A Novel Sortase, SrtC2, from Streptococcus …and anchors it to the cell wall by a two-step transpeptidation reaction (28, 56, 57). This involves recognition of the LPXTG motif and

JOURNAL OF BACTERIOLOGY, Sept. 2004, p. 5865–5875 Vol. 186, No. 170021-9193/04/$08.00�0 DOI: 10.1128/JB.186.17.5865–5875.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a SurfaceProtein Containing a QVPTGV Motif to the Cell Wall

Timothy C. Barnett, Aman R. Patel, and June R. Scott*Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia

Received 26 March 2004/Accepted 27 May 2004

The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires severalsurface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme tothe cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged Cterminus, which are thought to retard the protein in the cell membrane to facilitate recognition by themembrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GASstrains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchorsa subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS,SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobicregion and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of thisprotein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein ofGAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We concludethat srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.

Streptococcus pyogenes (group A streptococcus, GAS), is animportant gram-positive human pathogen capable of causing awide variety of diseases (11). The majority of these are mild,self-limiting, suppurative infections of the throat (pharyngitis)and skin (impetigo and pyoderma). However, these infectionsmay lead to serious poststreptococcal sequelae, including rheu-matic fever, glomerulonephritis, and reactive arthritis. In addi-tion, GAS have become notorious in recent years for their abilityto cause severe, invasive diseases with high mortality rates. Thesediseases include streptococcal toxic shock syndrome, septicemia,and the “flesh-eating disease,” necrotizing fasciitis. The abilityof these organisms to cause such a diverse disease spectrum isattributed, in part, to their production of a wide array of ex-tracellular virulence factors. For example, the toxins and su-perantigens which are secreted by GAS are responsible formany of the symptoms characteristic of different GAS-associ-ated diseases. In addition, cell-associated proteins that arecovalently attached to the bacterial surface are important foradherence to, and interaction with, the human host.

Surface proteins of gram-positive bacteria are usually se-creted through the cytoplasmic membrane via the Sec systemfollowing cleavage of an N-terminal signal sequence (34).These proteins are then either released into the extracellularmilieu or attached to the bacterial cell surface. Such surfaceproteins may be noncovalently associated with other surfacemolecules such as teichoic acids and lipoteichoic acids, an-chored directly to the cytoplasmic membrane by hydrophobicmembrane-spanning domains or as lipoproteins, or covalentlyattached to the cell wall cross-bridge by sortase enzymes (10).

Proteins anchored by sortase contain a cell wall-anchoringdomain near the C terminus. This consists of an LPXTG motiffollowed by a hydrophobic stretch of amino acids and a short,

positively charged tail (29, 39, 46). During secretion via the Secsystem, the hydrophobic region and charged tail retard trans-location across the cytoplasmic membrane (33, 45). The mem-brane-associated sortase enzyme recognizes the LPXTG motifand anchors it to the cell wall by a two-step transpeptidationreaction (28, 56, 57). This involves recognition of the LPXTGmotif and cleavage between the threonine and glycine residues,followed by the covalent linkage of the carboxyl group of thre-onine to an amino group of the cell wall precursor lipid II (40,44). In this reaction, the carboxyl group of threonine is co-valently attached to the amino group of the pentaglycinebranch of lipid II. The resulting molecule is thereby incorpo-rated covalently into the mature cell wall. The universal natureof this reaction in gram-positive organisms is reflected by theability of Staphylococcus aureus to recognize the cell wall-an-choring domains of proteins from several other gram-positivebacteria, including S. pyogenes, Listeria monocytogenes, Entero-coccus faecalis, and Streptococcus sobrinus (45).

Sortase enzymes are characterized by the presence of a con-served histidine (S. aureus SrtA histidine 120) and a conservedcysteine residue (S. aureus SrtA cysteine 184) in a TLXTC sig-nature motif (23, 36). These residues have been demonstratedto be essential for the activity of S. aureus SrtA (55). All gram-positive bacteria examined to date possess a major housekeep-ing sortase (SrtA), identified by either mutagenesis screens (1,28), PCR (7, 35), or BLAST searches (5, 18, 25, 26, 36). In S.aureus (27, 28, 30), L. monocytogenes (5, 18), S. pyogenes (1),Streptococcus gordonii (7), Streptococcus mutans (26), Strepto-coccus suis (35), and Streptococcus pneumoniae (25), this sor-tase has been shown to be responsible for anchoring the ma-jority of LPXTG-containing proteins to the cell surface.

Analysis of the available genome sequences suggested thatmost gram-positive bacteria encode one or more additionalsortases (36). In many cases, these are encoded close to genesencoding potential sortase substrates, as identified by a C-terminal LPXTG motif followed by a hydrophobic region and

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Emory University School of Medicine, RollinsResearch Center, Atlanta, GA 30322. Phone: (404) 727-0402. Fax:(404) 727-8999. E-mail: [email protected].

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charged tail. The anchoring of proteins encoded by genes clus-tered with an accessory sortase gene has been verified experi-mentally in four cases (1, 4, 30, 58). We previously showed thatsome strains of GAS encode two sortases, designated SrtA andSrtB (1). These enzymes recognize different subsets of proteinscontaining an LPXTG motif. A srtA null mutant is defective inthe surface localization of several proteins containing LPXTGfollowed by an acidic amino acid. SrtB, on the other hand, isrequired for the surface display of T6 protein, which containsthe sequence LPSTG followed by a serine rather than an acidicresidue. While the srtA gene is present in all GAS strainsexamined, srtB is present in only a limited number of strains.This agrees with the results of Bessen and Kalia (3), whoshowed that the sequence of the region of the GAS genomecontaining srtB differs among different strains. This region wasnamed the FCT (fibronectin and collagen binding and T anti-gen) region for the proteins that it encodes.

