Article

• The EMBO Journal (2008) 27, 2656 - 2668
• doi:10.1038/emboj.2008.185

Published online: 18 September 2008

• Subject Categories:

  • Membranes and Transport | Microbiology and Pathogens

Signal peptides direct surface proteins to two distinct envelope locations of Staphylococcus aureus

Andrea DeDent¹, Taeok Bae², Dominique M Missiakas¹ and Olaf Schneewind¹

1. Department of Microbiology, University of Chicago, Chicago, IL, USA
2. Department of Microbiology and Immunology, Indiana University School of Medicine Northwest, Gary, IN, USA

Correspondence to:

Olaf Schneewind, Department of Microbiology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Tel.: +1 773 834 9060; Fax: +1 773 834 8150; E-mail: oschnee@bsd.uchicago.edu

Received 3 March 2008; Accepted 25 August 2008

• Keywords:

  • crosswall,
  • signal peptides,
  • Staphylococcus aureus,
  • surface protein,
  • YSIRK/GS motif

Introduction

The murein sacculus of Gram-positive bacteria is a single large macromolecule, assembled from peptidoglycan precursor molecules and disassembled by lytic enzymes that exert an effect at division sites (Higashi et al, 1967; Strominger, 1968; Yamada et al, 1996; Baba and Schneewind, 1998). The murein sacculus serves as scaffold for covalent linkage of many different biological polymers, including carbohydrates, teichoic acids and proteins (Armstrong et al, 1959; Sanderson et al, 1962; Munoz et al, 1967; Schneewind et al, 1995). Cell wall-anchored products are displayed on the bacterial surface endowing each microbe with unique attributes that, among many different physiological functions, enable host infection (Navarre and Schneewind, 1999). An important feature of the murein sacculus is its exoskeletal nature and rigid enclosure of cells that would otherwise burst due to osmotic pressure (Salton, 1952). Many molecules move around or through the murein sacculus; however, once linked to peptidoglycan, compounds remain immobilized until liberated by lytic enzymes (Marraffini et al, 2006).

Sortases catalyse the covalent anchoring of proteins to the cell wall envelope and recognize surface protein substrates through C-terminal LPXTG motif sorting signals (Schneewind et al, 1992, 1993; Mazmanian et al, 1999). On cleavage of the polypeptide precursor between the threonine and glycine residues of the LPXTG motif, sortase forms an acyl enzyme intermediate with the C-terminal threonine (Navarre and Schneewind, 1994; Ton-That and Schneewind, 1999; Ton-That et al, 2000). Nucleophilic attack of the amino group of cell wall cross-bridges, pentaglycine in Staphylococcus aureus (Matsuhashi et al, 1965), generates the amide bond that links the C-terminal end of surface proteins to the bacterial cell wall envelope (Navarre et al, 1998; Ton-That et al, 1998; Perry et al, 2002). Recently, we asked whether anchored proteins are distributed uniformly over the surface of S. aureus (DeDent et al, 2007). Using immunofluorescence and confocal laser scanning microscopy, a ring-like distribution of protein A, the well-known immunoglobulin (Ig)-binding factor (Jensen, 1958), was detected on the staphylococcal surface (DeDent et al, 2007). In staphylococci where anchored proteins had been removed with protease, deposition of newly synthesized protein A occurred at 2–4 sites, which expanded until a ring-like distribution was achieved (DeDent et al, 2007).

Impetus for our current work is the report of Carlsson et al (2006) that surface proteins of Streptococcus pyogenes are directed to two different locations in the bacterial envelope by mechanisms that involve specific signal peptides. Classic experiments by Cole and Hahn revealed the division septum as the site of envelope deposition for M protein (Cole and Hahn, 1962; Hahn and Cole, 1963), Lancefield's antiphagocytic factor and molecular basis for typing of group A streptococci (Lancefield, 1928). In contrast, fibronectin-binding protein (protein F) (Hanski and Caparon, 1992) is deposited at the streptococcal cell poles (Carlsson et al, 2006). These localization patterns can be changed when signal peptides for M protein, which carries the YSIRK/GS motif, and protein F, which lacks this motif, are exchanged (Carlsson et al, 2006). YSIRK/GS motif signal peptides were first described for staphylococcal lipases, secreted glycerol ester hydrolases (Rosenstein and Götz, 2000). The sequence motif is otherwise found in signal peptides of cell wall-anchored surface proteins (Tettelin et al, 2001). Remarkably, many surface proteins of streptococci and staphylococci, but not those of bacilli, clostridia or actinomycetales, carry YSIRK/GS motif signal peptides, indicating that acquisition of distinct types of signal peptides may be restricted to these genera. Sequenced genomes of S. aureus reveal 21 surface proteins with sorting signals, 13 of which harbour signal peptides with the YSIRK/GS motif. Earlier studies revealed that the presence or absence of YSIRK/GS motif signal peptides does not affect sortase-mediated anchoring of surface proteins but impacts the rate of signal peptide processing (Bae and Schneewind, 2003). However, this work left unresolved whether staphylococci can distinguish between signal peptides and direct surface proteins to discrete locations in the bacterial envelope.

