leader peptide cleavage sites are shown as 
  asterisks , except for mcba , where the site is known 
  ( hyphen ) . in blue are sites of potential heterocycle 
  formation ( for mcba , known sites are blue ) . the percentage of amino 
  acid similarity to full - length saga ( as determined by clustalw alignment ) is 
  given . the cytolytic activity was tested for these substrates in 
  vitro using purified proteins and in vivo using the 
  sls - deficient strain , s. pyogenes saga , complemented 
  with the desired substrate . activity equal to wild type saga is designated as 
  ( + + + ) ; activity that is 3070% of wild type saga is ( + + ) ; detectable 
  activity that is less than 30% of saga is noted as ( + ) ; and nondetectable 
  activity is ( - ) . the activity for mcba is not applicable ( n.a . ) 
  because this secondary metabolite is a dna gyrase inhibitor , not a cytolysin . all of the 
  substrates contain the wild type saga leader peptide , except for the first 
  entry ( fxxxb mutant , saga - fia ) . the percentage of amino acid 
  similarity to the protoxin half of saga is shown . the second and third entries 
  are saga leader peptides fused to the protoxin of stapha and lista . sagx is an 
  artificially designed toxin , whereas the inverse and scrambled substrates 
  manipulate the sequence of saga between residues 3350 
  ( underlined ) . a , saga is converted into an active cytolysin , pro - streptolysin - s 
  ( pro - sls ) , by the actions of sagbcd ( a trimeric oxazole / thiazole 
  synthetase ) . a marginally conserved motif in the saga leader peptide , 
  fxxxb ( where b is a branched chain amino acid ) , is highlighted in 
  red . individual reactions catalyzed by sagc ( cyclodehydratase ) and 
  sagb ( fmn - dehydrogenase ) are shown . b , representative amino acid 
  sequences and cytolytic activity of saga - like substrates . shown in 
  red the putative leader peptide cleavage sites are shown as 
  asterisks , except for mcba , where the site is known 
  ( hyphen ) . in blue are sites of potential heterocycle 
  formation ( for mcba , known sites are blue ) . the percentage of amino 
  acid similarity to full - length saga ( as determined by clustalw alignment ) is 
  given . the cytolytic activity was tested for these substrates in 
  vitro using purified proteins and in vivo using the 
  sls - deficient strain , s. pyogenes saga , complemented 
  with the desired substrate . activity equal to wild type saga is designated as 
  ( + + + ) ; activity that is 3070% of wild type saga is ( + + ) ; detectable 
  activity that is less than 30% of saga is noted as ( + ) ; and nondetectable 
  activity is ( - ) . the activity for mcba is not applicable ( n.a . ) 
  because this secondary metabolite is a dna gyrase inhibitor , not a cytolysin . all of the 
  substrates contain the wild type saga leader peptide , except for the first 
  entry ( fxxxb mutant , saga - fia ) . the percentage of amino acid 
  similarity to the protoxin half of saga is shown . the second and third entries 
  are saga leader peptides fused to the protoxin of stapha and lista . sagx is an 
  artificially designed toxin , whereas the inverse and scrambled substrates 
  manipulate the sequence of saga between residues 3350 
  ( underlined ) . a , using a 
  peptide array synthesizer , the given sequences were generated with c - terminal 
  attachment to a cellulose membrane . the fxxxb motif was left intact 
  ( wt ) and mutated to ala for the substrates from s. pyogenes ( saga ) , 
  c. botulinum ( closa ) , e. coli ( mcba ) , and p. 
  furiosus ( paga ) . [ s]met - sagc was allowed to bind to the 
  array before extensive washing and exposure to film . the relative binding of 
  sagc to the array is indicated by the autoradiographic exposure . b , the saga leader 
  peptide alanine scan array was synthesized to obtain positional contributions 
  to sagc binding . the putative leader peptide of saga contains three alanines 
  ( 3 of 23 ) ; therefore 20 mutants of saga were tested in this assay . the residue 
  number , amino acid mutated to alanine , relative binding of 
  [ s]met - sagc ( autoradiography ) , and peptide loading control ( uv ) 
  are aligned . positions 24 , 8 , and 9 contain the fxxxb motif ; 
  positions 1719 comprise the tqv motif . c , a peptide array 
  comprised of fourteen 10-mer peptides of saga was prepared to evaluate the 
  binding contributions of each interaction site within the leader and the 
  protoxin c terminus . the entire length of saga is represented , except for the 
  middle cysteine - rich region . a , using a 
  peptide array synthesizer , the given sequences were generated with c - terminal 
  attachment to a cellulose membrane . the fxxxb motif was left intact 
  ( wt ) and mutated to ala for the substrates from s. pyogenes ( saga ) , 
  c. botulinum ( closa ) , e. coli ( mcba ) , and p. 