Using S. aureus SrtA as a BLAST query, Pallen et al. (36)identified two other potential sortase genes (slp3 and slp4) inthe genome sequences of GAS. In this study, we searched forthese sortase homologs in each of the GAS genome sequencesand found that slp3 was encoded in the FCT region of the M1strain SF370, while slp4 was present in the FCT region ofstrains belonging to serotypes M3, M5, and M18 (Fig. 1A).Further analysis revealed that slp3 and slp4 were likely to betwo different alleles of the same gene, which we have desig-nated srtC1 and srtC2, respectively (Fig. 1B). These genesappear to lie in an operon that also encodes one potentialsubstrate for SrtA and two potential cell wall-anchoring sub-strates with a motif that differs from LPXTG. We show herethat SrtC2 from an M3 strain is required for anchoring of oneof these proteins (SPyM3_100), which has a QVPTG motiffollowed by a hydrophobic region and charged tail. We alsodemonstrate that substituting LPSTG for QVPTG in this pro-tein prevented its anchoring by either SrtC2 or SrtA.

MATERIALS AND METHODS

Bacterial strains and plasmids. GAS strain JRS4 is a spontaneous streptomy-cin-resistant derivative of the serotype M6 strain D471 (48). JRS758 is a srtAmutant derivative of JRS4 (1). GAS strain AM3 (52) was used as a source ofDNA for PCR amplification of srtC2-specific sequences. For expression in GAS,DNA fragments were cloned into plasmid pNZ276 under the Lactococcus lactislacA promoter (12, 41). Escherichia coli strain C600 was used as a host formolecular cloning experiments.

Culture conditions. GAS strains were grown in Todd-Hewitt medium supple-mented with 0.2% yeast extract (Difco), and E. coli strains were grown in Luriabroth (LB) medium (47) at 37°C. For E. coli and GAS strains harboring pNZ276derivatives, chloramphenicol was added to the medium at concentrations of 20and 3 �g/ml, respectively. When appropriate, 1% (wt/vol) glucose was added toLB medium to repress expression from the lacA promoter by catabolite repres-sion.

DNA manipulations. Chromosomal DNA was isolated from GAS strains withthe MasterPure DNA extraction kit (Epicentre). Plasmid DNA was isolated withanion exchange columns or Qiaprep spin columns (Qiagen).

Survey for srtA, srtB, srtC1, and srtC2. Internal fragments of sortase genes wereamplified with primers srtA-F6 and srtA-R7 (srtA), srtB-F3 and srtB-R3 (srtB),srtC1-F12 and srtC1-R12 (srtC1), and srtC2-F11 and srtC2-R11 (srtC2) (Table1). PCR was performed with HotStart Taq (Qiagen) according to the manufac-turer’s protocol. For Southern hybridization analysis (50), PCR products fromSF370 (srtC1) and AM3 (srtC2) were labeled with [�-32P]dATP (Decaprimelabeling kit; Ambion) and used to probe HindIII-digested DNA.

Construction of bacterial strains expressing Orf100HA and SrtC2. A se-quence encoding a hemagglutinin (HA) tag (Sigma) was introduced into theSPyM3_0100 open reading frame (hereafter orf100) by a two-step overlapping

PCR protocol with the high-fidelity enzyme Herculase (Stratagene). PrimersCpa-F4 and OrfA-HA-R (Table 1) were used to amplify the 5� 297 bp of orf100and 2,214 bp of upstream sequence containing sipA2. Similarly, primers OrfA-HA-F and OrfB-R1 (Table 1) were used to amplify the 3� 735 bp of orf100 and2,317 bp of downstream sequence containing srtC2. Primers OrfA-HA-F andOrfA-HA-R contain additional 27-bp sequences that encode a 9-amino-acid HAtag and also form overlapping regions between the two PCR products. The PCRproducts were gel purified, combined, and used as the template for second-roundreactions.

A DNA fragment containing sipA2-orf100HA was amplified with nested PCRprimers LepA-PstI-F and OrfA-R3 (Table 1), digested with PstI and SmaI, andligated into pNZ276 digested with PstI and MscI to create pJRS1316. Similarly,a fragment containing sipA2-orf100HA-srtC2 was amplified with primers LepA-PstI-F and SrtC2-EcoRV-R (Table 1), digested with PstI and EcoRV (pJRS1317),and ligated into PstI- and MscI-digested pNZ276 to produce pJRS1317.

Construction of strains expressing Orf100HA-LPSTGE. The sequence of orf100encoding the QVPTGV motif in plasmids pJRS1316 and pJRS1317 was changedto a sequence encoding the LPSTGE motif of the M6 protein (21) to createplasmids pJRS1329 and pJRS1330, respectively. Mutagenesis was performedwith the QuikChange mutagenesis system (Stratagene) according to the manu-facturer’s protocol, along with mutagenic primers OrfA-LPSTGE-F and OrfA-LPSTGE-R (Table 1).

Cell fractionation and immunoblot studies. Cell wall and culture supernatantfractions were prepared by the method of Pancholi and Fischetti (37). Immuno-precipitation of cell wall extracts was performed with anti-HA affinity matrix(Roche) according to the manufacturer’s protocol. Protein extracts were sepa-rated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels or 4 to 12% gradient gels (Invitrogen) and transferred to anitrocellulose membrane (Nitrobind; Osmonics Inc.) for immunoblot analysis.

Whole-cell immunoblots and detection of proteins with antiserum were per-formed as described previously (6). The HA-7 monoclonal antibody (Sigma) wasused at a 1:1,000 dilution. The 10B6 monoclonal antibody (24) was used at a1:2,000 dilution.

