BACKGROUND
streptococcus pyogenes is thought to be responsible for more than  <dig>  deaths worldwide each year  <cit> .
pathogenesis involves several proteins localized to the extracellular environment.
these secreted proteins, or exoproteins, can be experimentally defined as those present in culture supernatant fluids.
exoproteins have a variety of functions and due to their localization most, if not all, interact with host molecules.
some have immunomodulatory effects, such as superantigens, which disrupt the immune response to infection by non-specifically stimulating t lymphocytes  <cit> .
others are cytolysins, such streptolysins o  and s , and many are hydrolytic enzymes that degrade host macromolecules to generate catabolic substrates or to promote tissue invasion.
examples of the latter include, hyaluronidase , which is required for growth using hyaluronic acid as the sole carbon source  <cit> ; a secreted protease, speb, which is thought to promote dissemination by degrading a variety of extracellular matrix proteins, as well streptococcal various adhesins  <cit>  and other secreted virulence factors such as nucleases and streptokinase  <cit> .
proteolysis can also liberate peptides and amino acids for catabolism.
in addition, secreted nucleases promote dissemination by degrading nucleic acids present in neutrophil extracellular entrapment, or nets  <cit> .
finally, secreted proteases and secreted nucleases are also likely to work together to disperse s. pyogenes biofilms, which are composed of both proteins and extracellular dna  <cit> .
the regulation of exoprotein production is complex and involves a variety of transcriptional regulatory proteins, many of which are influenced by the availability of various metabolic substrates  <cit> .
because s. pyogenes is auxotrophic for most amino acids, the pathogen's ability to respond to amino acid depletion is likely to be critical for survival within the human host.
the response involves both the rela-dependent pathway mediated by accumulation of ppgpp  <cit>  and a rela-independent pathway  <cit> , mediated, at least in part, by the transcriptional regulator cody  <cit> .
cody is present in the genomes of many low g + c gram-positive bacteria and mediates changes in expression in response to the availability of amino acids  <cit> .
the protein binds to intracellular branched amino acids, which increases its affinity for dna binding, typically resulting in the repression of gene expression.
when branched chain amino acids are depleted, dna affinity decreases allowing the initiation of transcription.
although usually considered to be a repressor, cody activates expression of acetate kinase  <cit>  and bsff, which is a small rna in b. subtilis <cit> .
in s. pyogenes, cody controls the expression of genes involved in the response to nutritional stress, including genes encoding exoproteins.
the transcript levels of  <dig> genes were previously compared between a wild-type strain of s. pyogenes and a cody mutant derivative by using quantitative reverse transcriptase pcr   <cit> .
eleven of the genes were predicted to encode secreted proteins.
the expression of four of these genes  was greater in the wild-type strain compared to the mutant strain, while the expression of the remaining seven was less .
subsequently, by using dna microarrays, inactivation of cody in s. pyogenes was found to alter the transcription of approximately 17% of genes in the chromosome, including several that encoded exoproteins  <cit> .
together, the results indicate that cody is a global regulator controlling the transcription of a variety of genes, including some encoding exoproteins, which are likely to influence host-pathogen interactions  <cit> .
the purpose of this study was to compare the exoproteins of a wild-type strain of s. pyogenes to a cody mutant strain to identify potential differences derived either at the transcriptional or post-transcriptional level.
the results confirmed, at the protein level, several differences in expression previously predicted by transcript analyses and identified additional exoproteins with altered abundance following the deletion of cody.