  furiosus ( paga ) . [ s]met - sagc was allowed to bind to the 
  array before extensive washing and exposure to film . the relative binding of 
  sagc to the array is indicated by the autoradiographic exposure . b , the saga leader 
  peptide alanine scan array was synthesized to obtain positional contributions 
  to sagc binding . the putative leader peptide of saga contains three alanines 
  ( 3 of 23 ) ; therefore 20 mutants of saga were tested in this assay . the residue 
  number , amino acid mutated to alanine , relative binding of 
  [ s]met - sagc ( autoradiography ) , and peptide loading control ( uv ) 
  are aligned . positions 24 , 8 , and 9 contain the fxxxb motif ; 
  positions 1719 comprise the tqv motif . c , a peptide array 
  comprised of fourteen 10-mer peptides of saga was prepared to evaluate the 
  binding contributions of each interaction site within the leader and the 
  protoxin c terminus . the entire length of saga is represented , except for the 
  middle cysteine - rich region . the fxxxb leader motif is necessary but not sufficient for sagc 
  recognition  although the fxxxb motif is an important 
  determinant for directing sagc substrate binding , it does not contribute all 
  of the interaction energy . this is illustrated by the observation that sagc 
  bound to the saga - wt leader peptide more efficiently than the other wt 
  peptides ( fig . furthermore , simple incorporation of fxxxb into the stapha and lista 
  leader peptide did not provide sufficient affinity to detect sagc binding by 
  this method ( data not shown ) . these observations led to the hypothesis that 
  additional residues of saga contribute to sagc binding . to elucidate these binding determinants , a second array was synthesized 
  that consisted of an alanine scan of the saga leader peptide 
  ( fig . each 
  residue of saga that is not naturally found as alanine was individually 
  mutated to alanine and tested for sagc binding as above . as expected , mutation 
  to alanine did not disrupt sagc affinity at every location . however , the 
  binding levels were significantly reduced when residues comprising the 
  fxxxb motif were mutated ( phe and ile ) . it 
  was also found that residues adjacent to the fxxxb sequence 
  ( leu , lys , and leu ) and another binding 
  site ( thr , gln , and val ) were also 
  critical for sagc binding ( fig . an additional array was used to ascertain the contribution of each 
  interaction site found in the leader peptide and to also assess whether the 
  c - terminal region of saga plays a role in binding . a panel of fourteen 10-mer 
  peptides were included in the array that scan the full - length of saga , except 
  for the oxidatively prone , and synthetically challenging , cysteine - rich region 
  ( fig . 2c ) . after 
  binding sagc to this array , it was found that only the first peptide was 
  capable of providing enough binding information to retain sagc through the 
  washing steps . peptide 1 comprises residues 110 of saga and not only 
  contains the fxxxb motif but also the important adjacent residues 
  ( leu , lys , phe , ile , and 
  leu ) . peptide 5 contains ile and leu but 
  only the first residue of the tqv site ( the ) . this peptide is 
  insufficient at retaining sagc . taken together , these findings demonstrate 
  that separate sites within the n - terminal leader peptide synergize to provide 
  sagc with a high affinity binding site . binding parameters for the saga - wt , saga - fia ( fxxxb double alanine 
  mutant ) , and saga - tqv ( triple alanine mutant ) leader peptides with sagc were 
  then measured by surface plasmon resonance to quantify the contribution of 