RESULTS

Identification of an operon containing a third sortase ho-molog from GAS. To determine whether the two sortase genehomologs (slp3 and slp4) identified by Pallen et al. (36) encodesortases that anchor proteins to the cell wall, we searched forboth genes in each of the available GAS genome sequences.We found that the genome of the M1 strain SF370 containsslp3, while slp4 is present in the sequences of the strains be-longing to serotypes M3, M5, M12, M18, and M49. Further-more, the slp homolog is always located in the FCT region (3),defined as the region between genes homologous to Spy0123and SPy0136 of the M1 strain SF370 (Fig. 1A). Further analysisrevealed that with the exception of the M6 strain JRS4, all sixstrains for which the FCT region sequence is available have aslp3 or slp4 homolog.

Alignment of the amino acid sequences encoded by slp3 andslp4 indicated that they are likely to be two different alleles ofthe same gene. We have named them srtC1 and srtC2, respec-tively (Fig. 1B). The M1 strain contains srtC1, while the othersequenced strains have srtC2. The srtC1 gene from SF370 en-codes a protein predicted to be 46.5% identical and 66.4%similar to the protein encoded by srtC2 from MGAS315, 12.4%identical and 24.6% similar to SrtA from SF370, 15% identicaland 26.8% similar to SrtB from the M6 strain JRS4, and 27.8%identical and 49.6% similar to SrtB from S. aureus. SrtC1 andSrtC2 both possess a conserved histidine residue (at positions149 and 151, respectively) and a conserved cysteine residue ina region that resembles the TLXTC signature sequence char-acteristic of sortase enzymes (ALSTC for SrtC1 and AFSTCfor SrtC2).

In the SF370 (M1) genome, srtC1 appears to lie in an operon

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containing three genes (cpa, Spy128, and Spy130) that encodeproteins with potential cell wall-anchoring domains and a pu-tative signal peptidase gene (sipA1) (Fig. 1A). The C terminusof Cpa from SF370 contains the sequence VVPTG followed bya hydrophobic region and a charged tail. Similarly, the C ter-

minus of Spy128 from SF370 contains the sequence EVPTG,while Spy130 contains LPSTGE preceding a hydrophobic re-gion and a charged tail. Spy130 is predicted to be a substratefor SrtA because it contains an LPXTG motif followed by anacidic residue (1). However, based on our previous results, Cpa

FIG. 1. FCT regions of different GAS strains. (A) Genomic organization of the FCT regions from different GAS strains. In M1 strains, srtC1is located in a potential operon with genes encoding a putative signal peptidase (sipA1) and three potential sortase substrate proteins, cpa, SPy128,and SPy130. A similar gene arrangement is seen in other strains. A divergently transcribed gene encoding a potential regulator of this operon (rofAand nra) is located upstream of cpa. Homologous genes are shaded alike. Potential sortase recognition motifs are given in parentheses after genenames. The sequences for M1 (13), M3 (2), M5 (genome sequence at http://www.sanger.ac.uk, GenBank accession no. NC_002958), M6 (3), M12(3), M18 (49), and M49 (3, 42) have been published. The star denotes that the srtB gene from M12 contains an internal stop codon (codon 102).(B) Multiple sequence alignment of SrtC amino acid sequences from the above strains. Alignment was performed with ClustalW software.

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and Spy128 are unlikely to be substrates for SrtA or SrtBbecause they do not contain an LPXTG motif preceding thehydrophobic region. Instead, these predicted proteins containrelated sequences that could serve as substrates for anothersortase, possibly SrtC1, which is encoded in the same operon.

Upstream of these genes and divergently transcribed fromthem is a positive regulatory gene, rofA (17). Downstream ofSpy130 are two genes transcribed in the other direction thatexhibit homology to IS66 family transposases (SPy131 andSPy133), and these are followed by srtB (1).

The arrangement of genes in the FCT region in strainscontaining srtC2 is similar to that in the M1 strain SF370 (Fig.1A). For example, in the M3 strain MGAS315, srtC2 appearsto lie in an operon encoding three potential sortase substratesthat exhibit homology to proteins encoded by M1 genes cpa(SPyM3_0098; 48.5% identity, 60.4% similarity), SPy0128(SPyM3_100; 36.4% identity, 47.7% similarity), and SPy0130(SPyM3_0102; 28.8% identity, 45% similarity) as well as asignal peptidase (sipA2; 39.4% identity, 51.6% similarity). Up-stream of cpa is a divergently transcribed gene that has homol-ogy to nra, which encodes a negative regulator of cpa in anM49 strain (42). Cpa and Spy128 homologs from strains en-coding srtC2 all contain the sequences VPPTG and QVPTG,respectively, followed by a hydrophobic region and a chargedtail, and are possible substrates for SrtC2. A homolog of Spy130from these strains contains the sequence LPLAGE, whichcould be a substrate for SrtA. However, the genes downstreamof SPyM3_0102 are different from those downstream of SPy0130in SF370. Downstream of SPyM3_0102 is SPyM3_0103, encod-ing a putative AraC-type regulator, and prtF2, encoding a fibro-nectin binding protein (20).

To determine the relative distribution of srtC1 and srtC2among different strains of GAS, we developed a PCR assaywith specific primers (srtC1-F12 and srtC1-R12; srtC2-F11 andsrtC2-R11) (see Materials and Methods). The results of thisanalysis are summarized in Table 2. Confirming and extendingthe analysis of available sequences above, the PCR resultsshowed that the srtC1 gene is present in and unique to M1

strains, while srtC2 is present in strains belonging to serotypesM2, M3, M5, M12, M18, M22, M49, and M50. These strainsinclude representatives that are opacity factor positive andnegative and include some that are very genetically distant, asdetermined by multilocus enzyme electrophoresis (31). Strainsbelonging to serotypes M4 and M6 and strains 64/14 andMGAS273 do not contain a srtC1 or srtC2 allele, as determinedby PCR analysis (Table 2).