RESULTS
analysis of exoproteins by sds-page
as an initial step to identify differences in exoprotein production between a cody mutant and a wild-type strain of s. pyogenes, the strains were grown to the stationary phase of growth and culture supernatant proteins  were analysed by using sds-page gel electrophoresis.
there was no difference in either the growth rate or growth yield of the two strains .
separation of csps by using sds-page showed several differences in the amounts of specific proteins .
seven protein bands were excised from the gel and analysed with tandem mass spectrometry .
the results indicated that hyalurondidase , which degrades hyaluronic acid present in the extracellular matrix of host tissue and the bacterial capsule, a 5’-nucleotidase , a secreted protein with similarity to amidases , and a hypothetical protein possessing a type ii secretion signal  were more abundant in the supernatant fluid obtained from the wild-type strain .
in addition, the oligopeptide transport protein oppa  and a zinc binding protein adca  were less abundant in supernatant obtained from the wild-type strain .
in some instances, ms/ms analyses of the excised protein bands detected peptides corresponding to more than one protein  indicating that sds-page was insufficient to completely separate the proteins.
for example, protein band  <dig>  contained an equal number of peptides corresponding to the secreted protease speb  and camp factor .
analysis of exoproteins by two-dimensional gel electrophoresis
to better resolve the exoproteins 2-de was used and images of representative gels are shown in figure  <dig>  the production of most exoproteins was not influenced by cody deletion, however several differences were noted .
differentially expressed proteins were excised from the gels and identified with ms/ms .
in some instances proteins were differentially expressed in the representative gels shown in figure  <dig> but not in the other biological replicates we identified only those proteins that were differentially expressed in all three biological replicates.
a the number corresponds to the protein spot in figure  <dig>
b the open reading frame annotation based on the complete genome sequence of strain nz <dig> .
c the ratio cody/wt; a – indicated the protein was not detected in gels from one strain.
d nd, not detected.
one of the most striking differences was the abundance of three positional variants of speb, which is a well-characterized cysteine protease that is secreted as a zymogen.
specifically, the spots designated  <dig>   <dig>  and  <dig> were 18-, 9-, and 2-fold more abundant, respectively in the cody mutant strain compared to the wild-type strain .
the results were consistent with previous reports indicating that speb transcripts were more abundant in the cody mutant strain when cultured with rich media, or blood  <cit> .
increased extracellular nuclease activity is associated withcodydeletion
the genome of strain nz <dig> encodes two secreted dnases.
streptodornase b , also known as mitogenic factor  <dig> , is encoded within the bacterial chromosome.
the other secreted nuclease, spd- <dig>  is encoded within a prophage  <cit> .
three sdab isoforms  were 6-, 8-, and 2-fold more abundant in the cody mutant strain compared to the parental strain .
in contrast, spd- <dig>  was only detected in csps prepared from the wild-type strain .
thus, the overall effect of cody deletion on extracellular nuclease activity remained unclear since sdab was more abundant in the mutant but spd- <dig> was less abundant.
to address this issue, csps were isolated from the strains following 24 h culture with cdm and dnase activity was determined.
the results showed that deletion of cody increased dnase activity .
biofilm formation in cdm, but not rich medium, is influenced bycodydeletion
static biofilms formed by s. pyogenes are dispersed by the addition of exogenous proteases and dnases, indicating the matrix is composed of both protein and dna  <cit> .
based on differences in the production of the secreted protease speb and extracellular dnases between the two strains, and the influence of cody on biofilm formation in related species  <cit> , it was of interest to determine if deletion of cody altered biofilm formation of s. pyogenes.
static biofilm assays showed that deletion of cody consistently reduced biofilm formation compared to the parental strain when cultured with cdm ; although both strains still formed biofilms.
there was no difference in biofilm formation when the strains were cultured with thy medium .
together the results indicate that cody has a relatively minor, but reproducible, influence s. pyogenes biofilm formation under specific environmental conditions, perhaps due to changes in extracellular nuclease activity.
deletion ofcodyaffects the production of a putative zinc permease and camp factor
a constellation of at least four variants of the uncharacterized protein adca  was more abundant in supernatants from the cody mutant strain .
a significant difference in adca transcripts was not previously identified using dna microarrays in either the exponential or stationary phases of growth  <cit> .
the predicted  <dig> amino acid protein  has a putative signal peptide, a histidine rich motif, and is annotated as a zinc binding transporter  <cit> .