Signal peptides of staphylococcal surface proteins

Amino-acid sequences of signal peptides from staphylococcal surface proteins were aligned according to their predicted or known signal peptidase cleavage sites (Figure 1). The YSIRK/GS motif is positioned 18–20 residues upstream of the signal peptide cleavage site in 13 of these molecules. It is noted that the number of residues between the N-terminal formyl-methionine and the YSIRK/GS motif varies between each surface protein, whereas the spatial distance between the sequence motif and the signal peptidase cleavage site appears conserved and may be of biological significance. All motif sequences examined span 12 residues, including YSIRK, followed by a 3-residue spacer with 1 hydrophobic residue at the third position, glycine (G), a 2-residue spacer with polar and hydrophobic residues, and serine (S). Most of the residues between the second serine of the YSIRK/GS motif and the signal peptidase cleavage site are hydrophobic in nature, in agreement with the signal peptide properties of these domains (Emr et al, 1978; von Heijne, 1992). We observed some sequence variation in the tyrosine (Y, replaced with either phenylalanine or histidine), serine (glycine or alanine) and lysine (arginine) residues of the YSIRK peptide, whereas the IR and GS residues are absolutely conserved. A second group of signal peptides from seven different surface proteins lacked the YSIRK/GS motif (Figure 1).

Figure 1.

Signal peptides of Staphylococcus aureus surface proteins. The N-terminal signal peptides of S. aureus surface proteins containing a C-terminal cell wall sorting signal were aligned at their predicted signal peptidase cleavage sites (A/X). The signal peptides of 13 surface proteins harbour the YSIRK/GS motif. Identical residues are highlighted in yellow and summarized as ^*, conserved residues marked in grey or summarized with a colon (:). The YSIRK/GS motif was absent in the remaining seven proteins; following alignment, no characteristic sequence feature could be identified in these signal peptides. The signal peptide of SasA was abbreviated; the sequence RQKAFHDSLANEKTRVRLYKSGKNWVKSGIKEIEMFKIMGLP was omitted at the position indicated by two backslashes ().

View full figure (286 KB)

Distribution of ClfA and SasF on the staphylococcal surface

Antibodies were raised against purified recombinant S. aureus surface protein A (SasA), D (SasD), F (SasF), K (SasK), clumping factor A (ClfA), serine-aspartate repeat protein C (SdrC) and D (SdrD), as well as fibronectin-binding protein B (FnbpB). Antibody binding to proteins on the staphylococcal surface was detected with Alexafluor 647-conjugated secondary antibodies and fluorescence microscopy (red staining; Figure 2). Our experiments used the spa mutant strains SEJ1 (derived from RN4220) or SEJ2 (derived from strain Newman) to avoid antibody binding to protein A (Stranger-Jones et al, 2006). Unlike streptococci, where cell division planes lie parallel (Swanson and McCarty, 1969), S. aureus divides perpendicular to previous division planes (Tzagoloff and Novick, 1977). Because of incomplete cell wall separation, differential interference contrast microscopy revealed single cells, diplococci or grape-like clusters of staphylococci (Figure 2) (Giesbrecht et al, 1998). BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-vancomycin, a glycopeptide that binds with high affinity to D-Ala-D-Ala pentapeptide precursors of peptidoglycan synthesis (Walsh, 1993), was used to reveal the bacterial murein sacculus and cross wall, a layer of newly synthesized peptidoglycan that separates dividing staphylococcal daughter cells (green staining; Figure 2) (Schuhardt et al, 1969; Giesbrecht et al, 1998).

Figure 2.