To rule out the possibility that these strains possessed asrtC1 or srtC2 gene that was not able to be amplified with thePCR primers used above due to sequence divergence, we ex-amined each of these strains by Southern hybridization withprobes internal to srtC1 from strain SF370 and srtC2 fromstrain AM3. Under high-stringency hybridization conditions(50% formamide, 42°C), the srtC1 and srtC2 probes hybridized

TABLE 1. Primers used in this study

Primer Sequencea

srtA-F6 5�-CAAACCTATCCGAAATACATTAATTGCTCG-3�srtA-R7 5�-gcgaattcTGTTAAATAGATGGACATCC-3�srtB-F3 5�-GGTGTGGCAAAAGGCTAAGG-3�srtB-R3 5�-GCACACACTACTTCTGCCC-3�srtC1-F12 5�-CTACTAACTATCAACAATATAAGAAAAAGGG-3�srtC1-R12 5�-GCTTGATATGGTTTAAAAATTCATTTTTACTAG-3�srtC2-F11 5�-GCATTTAAAACAGCTCAACAACAGCC-3�srtC2-R11 5�-GCCTTTGTTGGTCTTGATTGG-3�OrfA-HA-F 5�-tatccatatgatgttccagattatgctAAACCAGATAGTAAAGTTAAAAGTACAGAG-3�OrfA-HA-R 5�-agcataatctggaacatcatatggataATCAGTATTAGTATAGCTGATAATCTGTTC-3�Cpa-F4 5�-CACAGCAACTACAACTAATGCG-3�OrfB-R1 5�-GGTATTTCTTCAAAAGACTATCTTGC-3�LepA-PstI-F 5�-gcatctgcagGTTTGGCTATTTGGTCGTAAAGG-3�OrfA-R3 5�-gcatcccgggATAAGAATGAGAGTATCAATGGC-3�SrtC2-EcoRV-R 5�-gcatgatatcATTTTCCAATATTTTCTCAAAAGTCTCC-3�OrfA-LPSTGE-F 5�-CGTTGTCACAAATAAGCGTGACACTttAccatCAACTGGTGaaGTAGGCACCCTTGCTCCATTTGCAG-3OrfA-LPSTGE-R 5�-CTGCAAATGGAGCAAGGGTGCCTACttCACCAGTTGatggTaaAGTGTCACGCTTATTTGTGACAACG-3

a Introduced restriction sites are in bold. Uppercase letters represent bases complementary to GAS sequences. Lowercase letters represent bases added or changedto facilitate cloning or mutagenesis. Underlined regions are bases incorporated to produce an HA tag (OrfA-HA-F and OrfA-HA-R) or the region corresponding tothe LPSTGE sequence of M6 protein (OrfA-LPSTGE-F and OrfA-LPSTGE-R).

TABLE 2. Distribution of sortase genes in different GAS strains

Strain M typeaGene(s) present Reference

or sourcesrtA srtB srtC1 srtC2

SF370 1 � � � � 13MGAS5005 1 � � � � 19MGAS166 1 � � � � 31T2/44 2 � � � � V. FischettiAM3 3 � � � � 52MGAS321 4 � � � � 31T5B/126/4 5 � � � � V. FischettiJRS4 6 � � � � 48MGAS303 6 � � � � 31CS24 12 � � � � 51MGAS300 18 � � � � 31MGAS317 22 � � � � 31MGAS162 22 � � � � 31AL168 22 � � � � 53NZ131 49 � � � � 9B514 50 � � � � 2264/14 NT � � � � 43MGAS273 U � � � � 31

a NT, nontypeable. U, unknown.



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to DNA from SF370 and AM3, respectively, but not to DNAfrom any of the strains that were negative for these genes byPCR (data not shown). We did not observe cross-hybridizationbetween srtC1 and srtC2. However, under stringency condi-tions low enough to detect cross-hybridization between srtC1and srtC2 (20% formamide, 37°C), we did observe hybridiza-tion of the srtC1 probe to a �3.0-kb HindIII fragment fromstrains 64/14 and MGAS273 (data not shown). This is unlikelyto be caused by hybridization to srtA- or srtB-specific sequencesbecause these genes are encoded on 1.1-kb and 5.0-kb HindIIIfragments, respectively, in strain 64/14 (1). Furthermore, nohybridization of the srtC1 probe to DNA from M6 strains JRS4and MGAS303, both of which possess srtA and srtB (1), wasdetected. Therefore, strains 64/14 and MGAS273 may encodean additional uncharacterized sortase related to srtC1 and srtC2.

Anchoring of Orf100 to the cell wall of GAS requires SrtC2.We reasoned that the protein encoded by SPyM3_0100 (here-after called orf100) could be a substrate for SrtC2 because itcontains the sequence QVPTG in place of an LPXTG motifnear its C terminus. To detect this protein (Orf100), we intro-duced a sequence encoding an HA tag into an orf100 gene ona plasmid, creating orf100HA. Because the N terminus of Orf100is probably required for secretion through the cell membraneand the C terminus is expected to be needed for cell wallanchoring, we added the tag at an internal region of the pro-tein that we identified as likely to be accessible to detectionwith an anti-HA monoclonal antibody (HA-7). We placed theHA tag in a region containing a stretch of hydrophilic aminoacids that is predicted to have a high probability of surfacelocalization, between amino acids 99 and 100 of the proteinencoded by orf100.

To assess the role of SrtC2 in the anchoring of Orf100HA tothe cell surface, we constructed two plasmids that differ only inthe presence of srtC2 (Fig. 2A). Plasmid pJRS1316 containsthe signal peptidase gene sipA2 and orf100HA, while pJRS1317contains sipA2, orf100HA, and srtC2, cloned into pNZ276 underthe lacA promoter from Lactococcus lactis (12, 41). These plas-mids were transformed into strain JRS4, a serotype M6 strainthat lacks the srtC2 locus.

The presence of Orf100HA on the surface of cells of strainJRS4 containing either pJRS1316 or pJRS1317 was first inves-tigated with a whole-cell immunoblot assay, which we haveused previously to determine cell wall anchoring of proteins bySrtA and SrtB (1). The monoclonal anti-HA antibody reactedwith whole cells of JRS4/pJRS1317, which encodes srtC2,whereas no HA was detected in whole cells of strain JRS4/pJRS1316, which lacks srtC2, or strain JRS4/pNZ276 (Fig. 2B).