it is part of the troa superfamily, the members of which are involved in the transport of zinc into the cytoplasm.
an adca orthologue in streptococcus pneumoniae is a zn2+ permease involved in the development of natural competence for dna transformation  <cit>  and the orthologue in s. pneumoniae and s. gordonii is required for both biofilm formation and competence  <cit> .
we note that while adca was more abundant in the mutant strain, which did not form significant biofilms when cultured with cdm, the protein was detected in samples from the wild-type strain and thus production may have been sufficient to support biofilm production.
in addition, two positional variants of camp factor  were less abundant in csps obtained from the cody mutant strain compared to the wild-type strain .
the results correlated with those obtained previously by measuring cfa transcripts  <cit> .
cfa is encoded as a  <dig> amino acid protein with a type ii signal peptide.
in a camp test, cfa acts synergistically with the β hemolysin of staphylococcus aureus to lyse erythrocytes.
the camp test was used to compare cfa activity between the two strains and the results showed that deletion of cody decreased cfa activity .
while it remains possible that potential differences in growth between the two strains on blood agar plates may contribute to the difference in camp factor activity the results are consistent with those obtained with proteomic analyses  and those obtained previously by measuring transcripts  <cit> .
discussion
s. pyogenes exoproteins contribute substantially to interactions with the human host.
production is regulated by several, apparently redundant, transcriptional regulatory circuits working together to control expression.
we used a proteomics approach to characterize the contribution of cody to the regulation of s. pyogenes exoproteins.
the purposes of this study were to clarify how previously identified differences in transcript levels between a wild-type and cody mutant strain are manifest at the protein level and to determine if cody deletion is associated with additional differences in the exoproteome due to post-transcriptional effects.
the results confirmed, at the protein level, previously identified differences between the strains in the production of speb, cfa, and sdab.
moreover, additional exoproteins were discovered to be regulated by cody, including the virulence associated secreted nuclease spd- <dig>  which is encoded by a prophage, a putative zinc binding transport protein adca, and hyla.
overall, the results contribute to defining the s. pyogenes exoproteome and the role cody plays in determining its composition.
the proteolytically active form of speb can degrade several streptococcal exoproteins  <cit> .
speb is secreted as a 40 kda zymogen.
it is subsequently converted to a 28 kda proteolytically active form following a multi-step process involving intra- and inter-molecular speb cleavages and at least two peptidyl-prolyl, cistrans isomerases .
we harvested exoproteins by tca/acetone precipitation prior to activation of the speb protease.
thus, under the conditions used in this study, only the zymogen form of speb was detected in the 2-de gels and not the proteolytic form .
in addition, no protease activity was detected in the culture supernatant samples .
finally, the abundance of most exoproteins was similar between the two strains, despite the significant increase in speb zymogen production in the cody mutant strain, indicating that the exoproteins were not being degraded by speb in the mutant strain.
the production of two secreted nucleases was affected by cody deletion.
the expression of sdab was greater in the mutant strain, which is consistent with results previously obtained by using quantitative pcr during the exponential, but not stationary, phase of growth in rich media  <cit> .
in contrast, the amount of the prophage-encoded spd- <dig> protein was less in a cody mutant .
this difference was not evident in a previous study in either the exponential or stationary phases of growth, respectively  <cit> .
while this may indicate the involvement of post-transcriptional mechanisms in the regulation of spd- <dig>  it is noteworthy that the culture conditions used to identify differences in transcripts associated with cody inactivation differed from those used here to identify changes at the protein level, which may account for the differences among the studies.
dnase assays showed more activity in the cody mutant, which was consistent with the increase in sdab production .
previously, sdab was reported to be the protein primarily responsible for extracellular dnase activity in a serotype m <dig> strain based on the absence of activity following sdab inactivation  <cit> .
the genome of strain nz <dig> encodes four proteins with hyaluronidase motifs; two of these, spy49_ <dig> and spy49_1465c, are encoded by prophage and do not possess a signal peptide.
