the general secretory ( sec ) pathway , conserved throughout bacteria , is the canonical translocation pathway and is responsible for translocating the vast majority of secreted proteins across the cytoplasmic membrane . like other general translocation pathways
, sec requires the synthesis of its cargo as preproteins with n - terminal signal peptides , which target them to the sec machinery and from which the mature protein must be released after translocation ( 1 , 2 ) .
the enzyme responsible for the liberation of most mature proteins translocated by sec is type i signal peptidase ( spase ) ( 35 ) .
accordingly , spase has been demonstrated to be essential in both gram - positive and gram - negative bacteria .
staphylococcus aureus is a striking example of the importance of spase , as it mediates the secretion of a diverse range of virulence factors , including proteins required for adhesion and colonization , evasion of the host immune response , scavenging of nutrients and minerals from the environment , and dissemination ( 6 ) .
the arylomycin family of natural products are potent inhibitors of spase ( 710 ) . as part of an effort to develop the arylomycins as therapeutics
, we and others have been exploring the optimization of their spectrum of activity ( 1113 ) .
an arylomycin with particularly promising activity against s.  aureus is arylomycin m131 ( fig . 
we have also developed the arylomycins as chemical biology probes to assess secretion in different bacteria , including staphylococcus epidermidis ( 14 ) and s.  aureus ( 6 ) .
as part of these efforts , we recently demonstrated that s.  aureus responds to arylomycin - mediated spase inhibition by increasing expression of the four adjacent genes , sa0337 to sa0340 , and that arylomycin resistance is conferred by loss - of - function mutations in sa0337 ( 15 ) .
( b ) genomic organization of the ayrrabc operon ( ayrr , sa0337 ; ayra , sa0338 ; ayrb , sa0339 ; ayrc , sa0340 ) and the region immediately upstream that includes sa0336 .
below is the sequence of the putative promoter region upstream of ayrr ending with its gtg translational start site . a 22-nt palindromic repeat ( arrows )
the positions of the putative 35 and 10 regions and ribosome binding site ( rbs ) are indicated in the coding strand .
the transcriptional start site is marked as + 1 , corresponding to 18  nt upstream from the translational start site .
( c ) rt - pcr analysis of the ayrrabc operon and divergent genes ( sa0334 to sa0336 ) .
gyrb was used as an external control , and gene expression was normalized using gmk .
( d ) gel shifts performed with 30  ng of each dna probe and an increasing concentration of ayrr as indicated at the top .
the region of dna corresponding to each gel shift probe is shown above the gel data , with the ayrr binding site shown in red .
filled triangles denote free dna probe , and open triangles denote the indicated dna - ayrr complexes .
all dna probes were amplified from wild - type n315 genomic dna with the exception of irpal , which was created by annealing synthetic oligonucleotides .
( e ) luminescence from the ayrr promoter ( expressed from parc1 ) in wild - type n315 and n315sa0336-ayrrabc ( labeled ayrr ) , expressed as counts per second per unit of optical density at 590 nm ( cps / od590 ) .
( f ) luminescence from n315 harboring parc1 in the presence and absence of arylomycin m131 ( 0.5 mic ) . here
, we demonstrate that the genes sa0337 to sa0340 constitute an operon and that sa0337 is a transcriptional repressor that controls its expression .
remarkably , derepression of these genes bypasses the need for spase , rendering spase nonessential and suggesting that the function of the operon is to mediate an alternate process by which translocated proteins are released from the cytoplasmic membrane .
we have thus named the repressor gene ayrr and the downstream genes ayrabc for their role in arylomycin resistance .
sequence analysis identifies ayra as a 6-transmembrane - domain protein of unknown function ( duf3169/pf11368 ) and ayrb and ayrc as two domains of an abc transporter , and we demonstrate that all three are required to bypass spase . finally , we demonstrate that ayrabc is able to mediate secretion of the proteins normally processed by spase , although in some cases with reduced efficiency , but it does so either with the signal peptide still attached or after cleavage at a site within the signal peptide .
the stop and start codons of ayrr and ayra overlap , as do those of ayrb and ayrc , while the stop and start codons of ayra and ayrb are separated by only 24  bp ( fig . 
using reverse transcription - pcr ( rt - pcr ) , we examined transcription of ayrrabc in the arylomycin - resistant strain arc0001 , which harbors a nonsense mutation in ayrr ( 15 ) , and its parental wild - type strain n315 .