  each binding motif . using a c - terminal hexahistidine tagged version of 
  saga - wt , -fia , and -tqv , the rates of association ( ka ) and 
  dissociation ( kd ) were first measured with sagc 
  ( table 1 ) . as expected 
  from the peptide array data , the effects of fxxxb and tqv mutation 
  were substantial . compared with saga - wt , the reduction in affinity was accounted for by an 
  increased kd ( ka was unchanged ) . sagc 
  affinity for saga - tqv was also reduced ( 24-fold ) . the major factor in the 
  saga - tqv mutant reduction in affinity is an increased kd ; 
  however , there is also a slight decrease in ka 
  ( < 2-fold ) upon comparison with saga - wt 
  ( table 1 ) . table 1contribution of saga interaction sites to cyclodehydratase binding 
  kinetics surface plasmon resonance was performed using leader peptides of 
  saga with a c - terminal his6 tag as the immobilized ligand . the 
  sequences of the peptides are as follows : saga - wt , 
  mlkftsnilatsvaettqvapgghhhhhh ; saga - fia , 
  mlkatsnalatsvaettqvapgghhhhhh ; saga - tqv , 
  mlkftsnilatsvaetaaaapgghhhhhh . the analytes tested for binding were 
  the cyclodehydratases from s. pyogenes ( sagc ) , c. botulinum 
  ( closc ) , and e. coli ( mcbb ) . the association rates 
  ( ka ) and dissociation rates ( kd ) were 
  recorded in duplicate at two concentrations and used to calculate the 
  dissociation constant ( kd ) . the  values are given as an 
  assessment of curve fitting accuracy.ligandanalyteka ( 10)kd ( 10)kdm ssnm saga - wt 
   sagc 
   3.7  0.4 
   0.25  0.03 
   6.7  0.8 
   0.04 
   saga - fia 
   sagc 
   3.7  0.3 
   3.5  0.3 
   95  7.0 
   0.04 
   saga - tqv 
   sagc 
   2.1  0.3 
   3.4  0.8 
   160  30 
   0.03 
   saga - wt 
   closc 
   3.8  0.3 
   3.1  0.7 
   82  14 
   0.10 
   saga - fia 
   closc 
   3.3  0.5 
   7.2  1.0 
   220  30 
   0.07 
   saga - tqv 
   closc 
   2.5  0.3 
   9.6  0.9 
   380  40 
   0.03 
   saga - wt 
   mcbb 
   2.5  0.3 
   22  4.0 
   880  140 
   0.58 
   saga - fia 
   mcbb 
   2.3  0.2 
   27  1.0 
   1200  100 
   0.25 
 
  saga - tqv 
 
  mcbb 
 
  2.0  0.3 
 
  41  5.2 
 
  2100  310 
 
  0.04 
   contribution of saga interaction sites to cyclodehydratase binding 
  kinetics surface plasmon resonance was performed using leader peptides of 
  saga with a c - terminal his6 tag as the immobilized ligand . the 
  sequences of the peptides are as follows : saga - wt , 
  mlkftsnilatsvaettqvapgghhhhhh ; saga - fia , 
  mlkatsnalatsvaettqvapgghhhhhh ; saga - tqv , 
  mlkftsnilatsvaetaaaapgghhhhhh . the analytes tested for binding were 
  the cyclodehydratases from s. pyogenes ( sagc ) , c. botulinum 
  ( closc ) , and e. coli ( mcbb ) . the association rates 
  ( ka ) and dissociation rates ( kd ) were 
  recorded in duplicate at two concentrations and used to calculate the 
  dissociation constant ( kd ) . closc and mcbb , from c. botulinum and e. 
  coli , respectively , were tested for saga binding . relative to sagc , 
  binding to the saga - wt leader peptide was reduced with both closc and mcbb 
  ( 12- and 130-fold , respectively ; 
  table 1 ) . this trend correlates 
  with the degree of amino acid similarity to sagc ( 59% for closc ; 28% for 
  mcbb ) . mutation of the saga fxxxb and tqv motifs led to additional 
  losses of affinity in the same manner as for sagc binding ( i.e. both 
  the fxxxb and tqv mutants effect kd ; the 
  ka is only slightly affected by the mutation of tqv ) . this 
  suggests that although each cyclodehydratase is likely fine - tuned for a single 
  endogenous substrate , the substrate binding mechanism is conserved across 
  species . . 