presumably, these proteins are released from the cell upon phage-induced lysis and degrade the hyaluronic capsule of s. pyogenes, which facilitates phage attachment and infection of streptococci  <cit> .
among the two chromosomally encoded proteins with hyaluronidase motifs, spy49_1236c , which does not possess a signal peptide was recently discovered to have β-n-acetylgucosaminidase activity and not hyaluronidase activity  <cit> .
thus the only gene product possessing a signal peptide was the hyaluronidase protein  detected in supernatant preparations from the wild-type and cody mutant.
deletion of cody decreased the abundance of two positional variation of hyla, as detected in 2-de gels, which correlated with results obtained with sds-page.
hyaluronidases are often thought of as spreading factors, facilitating dissemination of the pathogen; however, in murine models of s. pyogenes infection, hyla did not promote pathogen dissemination directly, but did increase the permeability of host tissue, which is likely to enhance toxin dissemination and thereby contribute to virulence  <cit> .
CONCLUSIONS
in summary, a proteomic approach was used to assess the role cody plays in the regulation of s. pyogenes exoproteins.
the results confirmed, at the protein level, that cody regulates several well-studied exoproteins, including the speb protease and camp factor.
in addition, we discovered new cody regulated exoproteins including hyla.
the results are important in understanding the roles various regulatory proteins play in controlling exoprotein production, which is intimately linked to the ability of the pathogen to adapt, and therefore survive, changing conditions encountered in its human host.

 streptococcus pyogenes is thought to be responsible for more than deaths worldwide each year. pathogenesis involves several proteins localized to the extracellular environment. these secreted proteins, or exoproteins, can be experimentally defined as those present in culture supernatant fluids. exoproteins have a variety of functions and due to their localization most, if not all, interact with host molecules. some have immunomodulatory effects, such as superantigens, which disrupt the immune response to infection by non-specifically stimulating t lymphocytes. others are cytolysins, such streptolysins o and s, and many are hydrolytic enzymes that degrade host macromolecules to generate catabolic substrates or to promote tissue invasion. examples of the latter include, hyaluronidase, which is required for growth using hyaluronic acid as the sole carbon source; a secreted protease, speb, which is thought to promote dissemination by degrading a variety of extracellular matrix proteins, as well streptococcal various adhesins and other secreted virulence factors such as nucleases and streptokinase. proteolysis can also liberate peptides and amino acids for catabolism. in addition, secreted nucleases promote dissemination by degrading nucleic acids present in neutrophil extracellular entrapment, or nets. finally, secreted proteases and secreted nucleases are also likely to work together to disperse s. pyogenes biofilms, which are composed of both proteins and extracellular dna. the regulation of exoprotein production is complex and involves a variety of transcriptional regulatory proteins, many of which are influenced by the availability of various metabolic substrates. because s. pyogenes is auxotrophic for most amino acids, the pathogen's ability to respond to amino acid depletion is likely to be critical for survival within the human host. the response involves both the rela-dependent pathway mediated by accumulation of ppgpp and a rela-independent pathway, mediated, at least in part, by the transcriptional regulator cody. cody is present in the genomes of many low g + c gram-positive bacteria and mediates changes in expression in response to the availability of amino acids. the protein binds to intracellular branched amino acids, which increases its affinity for dna binding, typically resulting in the repression of gene expression. when branched chain amino acids are depleted, dna affinity decreases allowing the initiation of transcription. although usually considered to be a repressor, cody activates expression of acetate kinase and bsff, which is a small rna in b. subtilis. in s. pyogenes, cody controls the expression of genes involved in the response to nutritional stress, including genes encoding exoproteins. the transcript levels of genes were previously compared between a wild-type strain of s. pyogenes and a cody mutant derivative by using quantitative reverse transcriptase pcr. eleven of the genes were predicted to encode secreted proteins. the expression of four of these genes was greater in the wild-type strain compared to the mutant strain, while the expression of the remaining seven was less. subsequently, by using dna microarrays, inactivation of cody in s. pyogenes was found to alter the transcription of approximately 17% of genes in the chromosome, including several that encoded exoproteins. together, the