as expected , no differences in transcript levels were observed for control genes ( sa0334 to sa0336 and gyrb ) , but transcript levels of ayrr , ayra , ayrb , and ayrc in arc0001 were each increased ~8-fold compared to those in the wild type ( fig . 
moreover , analysis of our previously published rna - seq data ( 15 ) reveals reads that when overlapped align continuously across the entire ayrrabc locus , including the intergenic sequences ( see fig . 
collectively , these results suggest that ayrrabc is an operon and demonstrate that the mutation in ayrr that renders s.  aureus resistant to spase inhibition results in operon derepression .
ayrr encodes a helix - turn - helix motif protein annotated as an xre family transcriptional regulator with homology to the phage  cro repressor .
sequence analysis revealed an almost perfect 22-nucleotide ( nt ) palindrome , tttgacaaatatagttgtcaaa , upstream of ayrr which overlaps the 35 promoter element ( fig . 
such palindromes commonly compose the binding site of transcriptional regulators ( 16 ) , and cro regulates its own transcription by binding such a palindrome ( 17 , 18 ) . to determine if ayrr binds its upstream palindrome , a gel shift assay was employed using various dna fragments ( fig . 
the data clearly demonstrate that ayrr selectively binds the palindrome . to test the functional significance of this binding
, we constructed the transcriptional reporter plasmid parc1 , which harbors the intergenic region between sa0336 and ayrr upstream of the genes encoding luciferase ( luxcdabe ) ( 19 ) .
wild - type n315 and a strain lacking the entire region from sa0336 to ayrc ( n315sa0336-ayrrabc ) were then transformed with either the empty luxcdabe vector or parc1 , and luminescence was assayed in actively growing cultures ( fig . 
luminescence was significantly lower for n315 harboring parc1 , consistent with repression by the genomically encoded ayrr protein .
as an additional control , the ability of spase inhibition to release repression was tested by conducting the same experiment in the presence of a subinhibitory concentration of arylomycin m131 ( 0.5 mic ) .
two  hours after arylomycin addition , the luminescence signal observed with wild - type n315 cells harboring parc1 was 7-fold higher than that observed in the absence of arylomycin ( fig . 
these results confirm that ayrr acts as a repressor of ayrrabc and that spase inhibition induces derepression .
ayra encodes a putative membrane protein , predicted to possess a duf3169 domain of unknown function .
proteins in the duf3169 family are found in both staphylococcus and streptococcus species , with one homolog present per genome in sequenced strains , and while they share only ~30% sequence identity , six predicted transmembrane domains and a d - e(a / g)e motif located in the loop connecting the fourth and fifth predicted transmembrane segments are highly conserved .
interestingly , each ayra homolog identified appears to be immediately downstream of a gene that is homologous to ayrr .
the downstream genes ayrb and ayrc are predicted to encode the two domains of a type 2 family abc transporter , with ayrc encoding the transporter domain and ayrb encoding the atp - binding cassette domain ( conserved domain cd03230 ) .
although ayra could be deleted in an otherwise wild - type strain ( n315 ) , we were unable to delete ayra in arc0001 .
thus , to test whether ayra is required to tolerate spase inhibition , we attempted to evolve arylomycin resistance in n315ayra .
as a control , we constructed and analyzed a strain lacking sa0336 , which is predicted to encode a hypothetical protein and which is not part of the ayrrabc operon .
high - level arylomycin m131 resistance ( > 16  g / ml ) was evolved in n315sa0336 with frequencies indistinguishable from the wild - type parental strain ( 2.0  10 ) ( 15 ) ( see table  s2  in the supplemental material ) .
in addition , sequencing revealed that 8 out of 8 of the resistant strains examined contained mutations in ayrr ( see table  s3  in the supplemental material ) , similar to the behavior observed with wild - type cells ( 15 ) .
in contrast , n315ayra developed high - level resistance with a significantly reduced frequency ( 2.9  10 ) ( see table  s2 ) , and for the single resistant clone isolated , sequencing revealed a wild - type ayrr .
deletion of ayrbc in arc0001 ( arc0001ayrbc ) resulted in full resensitization to arylomycin m131 , demonstrating that the abc transporter is required to tolerate spase inhibition ( see table  s2  in the supplemental material ) .