 a , compilation of the cytolytic data for saga point mutations after 
  reaction with sagbcd . residues that are c - terminal to the predicted leader 
  peptide cleavage site ( vertical line ) of saga ( 2453 ) are 
  shown . b , the activity of each saga mutant was tested by the 
  erythrocyte lysis assay in vitro using purified proteins and in 
  vivo by genetic complementation . 2c / s , c31s / c32s ; 3c / s , c26s / c27s / c28s ; 7c / s , all 
  seven cysteines mutated to serine ; 4t / v , all four threonines mutated to 
  valine . c , the intrinsic activity of saga substituted with proline 
  was tested using purified protein . toxicity 
  ( tox ) was observed for some saga proline mutants , which prevented 
  recombinant expression . a synthetic peptide was prepared to test proline 
  substitution at each serine found to be important for saga activity . identification of saga residues necessary for cytolytic activity . 
 a , compilation of the cytolytic data for saga point mutations after 
  reaction with sagbcd . residues that are c - terminal to the predicted leader 
  peptide cleavage site ( vertical line ) of saga ( 2453 ) are 
  shown . b , the activity of each saga mutant was tested by the 
  erythrocyte lysis assay in vitro using purified proteins and in 
  vivo by genetic complementation . 2c / s , c31s / c32s ; 3c / s , c26s / c27s / c28s ; 7c / s , all 
  seven cysteines mutated to serine ; 4t / v , all four threonines mutated to 
  valine . c , the intrinsic activity of saga substituted with proline 
  was tested using purified protein . toxicity 
  ( tox ) was observed for some saga proline mutants , which prevented 
  recombinant expression . a synthetic peptide was prepared to test proline 
  substitution at each serine found to be important for saga activity . mutational analysis of heterocyclizable residues of saga 
  numerous attempts at identifying sites of heterocycle formation on saga by 
  mass spectrometry have failed . as a result , a site - directed mutagenesis 
  approach was initiated to determine functionally important residues of saga 
  with the assumption that the lack of cytolytic activity by particular saga 
  point mutants is suggestive of heterocycle locations . to begin this study , 
  every heterocyclizable residue ( cysteines , serines , and threonines ) located on 
  the protoxin half of saga ( cysteines and serines were mutated to alanines , whereas threonines were 
  mutated to valines , a closer structural mimic . the lytic activity of these 
  proteins was assessed in vitro after treatment with purified sagbcd 
  and through genetic complementation of s. pyogenes 
  saga . each mutation was classified as having no effect ( mutant 
  sls was fully active ) , a contributing effect ( reduced activity ) , or a critical 
  effect ( inactive ) . it is important to note that our method of testing the 
  cytolytic activity of saga point mutants through genetic complementation 
  measures activity and secretion in a simultaneous fashion . this analysis revealed that not all sites of 
  potential heterocycle formation were important for activity , but most 
  mutations had a measurable impact on function . two mutations , c32a and s39a , 
  completely abolished the cytolytic activity of sls in both assays and are thus 
  highly likely sites of thiazole / oxazole formation . moreover , it was found that 
  serine and cysteine could substitute for one another at these two critical 
  sites ( c32s and s39c ) with a small reduction of in vitro activity . however , serine can not be used to replace multiple adjacent cysteines as 
  indicated by the lack of activity in c31s / c32s ( 2c / s ) , c26s / c27s / c28s ( 3c / s ) , 
  and a total serine ( 7c / s ) mutant ( fig . 