To confirm this result and to determine whether Orf100HA iscovalently attached to the cell wall, cells were fractionated intocell wall and culture supernatant fractions, boiled in SDS-PAGE sample buffer, and examined by Western immunoblot(Fig. 2C). The cell wall fractions were obtained by digestion ofthe cells with phage lysin, an amidase that cleaves the amidebond between N-acetylmuramic acid and L-alanine in the pep-tidoglycan cross bridge (14) (Fig. 2C), or by digestion of thecells with the muramidases mutanolysin and lysozyme (datanot shown). The supernatant was then immunoprecipitatedwith an anti-HA affinity matrix (Roche). Cell wall extractionwith phage lysin or mutanolysin/lysozyme releases peptidogly-can fragments of various sizes (presumably due to partial di-gestion) attached to covalently anchored cell wall proteins(15). Thus, following SDS-PAGE analysis, a cell wall-anchored

FIG. 2. Anchoring of Orf100HA to the cell surface requires SrtC2. (A) Regions of the srtC2 operon cloned into pNZ276 under the Placpromoter. Plasmid pJRS1316 contains sipA2 and orf100HA, while pJRS1317 contains sipA2, orf100HA, and srtC2. (B) Whole-cell immunoblotanalysis for surface localization of Orf100HA in JRS4 strains harboring pNZ276, pJRS1316, and pJRS1317. Strains were grown overnight andresuspended in saline at an optical density at 600 nm of 2.0. Serial twofold dilutions were spotted onto a nitrocellulose membrane and reacted withmonoclonal antibody HA-7. (C) Western immunoblot of cell wall and culture supernatant extracts of JRS4 containing pJRS1316 or pJRS1317reacted with HA-7 monoclonal antibody. Cell wall extracts were prepared with phage lysin (14) and immunoprecipitated prior to SDS-PAGE.Proteins were separated by SDS-PAGE on 4 to 12% gradient gels, transferred to nitrocellulose, and detected with HA-7 monoclonal antibody. Thesizes of molecular mass standards (in kilodaltons) are indicated to the left. The heavy (H) and light (L) chains of the antibody used forimmunoprecipitation are also indicated.



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protein appears as a ladder of fragments migrating more slowlythan expected for the unattached mature protein.

Immunoprecipitated phage lysin extracts (Fig. 2C) andmutanolysin/lysozyme extracts (data not shown) from JRS4/pJRS1317 (containing srtC2) showed the expected ladder ofproteins that react with anti-HA, while extracts of JRS4/pJRS1316 (without srtC2) did not. This suggests that Orf100HA

is covalently anchored to the GAS cell wall only when SrtC2 ispresent. A similar ladder of proteins was observed followingextraction of cell wall material from the M3 strain AM3 con-taining pJRS1317 (data not shown). The mobility of this ladderof HA-containing bands corresponds to molecular massesranging from about 80 to �220 kDa. In addition, near the topof the gel, a smear of very high molecular weight material thatreacted with the HA antiserum is visible in the cell wall frac-tion of JRS4/pJRS1317. The nature of this material was notinvestigated further, although it was also present in the super-natant of this strain in either the exponential or stationaryphase of growth (Fig. 2C and data not shown).

Phage lysin extracts of both JRS4/pJRS1316 and JRS4/pJRS1317 contained a major anti-HA reactive band at thelocation expected for a protein of about 37 kDa. We believethis to represent an intermediate step in the cell wall attach-ment process probably detected because orf100HA is overpro-duced in our strains. Because it is overproduced, some mightappear in the cell wall fraction. This phenotype is similar tothat seen with S. aureus protein A from mutants that lack anLPXTG motif (46) and for protein A in an S. aureus srtAmutant (27). In both of these cases, protein A is not recognizedby SrtA and is missorted into the cytoplasm, membrane, andcell wall fractions.

Altering the QVPTGV motif near the C terminus pre-vents anchoring of Orf100 to the cell wall by SrtC2. The cellwall-anchoring motif recognized by sortase usually consists ofLPXTG followed by a hydrophobic stretch of amino acids anda positively charged tail. Orf100 lacks the LPXTG sequenceand instead contains QVPTGV followed by a hydrophobicregion and a charged tail. To test whether the QVPTGV se-quence is necessary for cell wall anchoring by SrtC2, this se-quence was replaced with the LPSTGE sequence of proteinM6, a GAS protein whose cell wall anchoring is SrtA de-pendent (1). For this purpose, the sequence encoding theQVPTGV sequence encoded in plasmids pJRS1316 andpJRS1317 was replaced with a sequence encoding LPSTGE,creating plasmids pJRS1329 (no srtC2) and pJRS1330 (withsrtC2), respectively. The sequences of the orf100HA mutant genesencoding the LPSTGE motif in pJRS1329 and pJRS1330 wereverified by DNA sequencing (data not shown). These plasmidswere transformed into JRS4 and its srtA derivative JRS758 (1).

The whole-cell immunoblot assay was used to examine sur-face display of Orf100HA and its mutant derivative containingan LPSTGE motif, as described above (Fig. 3A). As seen above,the Orf100HA protein with the native QVPTGV sequence was

FIG. 3. Role of QVPTG motif in SrtC2-dependent anchoring ofOrf100HA to the cell surface. Strains JRS4 and JRS758 were trans-formed with pJRS1329, pJRS1330, pJRS1317, and pJRS1316 and as-sayed for the presence of Orf100HA as described for Fig. 2. (A) Whole-cell immunoblot analysis. (B) Western immunoblot of cell wallextracts. (C) Western immunoblot of culture supernatant fractions.