moreover , deletion of ayrbc in n315 ( n315ayrbc ) results in the same reduced frequency of resistance to arylomycin m131 that was observed with n315ayra ( see table  s2 ) , and again , sequencing revealed wild - type ayrr in the single resistant mutant isolated . collectively , these results demonstrate that each member of the ayrrabc operon is required to tolerate spase inhibition .
because derepression of ayrrabc in s. aureus confers high - level resistance to spase inhibition , we speculated that it may render spase nonessential . using the same chromosomal integration approach
, we attempted to delete the spase gene spsb in arc0001 and its parental wild - type strain , n315 .
not surprisingly , attempts to delete spsb in the wild - type strain resulted in the recovery of the wild - type sequence .
in addition to confirming the absence of spsb at its chromosomal locus via sequencing , we demonstrated that spsb was not present elsewhere in the genome of arc0001spsb via pcr and rt - pcr of genomic dna and rna , respectively , using primers internal to the spsb gene ( see table  s4  in the supplemental material ) .
deletion of spsb was also verified phenotypically via an examination of the susceptibility of n315 and arc0001spsb to gentamicin and the -lactam antibiotics cefoxitin , oxacillin , and penicillin g. previously , we and others demonstrated that the activity of these antibiotics is uniquely synergistic with that of the arylomycins ( 13 , 20 ) . as controls , we also examined susceptibilities to cccp , vancomycin , daptomycin , erythromycin , trimethoprim , and tetracycline . for each control antibiotic tested , the observed susceptibilities of n315 were virtually identical to those observed for n315 and arc0001 ( 15 ) .
however , the susceptibility of arc0001spsb to gentamicin and each -lactam was 8- to 64-fold greater than that of n315 or arc0001 ( see table  s5  in the supplemental material ) , consistent with the absence of spase activity .
we next evaluated protein secretion in n315 , arc0001 , and arc0001spsb using one - dimensional ( 1d ) sds - page ( fig .  2 ) .
identical patterns of secretion were observed for the wild type and arc0001 , which suggests that arc0001 still employs spase even though the ayrrabc operon is derepressed .
in contrast , a small but clearly significant alteration in the pattern of secretion was apparent for arc0001spsb . to confirm that this altered pattern of secretion was the result of the loss of spase activity ,
secretion was examined in the presence of arylomycin m131 ( 4 mic ) . as expected ,
wild - type cells showed drastically reduced levels of secretion in the presence of the arylomycin .
however , treatment of arc0001 cells with the arylomycin did not reduce secretion and resulted in a secretion pattern indistinguishable from that of arc0001spsb , clearly revealing that the altered pattern does indeed result from the absence of spase activity .
moreover , the pattern and quantity of secreted proteins observed with arc0001spsb were unchanged by the addition of the arylomycin , confirming that secretion did not depend on spase function .
the extracellular proteomes of n315 , arc0001 , and arc0001spsb in the absence and presence of arylomycin m131 . to minimize cell lysis
, strains were grown until wild - type n315 reached an od590 of 4.0 , corresponding to late exponential growth .
strains were then balanced to an od590 of 4.0 , pelleted by centrifugation , and washed twice with medium before resuspension in fresh medium .
strains were then grown in the presence or absence of 4 mic of arylomycin m131 ( n315 ) for 20  min .
proteins in the supernatant were precipitated overnight with 10% ( wt / vol ) tca , washed with acetone , resuspended in 1 loading dye , and visualized by sds - page .
arrows denote bands where significant differences are observed in the extracellular proteomes of wild - type n315 and arc0001spsb .
the secreted proteomes of arc0001spsb and n315 were next compared in more detail by isolating proteins from the medium , digesting with trypsin , using reductive dimethylation ( redime ) to label the wild - type fragments with heavy isotopes and label the arc0001spsb fragments with light isotopes , and then analyzing the combined samples via multidimensional mass spectrometry ( mudpit ) ( 21 , 22 ) . in total ,
tryptic peptides corresponding to 38 proteins that are encoded with signal peptides were identified ( table  1 ) , which is consistent with previous studies of the s.  aureus secretome ( 6 ) . the quantity of secreted proteins was then determined from the ratio of parent ion peak areas , which demonstrated that while tryptic peptides for the same proteins were detected from each strain , several were detected at different levels ( table  1 ) .