  3b ) . the one case were the assays did not concur 
  was with the c24a mutation . we observed substantial lytic activity in 
  vitro but no activity in genetically complemented bacteria 
  ( fig . we have 
  previously shown that leader peptide removal is not required for in 
  vitro activity ( 3 ) . thus , 
  a reduction of activity in genetic complementation experiments may result from 
  reduced leader peptide processing and/or secretion . these results confirm that 
  many residues within saga are important for activity and predict that sls is 
  highly post - translationally modified . proline can substitute for thiazoles and oxazoles in the saga 
  propeptide  we rationalized that because thiazoles and oxazoles are 
  five - membered , nitrogen - containing heterocycles that restrict peptide backbone 
  conformation , proline may be capable of serving as a structural mimic . therefore , the saga - c32p and -s39p mutants were prepared and treated with 
  purified sagbcd . 3b ) and 
  encouraged the construction of saga mutants that were cytolytic in the absence 
  of sagbcd . thus , proline was systematically incorporated into select 
  heterocyclizable sites of saga that were previously determined to be important 
  for activity ( positions 32 , 34 , 39 , 46 , 48 ) . this proline - substituted saga 
  panel was then tested for lytic activity in the absence of sagbcd 
  ( fig . wild type 
  saga and two single substituted proline mutations ( c32p and s39p ) did not 
  exhibit intrinsic lytic activity . in contrast , two double proline substituted 
  mutants showed dose - dependent activity without sagbcd treatment ( c32p / s39p and 
  s34p / s39p ) ( fig . a triple mutant , c32p / s34p / s39p , predicted to 
  further enhance lytic activity , killed e. coli upon induction of 
  protein expression and yielded no protein . likewise , additional permutations 
  revealed that if either cys or ser are substituted 
  in combination with ser , ser , and ser , 
  e. coli toxicity was encountered . to support the claim that toxicity 
  arose because of sls - like activity , a synthetic peptide comprised of the 
  c - terminal 21 residues of saga was synthesized with prolines at positions 34 , 
  39 , 46 , and 48 . this peptide , although devoid of cysteine , was intrinsically 
  lytic in a dose - dependent manner ( fig . the observation that select 
  proline mutations endow saga with intrinsic lytic activity bolsters the 
  conclusion that the above sites are heterocyclized in sls . a , 4-week - old hairless crl : skh1(hrhr)br mice were 
  challenged subcutaneously with 10 colony - forming units of s. 
  pyogenes m1 saga complemented with saga - wt 
  ( left flank ) and saga - s39a ( right flank ) . gross 
  appearance of a typical mouse and lesion size ( mm ) are shown at 4 
  days post - inoculation . b , 
  histological evaluation ( hemotoxilyn / eosin staining ) of excised tissue from 
  site of injection . magnification is given at 40 and 100. ser of saga is required for virulence in a mouse infection 
  model . a , 4-week - old hairless crl : skh1(hrhr)br mice were 
  challenged subcutaneously with 10 colony - forming units of s. 
  pyogenes m1 saga complemented with saga - wt 
  ( left flank ) and saga - s39a ( right flank ) . gross 
  appearance of a typical mouse and lesion size ( mm ) are shown at 4 
  days post - inoculation . b , 
  histological evaluation ( hemotoxilyn / eosin staining ) of excised tissue from 
  site of injection . magnification is given at 40 and 100. key heterocycles of sls are required for s. pyogenes virulence in 
  mice  to determine the importance of heterocyclic conversion of sls 
  for disease progression , mice were subcutaneously injected with s. 
  pyogenes m1 saga mutant complemented with either 
  saga - wt , saga - s39a , or saga-7c / s . seven of eight 
  mice infected with the saga - wt complemented strain developed 
  significant necrotic ulcers and swelling , whereas no change was observed in 
  any infections of saga - s39a ( fig . histological 
  examination of tissues from wt and the mutant infections shows that wt 
  infections exhibit more extensive tissue disruption and a higher infiltration 
  of neutrophils ( fig . these results demonstrate that a single saga point 
  mutation predicted to interfere with heterocycle formation markedly reduces 
  the virulence of s. pyogenes in a murine model of skin infection . for saga to be converted into the active sls cytolysin , the sagbcd 
  synthetase complex must first recognize the saga substrate . heterocycles must 
  then be installed at the proper locations to elicit biological activity . by 
  using two independent assays , we analyzed the substrate permissivity of sagbcd 
  using in vitro biochemical assays and genetic reconstitution . the 
  substrate tolerance of sagbcd was initially assessed using substrates from 
  other members of the firmicutes phylum that were suspected of producing 
  sls - like cytolysins . to begin , we chose the substrate from c. 