results indicate that cody is a global regulator controlling the transcription of a variety of genes, including some encoding exoproteins, which are likely to influence host-pathogen interactions. the purpose of this study was to compare the exoproteins of a wild-type strain of s. pyogenes to a cody mutant strain to identify potential differences derived either at the transcriptional or post-transcriptional level. the results confirmed, at the protein level, several differences in expression previously predicted by transcript analyses and identified additional exoproteins with altered abundance following the deletion of cody.  analysis of exoproteins by sds-page as an initial step to identify differences in exoprotein production between a cody mutant and a wild-type strain of s. pyogenes, the strains were grown to the stationary phase of growth and culture supernatant proteins were analysed by using sds-page gel electrophoresis. there was no difference in either the growth rate or growth yield of the two strains. separation of csps by using sds-page showed several differences in the amounts of specific proteins. seven protein bands were excised from the gel and analysed with tandem mass spectrometry. the results indicated that hyalurondidase, which degrades hyaluronic acid present in the extracellular matrix of host tissue and the bacterial capsule, a 5’-nucleotidase, a secreted protein with similarity to amidases, and a hypothetical protein possessing a type ii secretion signal were more abundant in the supernatant fluid obtained from the wild-type strain. in addition, the oligopeptide transport protein oppa and a zinc binding protein adca were less abundant in supernatant obtained from the wild-type strain. in some instances, ms/ms analyses of the excised protein bands detected peptides corresponding to more than one protein indicating that sds-page was insufficient to completely separate the proteins. for example, protein band contained an equal number of peptides corresponding to the secreted protease speb and camp factor. analysis of exoproteins by two-dimensional gel electrophoresis to better resolve the exoproteins 2-de was used and images of representative gels are shown in figure  the production of most exoproteins was not influenced by cody deletion, however several differences were noted. differentially expressed proteins were excised from the gels and identified with ms/ms. in some instances proteins were differentially expressed in the representative gels shown in figure  but not in the other biological replicates we identified only those proteins that were differentially expressed in all three biological replicates. a the number corresponds to the protein spot in figure  b the open reading frame annotation based on the complete genome sequence of strain nz. c the ratio cody/wt; a – indicated the protein was not detected in gels from one strain. d nd, not detected. one of the most striking differences was the abundance of three positional variants of speb, which is a well-characterized cysteine protease that is secreted as a zymogen. specifically, the spots designated and were 18-, 9-, and 2-fold more abundant, respectively in the cody mutant strain compared to the wild-type strain. the results were consistent with previous reports indicating that speb transcripts were more abundant in the cody mutant strain when cultured with rich media, or blood. increased extracellular nuclease activity is associated withcodydeletion the genome of strain nz encodes two secreted dnases. streptodornase b, also known as mitogenic factor, is encoded within the bacterial chromosome. the other secreted nuclease, spd- is encoded within a prophage. three sdab isoforms were 6-, 8-, and 2-fold more abundant in the cody mutant strain compared to the parental strain. in contrast, spd- was only detected in csps prepared from the wild-type strain. thus, the overall effect of cody deletion on extracellular nuclease activity remained unclear since sdab was more abundant in the mutant but spd- was less abundant. to address this issue, csps were isolated from the strains following 24 h culture with cdm and dnase activity was determined. the results showed that deletion of cody increased dnase activity. biofilm formation in cdm, but not rich medium, is influenced bycodydeletion static biofilms formed by s. pyogenes are dispersed by the addition of exogenous proteases and dnases, indicating the matrix is composed of both protein and dna. based on differences in the production of the secreted protease speb and extracellular dnases between the two strains, and the influence of cody on biofilm formation in related species, it was of interest to determine if deletion of cody altered biofilm formation of s. pyogenes. static biofilm assays showed that deletion of cody consistently reduced biofilm formation compared to the parental strain when cultured with cdm; although both strains still formed biofilms. there was no difference in biofilm formation when the strains were cultured with thy medium. together the results indicate that cody