sixteen of the 38 secreted proteins were detected in both strains at similar levels ( within 2-fold ) , while 20 were detected at 2- to 8-fold - reduced levels ( for example , the proteases staphylokinase and sspp , the complement inhibitor sak , and a hypothetical surface protein sa2285 ) .
two proteins ( sa0663 and sdre ) were detected at 6- to 8-fold - elevated levels in the spsb strain , while prsa was detected at a 16-fold - elevated level .
prsa is a peptidyl - prolyl isomerase involved in protein folding of secreted proteins in the extracellular environment ( 23 ) , and we previously demonstrated that it is upregulated at the transcriptional level by spase inhibition .
comparison of the secreted proteomes of wild - type n315 and arc0001spsb reported as the average for all detected tryptic peptides for a given protein .
data are averages and standard errors of the means for three independent biological samples . underlined amino acids correspond to tryptic peptides detected in arc0001spsb which contain part of the spase signal sequence .
as expected for secretion mediated by spase , none of the tryptic peptides isolated from the wild - type sample contained any part of the signal peptide ; however , peptide fragments for eight of the proteins were found with n termini that precisely matched the predicted spase cleavage site ( see fig . 
in contrast , with arc0001spsb , tryptic peptides containing portions of the n - terminal leader peptides of 12 of the 38 proteins were detected ( table  1 ; also , see fig . 
these data suggest that ayrabc mediates secretion of the same proteins as does spase but either via cleavage at a more n - terminal site or perhaps by extricating the entire intact preprotein from the cytoplasmic membrane .
spase functions at the terminal step of the general secretory pathway by releasing translocated proteins from the cytoplasmic membrane at a defined cleavage site .
we have provided evidence of an inducible alternative terminal step in s.  aureus encoded by the ayrrabc operon , which under normal conditions is repressed by ayrr but which is derepressed by spase inhibition .
the natural physiological role of the pathway may be to support spase function at times when elevated secretion is required and the capacity of spase has been exceeded .
alternatively , it may have evolved to facilitate survival in the presence of an spase inhibitor .
the latter possibility is consistent with the fact that spase is the only component of the general secretory pathway that is exposed on the outer surface of the cytoplasmic membrane , making it uniquely susceptible to extracellular inhibitors .
the evolution of four different families of arylomycins ( 10 ) further suggests that that spase inhibition may have represented a significant selection pressure , as does the existence of multiple , redundant spases in many gram - positive bacteria .
hints as to the mechanism by which ayrabc mediates the release of proteins from their attachment to the cell may be gleaned from an analysis of the individual genes .
it is possible that the abc transporter functions as part of the translocation process , perhaps augmenting or even replacing other components of the general secretory pathway .
however , bacterial abc transporters have been identified that extract lipophilic moieties from the cytoplasmic membrane ( 24 ) , and ayrbc may function after sec translocation to extract intact preproteins or proteins after intramembrane cleavage .
the role of ayra , which encodes a membrane protein of unknown function , is even less clear , but it is interesting to speculate that it could be a protease responsible for intramembrane cleavage , if one is required , which is consistent with its predicted membrane localization .
it is also interesting that ayrr homologs in other species appear to control the regulation of proteases ( 25 , 26 ) .
a more complete understanding of the pathway awaits detailed analysis of these proteins as well as the signal responsible for derepression of the operon , which is in progress .
undoubtedly , understanding the mechanistic details of this alternate terminal step of the sec pathway will lead to a greater understanding of secretion and the response to secretion stress .
the stop and start codons of ayrr and ayra overlap , as do those of ayrb and ayrc , while the stop and start codons of ayra and ayrb are separated by only 24  bp ( fig . 
using reverse transcription - pcr ( rt - pcr ) , we examined transcription of ayrrabc in the arylomycin - resistant strain arc0001 , which harbors a nonsense mutation in ayrr ( 15 ) , and its parental wild - type strain n315 .
as expected , no differences in transcript levels were observed for control genes ( sa0334 to sa0336 and gyrb ) , but transcript levels of ayrr , ayra , ayrb , and ayrc in arc0001 were each increased ~8-fold compared to those in the wild type ( fig . 