Lanes: 1, JRS4/pJRS1329; 2, JRS758/pJRS1329; 3, JRS4/pJRS1330; 4,JRS758/pJRS1330; 5, JRS4/pJRS1317; 6, JRS758/pJRS1317; 7, JRS4/pJRS1316; 8, JRS758/pJRS1316. The sizes of molecular mass stan-dards (in kilodaltons) are indicated to the left.



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attached to the cell wall only in the presence of srtC2 (Fig. 3A,rows 5 and 6 versus 7 ad 8). However, the Orf100HA with theLPSTGE motif in place of QVPTGV was not detected in sig-nificant amounts on the GAS surface, even when the srtC2gene was present (Fig. 3A, rows 3 and 4). This indicates thatchanging the QVPTG motif prevents srtC2-dependent anchor-ing of Orf100HA.

The effect on cell wall anchoring by SrtA as well as the effectof SrtC2 was examined with the srtA mutant strain JRS758.The LPSTGE version of Orf100HA was not found in significantamounts on the GAS surface in either the SrtA-producingstrain JRS4 or the srtA mutant strain JRS758 (Fig. 3A, rows 1and 3 and rows 2 and 4). Thus, the LPSTGE motif is not suf-ficient for SrtA anchoring, even of a protein in which it pre-cedes a hydrophobic region and charged tail that is recognizedby SrtC2. It was also noticeable that more Orf100HA appearedto be anchored by SrtC2 in an srtA mutant than in an srtA�

background (compare rows 5 and 6) (see below and Discus-sion).

To determine where Orf100HA and its mutant form werelocated in each strain tested above, cell wall (Fig. 3B) and cul-ture supernatant (Fig. 3C) fractions were prepared, boiled inSDS-PAGE sample buffer, and examined for the presence ofOrf100HA by Western immunoblot. Orf100HA containing itsnative QVPTGV motif appeared as a ladder of bands, char-acteristic of cell wall-attached proteins in the presence of srtC2(Fig. 3B, lanes 5 and 6) and not in the absence of srtC2 (Fig.3B, lanes 7 and 8). As seen previously, the Orf100HA ladderranged in apparent size from �80 to �220 kDa (Fig. 3B, lanes5 and 6).

When the QVPTGV sequence was replaced with LPSTGE,we detected no Orf100HA attached to cell wall fragments in thepresence of either srtC2 (Fig. 3B, compare lanes 3 and 5 or 4and 6) or srtA (Fig. 3B, lanes 1 and 3). In the cell wall extracts,the large amount of Orf100HA that migrated as a band of about37 kDa (seen previously; Fig. 2B) was visible in all cases exceptin the extracts from strains containing srtA. Whether or notsrtC2 was present, in the presence of srtA this HA-containingband was found instead in the culture supernatant (Fig. 3B andC, lanes 1 and 3).

To test whether secretion of this protein was due to recog-nition and cleavage of the LPSTGE motif by SrtA, we exam-ined each of the cell wall and culture supernatant fractionsfrom strains JRS4/pJRS1330 and JRS758/pJRS1330 by SDS-

PAGE and Western immunoblot (Fig. 4). The cell wall-asso-ciated protein in JRS758/pJRS1330 had an apparent molecularmass of approximately 37 kDa, while the secreted protein inJRS4/pJRS1330 had a slightly lower molecular mass (�35kDa). This is consistent with the interpretation that the speciesreleased into the supernatant by SrtA was cleaved at theLPSTGE motif, removing the �3-kDa hydrophobic region andcharged tail.

As seen previously (Fig. 2C) in the Western immunoblotanalysis of Orf100HA (Fig. 3B and 3C), some of this proteinwas present as a very large species. This material was apparentin both cell wall and culture supernatant fractions but onlyfrom strains containing the native Orf100HA (QVPTGV motif)and only in the presence of srtC2 (Fig. 3B and 3C, lanes 5 and6). This suggests that SrtC2 has attached the Orf100HA proteinto a large cell wall component which is partitioned into boththe cell wall and culture supernatant fractions.

As seen in the whole-cell immunoblots, more Orf100HA wasdetected in a srtA mutant than a srtA� background (Fig. 3B,compare lanes 5 and 6). We reasoned that this might be due toan increased amount of cell wall precursor available to SrtC2 inthe absence of SrtA-anchored cell wall proteins. If SrtA andSrtC2 compete for available cell wall precursors, overexpres-sion of orf100HA and srtC2 in a srtA� strain might result indecreased amounts of a SrtA-anchored protein on the cellsurface. To test this, JRS4 strains containing plasmids pNZ276,pJRS1316, and pJRS1317 were examined for the presence ofM protein on the cell surface by whole-cell immunoblot (Fig.5). As expected, the srtA mutant strain JRS758 exhibited nosurface-associated M protein. A similar amount of M proteinwas detected on the surface of the srtC2 mutant strain JRS4/pJRS1316 as in JRS4/pNZ276 (vector control). In contrast, thesrtC2� strain JRS4/pJRS1317 showed much less M proteinon the cell surface. Thus, overexpression and anchoring ofOrf100HA to the cell wall inhibit the efficient cell wall anchor-ing of M6 protein.

DISCUSSION

Some gram-positive bacteria have multiple sortases withnonredundant specificities. Analysis in silico identified multi-ple sortase gene homologs in the genomes of most gram-positive bacteria (36). The housekeeping sortase (SrtA) seemsto anchor most cell wall proteins, while a limited number of

FIG. 4. Orf100HA containing an LPSTGE motif is processed bySrtA. Cell wall and culture supernatant fractions from strains JRS4/pJRS1330 and JRS758/pJRS1330 were separated by SDS–12% PAGEand assayed for the presence of Orf100HA by Western immunoblot.The sizes of molecular mass standards (in kilodaltons) are indicated tothe left.