has a relatively minor, but reproducible, influence s. pyogenes biofilm formation under specific environmental conditions, perhaps due to changes in extracellular nuclease activity. deletion ofcodyaffects the production of a putative zinc permease and camp factor a constellation of at least four variants of the uncharacterized protein adca was more abundant in supernatants from the cody mutant strain. a significant difference in adca transcripts was not previously identified using dna microarrays in either the exponential or stationary phases of growth. the predicted amino acid protein has a putative signal peptide, a histidine rich motif, and is annotated as a zinc binding transporter. it is part of the troa superfamily, the members of which are involved in the transport of zinc into the cytoplasm. an adca orthologue in streptococcus pneumoniae is a zn2+ permease involved in the development of natural competence for dna transformation and the orthologue in s. pneumoniae and s. gordonii is required for both biofilm formation and competence. we note that while adca was more abundant in the mutant strain, which did not form significant biofilms when cultured with cdm, the protein was detected in samples from the wild-type strain and thus production may have been sufficient to support biofilm production. in addition, two positional variants of camp factor were less abundant in csps obtained from the cody mutant strain compared to the wild-type strain. the results correlated with those obtained previously by measuring cfa transcripts. cfa is encoded as a amino acid protein with a type ii signal peptide. in a camp test, cfa acts synergistically with the β hemolysin of staphylococcus aureus to lyse erythrocytes. the camp test was used to compare cfa activity between the two strains and the results showed that deletion of cody decreased cfa activity. while it remains possible that potential differences in growth between the two strains on blood agar plates may contribute to the difference in camp factor activity the results are consistent with those obtained with proteomic analyses and those obtained previously by measuring transcripts. discussion s. pyogenes exoproteins contribute substantially to interactions with the human host. production is regulated by several, apparently redundant, transcriptional regulatory circuits working together to control expression. we used a proteomics approach to characterize the contribution of cody to the regulation of s. pyogenes exoproteins. the purposes of this study were to clarify how previously identified differences in transcript levels between a wild-type and cody mutant strain are manifest at the protein level and to determine if cody deletion is associated with additional differences in the exoproteome due to post-transcriptional effects. the results confirmed, at the protein level, previously identified differences between the strains in the production of speb, cfa, and sdab. moreover, additional exoproteins were discovered to be regulated by cody, including the virulence associated secreted nuclease spd- which is encoded by a prophage, a putative zinc binding transport protein adca, and hyla. overall, the results contribute to defining the s. pyogenes exoproteome and the role cody plays in determining its composition. the proteolytically active form of speb can degrade several streptococcal exoproteins. speb is secreted as a 40 kda zymogen. it is subsequently converted to a 28 kda proteolytically active form following a multi-step process involving intra- and inter-molecular speb cleavages and at least two peptidyl-prolyl, cistrans isomerases. we harvested exoproteins by tca/acetone precipitation prior to activation of the speb protease. thus, under the conditions used in this study, only the zymogen form of speb was detected in the 2-de gels and not the proteolytic form. in addition, no protease activity was detected in the culture supernatant samples. finally, the abundance of most exoproteins was similar between the two strains, despite the significant increase in speb zymogen production in the cody mutant strain, indicating that the exoproteins were not being degraded by speb in the mutant strain. the production of two secreted nucleases was affected by cody deletion. the expression of sdab was greater in the mutant strain, which is consistent with results previously obtained by using quantitative pcr during the exponential, but not stationary, phase of growth in rich media. in contrast, the amount of the prophage-encoded spd- protein was less in a cody mutant. this difference was not evident in a previous study in either the exponential or stationary phases of growth, respectively. while this may indicate the involvement of post-transcriptional mechanisms in the regulation of spd- it is noteworthy that the culture conditions used to identify differences in transcripts associated with cody inactivation differed from those used here to identify changes at the protein level, which may account for the differences among the studies. dnase assays showed more activity in