1c ) . moreover , analysis of our previously published rna - seq data ( 15 ) reveals reads that when overlapped align continuously across the entire ayrrabc locus , including the intergenic sequences ( see fig . 
collectively , these results suggest that ayrrabc is an operon and demonstrate that the mutation in ayrr that renders s.  aureus resistant to spase inhibition results in operon derepression .
ayrr encodes a helix - turn - helix motif protein annotated as an xre family transcriptional regulator with homology to the phage  cro repressor .
sequence analysis revealed an almost perfect 22-nucleotide ( nt ) palindrome , tttgacaaatatagttgtcaaa , upstream of ayrr which overlaps the 35 promoter element ( fig . 
such palindromes commonly compose the binding site of transcriptional regulators ( 16 ) , and cro regulates its own transcription by binding such a palindrome ( 17 , 18 ) . to determine
if ayrr binds its upstream palindrome , a gel shift assay was employed using various dna fragments ( fig . 
to test the functional significance of this binding , we constructed the transcriptional reporter plasmid parc1 , which harbors the intergenic region between sa0336 and ayrr upstream of the genes encoding luciferase ( luxcdabe ) ( 19 ) .
wild - type n315 and a strain lacking the entire region from sa0336 to ayrc ( n315sa0336-ayrrabc ) were then transformed with either the empty luxcdabe vector or parc1 , and luminescence was assayed in actively growing cultures ( fig . 
luminescence was significantly lower for n315 harboring parc1 , consistent with repression by the genomically encoded ayrr protein .
as an additional control , the ability of spase inhibition to release repression was tested by conducting the same experiment in the presence of a subinhibitory concentration of arylomycin m131 ( 0.5 mic ) .
two  hours after arylomycin addition , the luminescence signal observed with wild - type n315 cells harboring parc1 was 7-fold higher than that observed in the absence of arylomycin ( fig . 
these results confirm that ayrr acts as a repressor of ayrrabc and that spase inhibition induces derepression .
ayra encodes a putative membrane protein , predicted to possess a duf3169 domain of unknown function .
proteins in the duf3169 family are found in both staphylococcus and streptococcus species , with one homolog present per genome in sequenced strains , and while they share only ~30% sequence identity , six predicted transmembrane domains and a d - e(a / g)e motif located in the loop connecting the fourth and fifth predicted transmembrane segments are highly conserved .
interestingly , each ayra homolog identified appears to be immediately downstream of a gene that is homologous to ayrr .
the downstream genes ayrb and ayrc are predicted to encode the two domains of a type 2 family abc transporter , with ayrc encoding the transporter domain and ayrb encoding the atp - binding cassette domain ( conserved domain cd03230 ) .
although ayra could be deleted in an otherwise wild - type strain ( n315 ) , we were unable to delete ayra in arc0001 .
thus , to test whether ayra is required to tolerate spase inhibition , we attempted to evolve arylomycin resistance in n315ayra .
as a control , we constructed and analyzed a strain lacking sa0336 , which is predicted to encode a hypothetical protein and which is not part of the ayrrabc operon .
high - level arylomycin m131 resistance ( > 16  g / ml ) was evolved in n315sa0336 with frequencies indistinguishable from the wild - type parental strain ( 2.0  10 ) ( 15 ) ( see table  s2  in the supplemental material ) .
in addition , sequencing revealed that 8 out of 8 of the resistant strains examined contained mutations in ayrr ( see table  s3  in the supplemental material ) , similar to the behavior observed with wild - type cells ( 15 ) .
in contrast , n315ayra developed high - level resistance with a significantly reduced frequency ( 2.9  10 ) ( see table  s2 ) , and for the single resistant clone isolated , sequencing revealed a wild - type ayrr .
deletion of ayrbc in arc0001 ( arc0001ayrbc ) resulted in full resensitization to arylomycin m131 , demonstrating that the abc transporter is required to tolerate spase inhibition ( see table  s2  in the supplemental material ) .
moreover , deletion of ayrbc in n315 ( n315ayrbc ) results in the same reduced frequency of resistance to arylomycin m131 that was observed with n315ayra ( see table  s2 ) , and again , sequencing revealed wild - type ayrr in the single resistant mutant isolated . collectively , these results demonstrate that each member of the ayrrabc operon is required to tolerate spase inhibition .
because derepression of ayrrabc in s. aureus confers high - level resistance to spase inhibition , we speculated that it may render spase nonessential . using the same chromosomal integration approach
, we attempted to delete the spase gene spsb in arc0001 and its parental wild - type strain , n315 .