  botulinum , which harbors the most highly related sag - like 
  genetic cluster ( closa - i ) known outside of the streptococcus 
  genus . sagbcd accepted closa as a noncognate substrate in vitro and 
  in genetically complemented s. pyogenes saga 
  bacteria , but the activity was reduced relative to saga 
  ( fig . the most likely explanation , in light of 
  our binding data , is that sagbcd process closa more slowly because of a 
  lowered substrate affinity . in vitro , however , under the conditions 
  employed , nonphysiological concentrations of synthetase and substrate could 
  easily result in an elevated level of substrate permissivity . other 
  explanations for reduced closa activity upon genetic reconstitution could be 
  that on a molar basis sls is more potent or that the leader peptidase and/or 
  export machinery process the toxin more slowly . if the latter was true , 
  toxicity would be an expected outcome , but analysis of growth curves indicated 
  that this is not the case ( data not shown ) . because closa and mcba were both known in vitro substrates of the 
  sagbcd synthetase ( 3 ) , we were 
  initially surprised when sagbcd rejected potential substrates more similar to 
  saga than mcba ( i.e. stapha and lista ; 
  fig . however , 
  chimeric substrates consisting of the saga leader peptide coupled with the 
  stapha or lista protoxin were fully active in vitro after treatment 
  with recombinant sagbcd . later experiments showed that a partially 
  conserved motif in the saga leader peptide , fxxxb , played a major 
  role in efficient cytolytic conversion 
  ( fig . 1c ) and sagc 
  affinity ( fig . 2 and 
  table 1 ) . additionally , the 
  saga leader peptide can be fused to properly designed artificial protoxins to 
  yield cytolysins after treatment with sagbcd . peptide array binding experiments uncovered that in addition to the 
  fxxxb motif and some adjacent residues , sagc also recognizes another 
  site ( tqv ) on the saga leader peptide ( fig . 
  2 ) . given that the kd was measured to be in 
  the low nanomolar range , multiple binding sites on saga would be presumed 
  necessary to drive such an avid interaction 
  ( table 1 ) . mutation of the 
  saga - tqv site led primarily to an increased kd with a 
  modest decrease of ka relative to saga - wt 
  ( table 1 ) . this is in contrast 
  to the saga - fia mutant , which only led to an increased kd . these binding trends also held for the saga binding interaction with the 
  cyclodehydratases from a closely related organism ( c. botulinum , 
  closc ) and a distantly related organism ( e. coli , mcbb ; 
  table 1 ) . we hypothesize this 
  relationship will be true of all sag - like clusters that harbor 
  substrates with fxxxb motifs in the leader peptide . elucidation of the sagc substrate recognition requirements was undertaken 
  to judge why certain substrates are accepted by the sagbcd synthetase and why 
  others are not . earlier studies have demonstrated that the sls precursor from 
  a fish pathogen , streptococcus iniae , was able to complement the 
  hemolytic activity of s. pyogenes saga 
  ( 25 , 
  26 ) . upon comparison with saga 
  from s. pyogenes , the s. iniae leader peptide has a single 
  mutation in the leader sequence ( k3q ) . thus , it should not be surprising that 
  saga from s. iniae was an efficient substrate of s. pyogenes 
  sagbcd . we conclude that closa was accepted by recombinant and endogenous 
  sagbcd because of a complete fxxxb site ( lkf and vl ) and a partial 
  tqv site ( val ) . previous results has also shown that sagbcd 
  accepts mcba as a substrate 
  ( 3 ) . this is supported by the 
  present work demonstrating that sagc directly binds to the mcba leader peptide 
  ( fig . relative to 
  saga , the sagc binding to mcba was greatly reduced , but the data provide a 
  satisfactory explanation ; the leader peptide of mcba contains partial 
  sagc - binding sites . the fxxxb sequence is present , but the important 
  surrounding residues are nonconservatively mutated . also , the gln of the tqv 
  site is present ( fig . the identification of the tqv site helps explain why simple inclusion of 
  fxxxb into the stapha and lista leader peptides did not result in 
  high affinity sagc binding . residues adjacent to the fxxxb motif ( lkf 
  and il ) also contribute to this interaction . of a total 23 residues in the 
  predicted saga leader peptide , eight this implies that upon sagc binding saga , nearly the 
  entire length of the leader peptide will be associated , possibly buried , in a 
  sagc - binding site . undoubtedly , the extended recognition capability of sagc 
  acts to prevent the synthetase complex from processing unwanted 
  peptides / proteins . 1a ) is highly 
  active , and it is possible that a tightly bound synthetase complex will cage 