the cody mutant, which was consistent with the increase in sdab production. previously, sdab was reported to be the protein primarily responsible for extracellular dnase activity in a serotype m strain based on the absence of activity following sdab inactivation. the genome of strain nz encodes four proteins with hyaluronidase motifs; two of these, spy49_ and spy49_1465c, are encoded by prophage and do not possess a signal peptide. presumably, these proteins are released from the cell upon phage-induced lysis and degrade the hyaluronic capsule of s. pyogenes, which facilitates phage attachment and infection of streptococci. among the two chromosomally encoded proteins with hyaluronidase motifs, spy49_1236c, which does not possess a signal peptide was recently discovered to have β-n-acetylgucosaminidase activity and not hyaluronidase activity. thus the only gene product possessing a signal peptide was the hyaluronidase protein detected in supernatant preparations from the wild-type and cody mutant. deletion of cody decreased the abundance of two positional variation of hyla, as detected in 2-de gels, which correlated with results obtained with sds-page. hyaluronidases are often thought of as spreading factors, facilitating dissemination of the pathogen; however, in murine models of s. pyogenes infection, hyla did not promote pathogen dissemination directly, but did increase the permeability of host tissue, which is likely to enhance toxin dissemination and thereby contribute to virulence.  in summary, a proteomic approach was used to assess the role cody plays in the regulation of s. pyogenes exoproteins. the results confirmed, at the protein level, that cody regulates several well-studied exoproteins, including the speb protease and camp factor. in addition, we discovered new cody regulated exoproteins including hyla. the results are important in understanding the roles various regulatory proteins play in controlling exoprotein production, which is intimately linked to the ability of the pathogen to adapt, and therefore survive, changing conditions encountered in its human host. 
because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium and chemically defined media. in addition, hyaluronidase, camp factor, a prophage encoded nuclease, and an uncharacterized extracellular protein were less abundant in supernatant fluids obtained from the cody mutant strain. overall, the results indicate that cody alters the abundance of a select group of s. pyogenes exoproteins, including dnases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules. cody is a transcriptional regulator that controls gene expression in response to amino acid availability. proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. enzymatic assays showed greater dnase activity in culture supernatants isolated in the post-exponential phase of growth from the cody mutant strain compared to the wild-type strain. no difference was observed with rich media but with cdm the biofilms formed by the cody mutant strain had less biomass compared to the wild-type strain. we compared the secreted proteins produced by wild-type s. pyogenes to a cody mutant in the post-exponential phase of growth. the production of the secreted cysteine protease speb, a secreted chromosomally encoded nuclease, and a putative adhesion factor were more abundant in supernatant fluids obtained from the cody mutant. the purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the cody gene. we used both one and two-dimensional gel electrophoresis to separate exoproteins.
 the production of streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. cody is a transcriptional regulator that controls gene expression in response to amino acid availability. the purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the cody gene.  we compared the secreted proteins produced by wild-type s. pyogenes to a cody mutant in the post-exponential phase of growth. we used both one and two-dimensional gel electrophoresis to separate exoproteins. proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. the production of the secreted cysteine protease speb, a secreted chromosomally encoded nuclease, and a putative adhesion factor were more abundant in supernatant fluids obtained from the cody mutant. in addition, hyaluronidase, camp factor, a prophage encoded nuclease, and an uncharacterized extracellular protein were less abundant in supernatant fluids obtained from the cody mutant strain. enzymatic assays showed greater dnase activity in culture supernatants isolated in the post-exponential phase of growth from the cody mutant strain compared to the wild-type strain. because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium and chemically defined media. no difference was observed with rich media but with cdm the biofilms formed by the cody mutant strain had less biomass compared to the wild-type strain.  overall, the results indicate that cody alters the abundance of a select group of s. pyogenes exoproteins, including dnases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules. 