not surprisingly , attempts to delete spsb in the wild - type strain resulted in the recovery of the wild - type sequence .
in addition to confirming the absence of spsb at its chromosomal locus via sequencing , we demonstrated that spsb was not present elsewhere in the genome of arc0001spsb via pcr and rt - pcr of genomic dna and rna , respectively , using primers internal to the spsb gene ( see table  s4  in the supplemental material ) .
deletion of spsb was also verified phenotypically via an examination of the susceptibility of n315 and arc0001spsb to gentamicin and the -lactam antibiotics cefoxitin , oxacillin , and penicillin g. previously , we and others demonstrated that the activity of these antibiotics is uniquely synergistic with that of the arylomycins ( 13 , 20 ) . as controls , we also examined susceptibilities to cccp , vancomycin , daptomycin , erythromycin , trimethoprim , and tetracycline . for each control antibiotic tested , the observed susceptibilities of n315 were virtually identical to those observed for n315 and arc0001 ( 15 ) .
however , the susceptibility of arc0001spsb to gentamicin and each -lactam was 8- to 64-fold greater than that of n315 or arc0001 ( see table  s5  in the supplemental material ) , consistent with the absence of spase activity .
we next evaluated protein secretion in n315 , arc0001 , and arc0001spsb using one - dimensional ( 1d ) sds - page ( fig .  2 ) .
identical patterns of secretion were observed for the wild type and arc0001 , which suggests that arc0001 still employs spase even though the ayrrabc operon is derepressed .
in contrast , a small but clearly significant alteration in the pattern of secretion was apparent for arc0001spsb . to confirm that this altered pattern of secretion was the result of the loss of spase activity ,
secretion was examined in the presence of arylomycin m131 ( 4 mic ) . as expected ,
wild - type cells showed drastically reduced levels of secretion in the presence of the arylomycin .
however , treatment of arc0001 cells with the arylomycin did not reduce secretion and resulted in a secretion pattern indistinguishable from that of arc0001spsb , clearly revealing that the altered pattern does indeed result from the absence of spase activity .
moreover , the pattern and quantity of secreted proteins observed with arc0001spsb were unchanged by the addition of the arylomycin , confirming that secretion did not depend on spase function .
the extracellular proteomes of n315 , arc0001 , and arc0001spsb in the absence and presence of arylomycin m131 . to minimize cell lysis
, strains were grown until wild - type n315 reached an od590 of 4.0 , corresponding to late exponential growth .
strains were then balanced to an od590 of 4.0 , pelleted by centrifugation , and washed twice with medium before resuspension in fresh medium .
strains were then grown in the presence or absence of 4 mic of arylomycin m131 ( n315 ) for 20  min .
proteins in the supernatant were precipitated overnight with 10% ( wt / vol ) tca , washed with acetone , resuspended in 1 loading dye , and visualized by sds - page .
arrows denote bands where significant differences are observed in the extracellular proteomes of wild - type n315 and arc0001spsb .
the secreted proteomes of arc0001spsb and n315 were next compared in more detail by isolating proteins from the medium , digesting with trypsin , using reductive dimethylation ( redime ) to label the wild - type fragments with heavy isotopes and label the arc0001spsb fragments with light isotopes , and then analyzing the combined samples via multidimensional mass spectrometry ( mudpit ) ( 21 , 22 ) . in total ,
tryptic peptides corresponding to 38 proteins that are encoded with signal peptides were identified ( table  1 ) , which is consistent with previous studies of the s.  aureus secretome ( 6 ) . the quantity of secreted proteins was then determined from the ratio of parent ion peak areas , which demonstrated that while tryptic peptides for the same proteins were detected from each strain , several were detected at different levels ( table  1 ) .
sixteen of the 38 secreted proteins were detected in both strains at similar levels ( within 2-fold ) , while 20 were detected at 2- to 8-fold - reduced levels ( for example , the proteases staphylokinase and sspp , the complement inhibitor sak , and a hypothetical surface protein sa2285 ) .
two proteins ( sa0663 and sdre ) were detected at 6- to 8-fold - elevated levels in the spsb strain , while prsa was detected at a 16-fold - elevated level .
prsa is a peptidyl - prolyl isomerase involved in protein folding of secreted proteins in the extracellular environment ( 23 ) , and we previously demonstrated that it is upregulated at the transcriptional level by spase inhibition . examination of signal sequences revealed no obvious correlation with observed differences in secretion .
comparison of the secreted proteomes of wild - type n315 and arc0001spsb reported as the average for all detected tryptic peptides for a given protein .