FIG. 5. Effect of SrtC2-dependent anchoring of Orf100HA on thesurface display of M6 protein. Whole-cell immunoblots of JRS4 strainscontaining plasmids pNZ276, pJRS1316 (srtC2 mutant), and pJRS1317(srtC2�) and strain JRS758 (srtA mutant) were prepared as describedfor Fig. 2 and reacted with monoclonal antibody 10B6.



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proteins that are not anchored by SrtA are anchored by acces-sory sortases. Usually, these accessory sortases are encoded inan operon along with the proteins they are thought to anchor.This has been shown for S. aureus SrtB (30), S. pyogenes SrtB(1), Corynebacterium diphtheriae SrtA (58), and L. monocyto-genes SrtB (4). In this work, we have characterized a thirdsortase from GAS that was identified with the S. aureus SrtAsequence as a BLAST search query (36). This sortase (SrtC2)is encoded in an operon along with three potential cell wall-anchored proteins, Cpa, Orf100, and SPyM3_0102. We haveshown here that the presence of srtC2 is required for cell wallanchoring of one of these proteins, Orf100.

The different sortases encoded in the genomes of gram-positive bacteria exhibit different specificities for the proteinsthey anchor. While all cell wall-anchored proteins contain ahydrophobic region followed by a charged C terminus, themotifs preceding this region appear to be sortase specific. Con-sequently, when more than one sortase has been characterizedfrom a single organism, there is usually no overlap in the pro-teins anchored. For example, the S. aureus housekeeping sor-tase (SrtA) recognizes and anchors proteins with an LPXTGmotif (27, 28, 30), while the accessory sortase (SrtB) anchors asingle protein with an NPQTN motif (30). Similarly, someGAS strains possess two sortases, SrtA and SrtB, which anchordifferent subsets of proteins with an LPXTG motif (1). SrtAanchors the majority of these proteins, which contain anLPXTG motif followed by an acidic residue, while SrtB an-chors a protein containing the sequence LPSTG followed by aserine and does not appear to anchor SrtA-dependent surfaceproteins.

The SrtC2-anchored protein (Orf100) investigated in thisstudy possesses a unique cell wall-anchoring domain, whichconsists of a QVPTG motif followed by a hydrophobic regionand charged tail. We have demonstrated that SrtC2 specificallyrecognizes this motif, since substituting the SrtA-dependentLPXTG motif for the QVPTG motif abolishes SrtC2 anchor-ing of Orf100. Thus, the multiple sortases encoded in the ge-nomes of S. aureus and GAS exhibit specificity for the proteinsthey anchor. Therefore, the multiple sortase homologs en-coded in the genomes of gram-positive bacteria that have beencharacterized to date appear to have unique functions.

The two steps of the cell wall-anchoring reaction requiredifferent signals. The cell wall-anchoring process involves atwo-step transpeptidation reaction (28, 56, 57). First, the sor-tase recognizes the LPXTG motif of the protein to be an-chored and cleaves it between the threonine and glycine resi-dues, forming an acyl-enzyme intermediate. In the second step,the protein intermediate is transferred to the cell wall precur-sor lipid II (40, 44) and subsequently incorporated into themature cell wall. From our work, it appears that the ability ofSrtA to transfer the intermediate to the cell wall precursorrequires signals present in the protein being anchored in ad-dition to the LPXTG motif. In this work, we found that thehydrophobic and charged regions of the Orf100 cell wall-an-choring domain together with the preceding QVPTG sequencewere sufficient to allow it to be anchored by SrtC2 (Fig. 6, partI). In contrast, a mutant version of Orf100 in which theQVPTGV sequence was replaced with the SrtA recognitionsequence LPSTGE was not anchored to the cell wall. Instead,this protein was secreted into the culture supernatant by a

mechanism that requires SrtA (Fig. 6, part II). It appears thatOrf100HA-LPSTGE is recognized and cleaved by SrtA, since thesecreted protein had a slightly smaller apparent molecularmass than the nonsecreted form found in the srtA mutantstrain. However, the cleaved Orf100HA-LPSTGE protein is notattached to the cell wall by SrtA. Thus, the second step in theSrtA-catalyzed transpeptidation reaction (transfer to the cellwall intermediate) may require a signal present N-terminal tothe LPXTG motif, and this signal is not needed for the firststep (recognition and cleavage of the LPXTG motif).

In S. aureus, it has also been observed that, for some pro-teins, cell wall anchoring by SrtA may require a signal N-terminal to the LPXTG motif (45), although this study did notdetermine whether this signal was required for one or bothsteps of the transpeptidation reaction. Proteins constructedwith fusions of the cell wall-anchoring domains of E. faecalisPrgB, S. pyogenes T6, and L. monocytogenes InlA to protein Awere efficiently anchored to the S. aureus cell wall, while pro-teins with the same cell wall-anchoring domains fused to en-terotoxin B were anchored with much lower efficiency (PrgBand T6) or not anchored at all (InlA). Therefore, a signalpresent N-terminal of the LPXTG motif in protein A butabsent from enterotoxin B is required for these proteins to beanchored to the staphylococcal cell wall by SrtA.

SrtA and SrtC2 probably anchor proteins to the same cellwall substrate. Evidence from this study suggests that anchor-ing of proteins to the GAS cell wall is inefficient and might belimited by the availability of the cell wall substrate. Althoughboth SrtC2 and Orf100 were overproduced in our strain, mostof the Orf100 was not anchored to the cell wall but instead waspresent as a band similar in size to that observed in the absenceof SrtC2 (�37 kDa) (Fig. 2C and 3B). This implies that theanchoring process is inefficient. Similarly, a significant amountof M protein produced by GAS is not anchored to the cell wallby SrtA but is found instead in the culture medium (1). Thus,it appears that anchoring by both SrtA and SrtC is inefficient inGAS.