  the activity until the metabolite is ready for export . however , the early work of bernheimer 
  ( 27 , 
  28 ) , bernheimer and schwartz 
  ( 29 ) , and koyama 
  ( 3032 ) 
  shed important light on the nature and action of sls . using gel filtration 
  analysis on partially purified sls , bernheimer 
  ( 33 ) predicted that the 
  molecular weight of the polypeptide would be 2.8 kda . this is quite 
  remarkable , given that a modern calculation of 2.7 kda is based on 
  modifications to saga , a peptide discovered over 30 years later . our results 
  suggest that in the mature structure of sls , oxazoles will be formed from saga 
  residues ser , ser , ser , and 
  ser ( fig . the work of kolter and co - workers 
  ( 1012 ) 
  on microcin b17 provides a literature precedent for heterocycle formation when 
  the preceding residue is glycine ( ser , ser , and 
  ser ) ( 34 , 
  35 ) . presumably , the increased 
  flexibility in the peptide backbone at these locations facilitates the orbital 
  alignment required for cyclodehydration . oxazole formation at the 
  abovementioned sites is further supported by the creation of 
  proline - substituted saga mutants that exhibit intrinsic cytolytic activity 
  ( fig . our mutagenesis data confirm a previous study identifying saga 
  cys and cys as residues important for the activity 
  of sls on blood agar plates 
  ( 2 ) . despite this , the fate of 
  the cysteine - rich region ( cys  cys ) remains 
  speculative . given the observation that c32s and c32p were cytolytic , we 
  expect cys to be converted to a thiazole . this implies that 
  there is either a polythiazole - specific electronic contribution to cytolysis 
  or that another type of modification is present in the mature structure of 
  sls . our current data can not distinguish between these two possibilities , 
  which will require extensive methodology improvements . we hypothesize that it 
  is unlikely free thiols , disulfides , or lanthionines will be present in the 
  final sls structure . this is because sls is known to be oxygen - stable 
  ( 36 , 
  37 ) , and cysteine was not 
  detected in an amino acid analysis study 
  ( 30 ) . moreover , the 
  saga - i cluster , which is sufficient to confer sls production to a 
  heterologous bacterial species 
  ( 4 ) , does not harbor the 
  appropriate dehydratases and cyclases to form lanthionine linkages 
  ( 4 , 
  38 , 
  39 ) . the murine infection study supports our previous data that sls heterocycle 
  formation is required for the virulence of s. pyogenes . furthermore , 
  we show that a single amino acid change , saga - s39a , renders this pathogen 
  avirulent in a skin infection model ( fig . 
  4 ) . without active sls , the bacteria exhibit undetectable 
  pathogenicity and are efficiently cleared by the murine immune system . it is 
  conceivable that in such a setting , antibodies could be raised against 
  inactive sls mutants . whether this represents a viable strategy for vaccine 
  development remains to be seen , but with the tactical role sls - like toxins 
  play in pathogenesis , further study is warranted . this study has elucidated the requisite features of sagbcd substrate 
  recognition and saga residues of functional importance . we conclude that 
  although the c terminus of saga undergoes enzymatic conversion , only the 
  n - terminal leader peptide substantially contributes to substrate recognition . conceivably , these principles could be used to guide the design of other 
  artificial toxins for antibiotic or anticancer applications . additionally , our 
  study has yielded a wealth of structure - activity data on a conserved family of 
  bacterial virulence factors and lays the foundation for the development of a 
  structure - based vaccine .
the human pathogen streptococcus pyogenes secretes a highly 
  cytolytic toxin known as streptolysin s ( sls ) . 
 sls is a key virulence 
  determinant and responsible for the -hemolytic phenotype of these 
  bacteria . despite over a century of research 
 , the chemical structure of sls 
  remains unknown . 
 recent experiments have revealed that sls is generated from 
  an inactive precursor peptide that undergoes extensive post - translational 
  modification to an active form . in this work , 
 we address outstanding questions 
  regarding the sls biosynthetic process , elucidating the features of substrate 
  recognition and sites of posttranslational modification to the sls precursor 
  peptide . 
 further , we exploit these findings to guide the design of artificial 
  cytolytic toxins that are recognized by the sls biosynthetic enzymes and 
  others that are intrinsically cytolytic . 
 this new structural information has 
  ramifications for future antimicrobial therapies .