. underlined amino acids correspond to tryptic peptides detected in arc0001spsb which contain part of the spase signal sequence .
as expected for secretion mediated by spase , none of the tryptic peptides isolated from the wild - type sample contained any part of the signal peptide ; however , peptide fragments for eight of the proteins were found with n termini that precisely matched the predicted spase cleavage site ( see fig . 
in contrast , with arc0001spsb , tryptic peptides containing portions of the n - terminal leader peptides of 12 of the 38 proteins were detected ( table  1 ; also , see fig . 
these data suggest that ayrabc mediates secretion of the same proteins as does spase but either via cleavage at a more n - terminal site or perhaps by extricating the entire intact preprotein from the cytoplasmic membrane .
spase functions at the terminal step of the general secretory pathway by releasing translocated proteins from the cytoplasmic membrane at a defined cleavage site .
we have provided evidence of an inducible alternative terminal step in s.  aureus encoded by the ayrrabc operon , which under normal conditions is repressed by ayrr but which is derepressed by spase inhibition .
the natural physiological role of the pathway may be to support spase function at times when elevated secretion is required and the capacity of spase has been exceeded .
alternatively , it may have evolved to facilitate survival in the presence of an spase inhibitor .
the latter possibility is consistent with the fact that spase is the only component of the general secretory pathway that is exposed on the outer surface of the cytoplasmic membrane , making it uniquely susceptible to extracellular inhibitors .
the evolution of four different families of arylomycins ( 10 ) further suggests that that spase inhibition may have represented a significant selection pressure , as does the existence of multiple , redundant spases in many gram - positive bacteria .
hints as to the mechanism by which ayrabc mediates the release of proteins from their attachment to the cell may be gleaned from an analysis of the individual genes .
it is possible that the abc transporter functions as part of the translocation process , perhaps augmenting or even replacing other components of the general secretory pathway .
however , bacterial abc transporters have been identified that extract lipophilic moieties from the cytoplasmic membrane ( 24 ) , and ayrbc may function after sec translocation to extract intact preproteins or proteins after intramembrane cleavage .
the role of ayra , which encodes a membrane protein of unknown function , is even less clear , but it is interesting to speculate that it could be a protease responsible for intramembrane cleavage , if one is required , which is consistent with its predicted membrane localization .
it is also interesting that ayrr homologs in other species appear to control the regulation of proteases ( 25 , 26 ) .
a more complete understanding of the pathway awaits detailed analysis of these proteins as well as the signal responsible for derepression of the operon , which is in progress .
undoubtedly , understanding the mechanistic details of this alternate terminal step of the sec pathway will lead to a greater understanding of secretion and the response to secretion stress .
the ayrrabc operon corresponds to the region from position 343,418 to 345,827 in the nctc8325 chromosome .
the major transcriptional start site corresponds to coordinate 434,400 , which is 18  nt upstream of the gtg start codon .
a small number of reads corresponded to coordinate 343,248 suggesting a secondary minor promoter region further upstream .
overlapping rna reads were detected for the entire ayrrabc locus , suggesting that they are cotranscribed .
download   figure  s1 , tif file , 0.8 mb solid - state synthesis of ayrr .
the entire ayrr protein was synthesized using standard methods for boc solid - state peptide synthesis employed in our laboratory ( r. adhikary , j. zimmermann , j. liu , p. e. dawson , f. e. romesberg , j. phys .
, the peptide was synthesized on 0.4 mmol of p - methyl - benzhydrylamine hcl ( mbha ) resin using 2.2 mmol of protected amino acid , 4 ml 0.5 m of the peptide coupling reagent hctu ( 2.0 mmol ) , and 700 l n , n - diisopropylethylamine ( diea ) ( ~4.1 mmol ) , with a coupling time of 20 min .
the first amino acid , boc - lys(fmoc)-oh , was coupled onto the mbha resin , and the fmoc group was removed by reaction with 20% piperidine - dimethylformamide ( vol / vol ) ; boc groups were deprotected with neat trifluoroacetic acid ( tfa ) for 2 min .