More SrtC2-anchored Orf100 was detectable on the GAScell surface of a srtA mutant strain than a srtA� strain (Fig.3A). This did not appear to result from an increase in ourability to detect Orf100 in the absence of surface proteinsanchored by SrtA because we observed an increased associa-tion of Orf100 with cell wall fragments following extraction ofcell wall material (Fig. 3B). Thus, we suggest that SrtA maycompete with SrtC for cell wall precursors to which proteinscan be covalently attached. The converse experiment alsoagrees with this idea. There was less detectable SrtA-anchoredM protein on the GAS surface in the strain overexpressingSrtC2 and Orf100 than in its parental strain (Fig. 5). Becauseit appears that SrtC2 competes with SrtA to anchor surfaceproteins, it seems likely that they anchor proteins to the samecell wall substrate.

Possible functions of genes encoded in the SrtC1 and SrtC2operons. Although not all strains of GAS encode a SrtC, thosestrains most commonly isolated both from invasive clinicalspecimens and from cases of pharyngitis encode either SrtC1(M1 strains) or SrtC2 (M3 and M12). Thus, it is plausible thatSrtC plays an important role in GAS pathogenesis for thestrains that are currently predominant.

For the GAS strains whose sequences are available, srtC is



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FIG. 6. Fates of the different Orf100 proteins investigated in this study. (A) Orf100 is synthesized in the cytoplasm and transported to thecytoplasmic membrane, where the signal peptide (Sig) is removed by a mechanism that may require SipA2. (B) Orf100 is tethered to the membraneby its C-terminal hydrophobic region and charged tail. (C) SrtC2 recognizes and cleaves the QVPTG motif. (D) SrtC2 links the cleaved proteinto the cell wall precursor. (E) Same as steps A and B. (F) SrtA recognizes and cleaves the LPSTGE motif, but other inherent properties ofOrf100HA-LPSTGE prevent it from being linked to the cell wall precursor. (G) Instead, this protein is released into the culture supernatant. Thisfigure was adapted from that of Navarre and Schneewind (34) and modified to include the results from this study.



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closely linked to, and probably cotranscribed with, severalother genes (Fig. 1A). The first gene is cpa, and, because thisencodes a protein that has a hydrophobic C-terminal regionfollowed by a charged tail, it is likely to be cell wall anchored.However, in place of an LPXTG motif, it contains the se-quence V(P/V)PTG and therefore may be anchored by SrtCinstead of SrtA or SrtB. Following cpa is a gene (sipA) withhomology to the signal peptidase (lepA) of GAS. Many gram-positive bacteria contain several sip homologs (8, 38, 54).Sometimes these have apparently redundant functions, whilein other cases their functions appear to be unique. It seemspossible that sipA of GAS may be involved in secretion throughthe cytoplasmic membrane of the cell wall-anchored proteinsencoded in this operon. Following sipA lies orf100, the productof which we have now demonstrated to be anchored to the cellwall by SrtC2. Immediately downstream of orf100 is srtC, fol-lowed by a gene (SPyM3_0102) that encodes a protein with anLPX(T/A)G motif followed by an acidic residue that is likely tobe anchored by SrtA.

The proteins encoded by orf100 and SPyM3_0102 do notexhibit homology to any characterized proteins in the Gen-Bank database. However, in an M49 strain, Cpa, encoded bythe first gene in this locus, was shown to bind collagen (42).Furthermore, another adhesin, protein F (20), which is pro-duced only in some GAS strains (32), is also encoded nearby.In M12 strains, protein F1 may be encoded in the same operonas srtC2. Thus, the FCT region seems to encode surface pro-teins, some of which have been shown to adhere to extracel-lular matrix proteins. This locus may also encode proteinsneeded for secretion and cell wall attachment to these GASsurface proteins. Consequently, it seems possible that this lo-cus may have an important role in the early steps of GASinfection involving binding to and colonization of the host. Thehomologous proteins encoded in the SrtC1 and SrtC2 operonsfound in different GAS strains are likely to have related func-tions. However, their differences may allow the different GASstrains to colonize different host microenvironments, such asdifferent types of host cells (skin or respiratory epithelium, forexample), or to specifically colonize individual hosts with dif-ferences in their extracellular matrix proteins or epithelial sur-face proteins.

Regulation of the SrtC operons. Although SrtA, which an-chors most cell wall proteins in gram-positive bacteria, is usu-ally expressed constitutively, secondary sortases are oftenfound in operons that are expressed only under specific envi-ronmental conditions. For example, expression of SrtB in S.aureus is dependent on the iron concentration (30). In GAS,the arrangement of srtC in an operon along with the proteinsit anchors to the cell wall should allow coordinated control ofthe production and surface display of these proteins underappropriate conditions.

The SrtC1 and SrtC2 operons (Fig. 1) appear to be differ-entially regulated by RofA and Nra, respectively. RofA is apositive regulator of protein F under anaerobic conditions inthe M6 strain JRS4 (16, 17), and under aerobic conditions,expression of protein F is induced by a RofA-independentmechanism (17). In an M49 strain, Nra has been reported to bea negative regulator of Cpa expression (42). Therefore, al-though the signals required for expression of the SrtC1 andSrtC2 operons may differ, both operons appear to be regu-

lated. Consequently, expression of these genes is likely to occuronly under appropriate environmental conditions. For exam-ple, this operon may be expressed in the high oxygen tensionpresent at the host surface but not in deep tissues. This wouldbe advantageous if the proteins encoded by the operon areneeded for colonization but not for spread of the GAS. Iden-tification of the signals required for induction of the srtC1 andsrtC2 operons and the function of the encoded cell wall-an-chored proteins should give clues to the role of these genes inthe biology of GAS.

ACKNOWLEDGMENTS

This work was supported by grant AI055605 from the NationalInstitutes of Health.

We thank Vince Fischetti for supplying strains, phage lysin, andmonoclonal antibody 10B6.

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A Novel Sortase, SrtC2, from Streptococcus …and anchors it to the cell wall by a two-step transpeptidation reaction (28, 56, 57). This involves recognition of the LPXTG motif and