an extra lysine residue was added at the c terminus of ayrr to conjugate biotin with the side chain of lysine .
the biotinyl - n - hydroxysuccinimide ester was generated by first dissolving 2 mmol n - hydroxysuccinimide and 2 mmol biotin in 5 ml dimethyl sulfoxide ( dmso ) and then adding 2 mmol n , n-diisopropyl carbodiimide .
after 30 min , the mixture was added to the resin and coupled for 1 h , followed by the addition of 2 mmol diea , which was coupled for an additional hour .
the peptide was cleaved from the dry resin support using anhydrous hf with 10% ( vol / vol ) anisole for 1 h at 0c .
the resulting polypeptide was purified via reverse - phase high - performance liquid chromatography ( hplc ) using a linear gradient of solvent a ( 0.1% [ vol / vol ] aqueous tfa ) and solvent b ( 90% [ vol / vol ] acetonitrile , 10% [ vol / vol ] water , 0.1% [ vol / vol ] tfa ) .
purity was assessed by analytical hplc ( a ) and mass spectrometry ( b and c ) .
the observed mass of the pure biotinylated peptide is 8,027.0 da ( calculated : 8,027.3 da ) ; shown are the mass spectrum ( b ) and reconstructed mass spectrum ( c ) .
download   figure  s2 , tif file , 0.1 mb a representation of tryptic peptides detected from the secreted proteome .
a schematic of a secreted protein depicting its signal peptide ( grey ) and mature protein ( white ) with the location of 3 tryptic peptides is shown : ( 1 ) tryptic peptides identified in the arc0001spsb strain which contain amino acids located in the signal peptide and which overlap the spase cleavage site ; the spase cleavage site is denoted by a dash in the amino acid sequence ; ( 2 ) tryptic peptides found in wild - type n315 that correspond to the n terminus of the mature spase processed secreted protein ; and ( 3 ) representative tryptic peptides from the mature protein . below the schematic are parent ion chromatograms of three representative proteins ( immunoglobulin g - binding protein a [ spa sa0107 ] , the putative surface protein sa2285 , and the staphylococcal complement inhibitor sa1754 ) , detected from the secreted proteome of wild - type ( blue trace ) or arc0001spsb ( red trace ) s.  aureus .
download   figure  s3 , tif file , 0.3 mb primers used in this study .
table  s1 , doc file , 0.01 mb mics and frequencies of resistance to arylomycin m131 .
table  s2 , doc file , 0.03 mb ayrr mutations that evolve to arylomycin m131 in n315sa0336 .
table  s3 , doc file , 0.01 mb amplification of spsb from rna and genomic dna .
table  s4 , doc file , 0.01 mb mics of n315 , arc0001 , and arc0001spsb table  s5 , doc file , 0.03 mb
 abstracttype i signal peptidase ( spase ) is essential for viability in wild - type bacteria because the terminal step of the bacterial general secretory pathway requires its proteolytic activity to release proteins from their membrane - bound n - terminal leader sequences after translocation across the cytoplasmic membrane . here 
 , we identify the staphylococcus  aureus operon ayrrabc ( sa0337 to sa0340 ) and show that once released from repression by ayrr , the protein products ayrabc together confer resistance to the spase inhibitor arylomycin m131 by providing an alternate and novel method of releasing translocated proteins . 
 thus , the derepression of ayrrabc allows cells to bypass the essentiality of spase . 
 we demonstrate that ayrabc functionally complements spase by mediating the processing of the normally secreted proteins , albeit in some cases with reduced efficiency and either without cleavage or via cleavage at a site n - terminal to the canonical spase cleavage site . 
 thus , ayrrabc encodes a secretion stress - inducible alternate terminal step of the general secretory pathway.importance   addressing proteins for proper localization within or outside a cell in both eukaryotes and prokaryotes is often accomplished with intrinsic signals which mediate membrane translocation and which ultimately must be removed . 
 the canonical enzyme responsible for the removal of translocation signals is bacterial type i signal peptidase ( spase ) , which functions at the terminal step of the general secretory pathway and is thus essential in wild - type bacteria . here 
 , we identify a four - gene operon in s.  aureus that encodes an alternate terminal step of the general secretory pathway and thus makes spase nonessential . 
 the results have important implications for protein secretion in bacteria and potentially for protein trafficking in prokaryotes and eukaryotes in general . 