Distribution of ClfA and SasF in the cell wall envelope of staphylococci. (A) S. aureus strains SEJ1 (spa), ACD1 (spa, clfAermB) and SEJ2 (spa) (Supplementary Table 1) were viewed with differential interference contrast (DIC) or fluorescence microscopy using BODIPY-vancomycin (green) or ClfA-specific antibodies followed by secondary goat anti-rabbit conjugated to Alexafluor 647 (red). ClfA is secreted through a signal peptide that harbours the YSIRK/GS motif (+YSIRK). (B) S. aureus strains SEJ1 (spa), ACD2 (spa, sasFermB) and SEJ2 (spa) were viewed with DIC or fluorescence microscopy using BODIPY-vancomycin (green) or SasF-specific antibodies followed by secondary goat anti-rabbit conjugated to Alexafluor 647 (red). SasF is secreted through a signal peptide that does not harbour the YSIRK/GS motif (-YSIRK). Two-dimensional two-colour and DIC images were acquired sequentially with separate laser lines and merged using the ImageJ software.

View full figure (180 KB)

Antibodies against ClfA detected a ring-like or hemispherical distribution of this protein on the surface of staphylococci (Figure 2A). Similar to staining reported for protein A (DeDent et al, 2007), distribution of ClfA for both SEJ1 and SEJ2 appeared irregular, and its ring-shaped contour in dividing cells was typically positioned perpendicular to vancomycin-stained cross walls (Figure 2A). As a control, bursa aurealis insertion into clfA (clfAermB) abrogated ClfA antibody binding, thereby revealing the specificity of this reagent. In contrast to ClfA, surface distribution of SasF assumed a much more discrete, punctate staining pattern, typically one, two or three locations in the staphylococcal envelope in both strains SEJ1 and SEJ2, whereas a ring-like contour surrounding the bacterial cell was not observed (Figure 2B). Sites of SasF deposition were found at different locations in the cell wall envelope but did not appear to colocalize with the cross wall.

Surface distribution of proteins with and without YSIRK/GS signal peptides

We sought to examine the universality of the observed surface distribution patterns for proteins with or without YSIRK/GS motif signal peptides. FnbpB, SdrC and SdrD each harbour a YSIRK/GS motif signal peptide. A ring-like or hemispherical surface distribution was observed for each of the three proteins (Figure 3A). The immune-fluorescent signals displayed for SdrD were very strong; this may in part conceal the uneven distribution of this protein over the bacterial surface (Figure 3A). The latter phenomenon was more readily detectable in fluorescence images of staphylococci stained with FnbpB- and SdrC-specific antibodies, which also assumed ring-shaped or hemispherical surface distribution of fluorescent signals (Figure 3A). Signal peptides of SasA, SasD and SasK lack the YSIRK/GS motif (Figure 1). The envelope of staphylococci that had been incubated with SasA-, SasD- or SasK-specific antibodies displayed discrete, punctate fluorescent signals (Figure 3B). In images from all three experiments, we observed one, two or three surface protein deposits within the bacterial envelope; however, there were also many cells without detectable signals (Figure 3B). We presume that the frequent absence of surface protein deposits is due to the acquisition of two-dimensional fluorescence images; surface protein deposits residing underneath staphylococcal cells are most likely not detectable with this technology. As controls, bursa aurealis insertion mutations in fnbpB, sasA, sasD, sasK, sdrC or sdrD abrogated immunoreactive signals of corresponding antibodies, thereby demonstrating their specificity (data not shown). Thus, surface proteins that are secreted through YSIRK/GS motif signal peptides (Spa, ClfA, FnbpB, SdrC and SdrD) are distributed in a ring-like manner in the staphylococcal envelope and perpendicular to cross wall, that is, the cell division plane. In contrast, surface proteins that are secreted by signal peptides without a YSIRK/GS motif are found in discrete, punctate deposits in the cell wall envelope.

Figure 3.

Distribution of surface proteins in the cell wall envelope of staphylococci. (A) S. aureus strain SEJ1 (spa) was analysed with differential interference contrast (DIC) or fluorescence microscopy using BODIPY-vancomycin (green) or specific antibodies (SdrD, SdrC and FnbpB) followed by secondary goat anti-rabbit conjugated to Alexafluor 647 (red). SdrD, SdrC and FnbpB are secreted through signal peptides that harbour the YSIRK/GS motif (+YSIRK). (B) S. aureus strain SEJ1 (spa) was viewed with DIC or fluorescence microscopy using BODIPY-vancomycin (green) or specific antibodies (SasA, SasD and SasK) followed by secondary goat anti-rabbit conjugated to Alexafluor 647 (red). SasA, SasD and SasK are secreted through signal peptides that do not harbour the YSIRK/GS motif (-YSIRK). Two-dimensional two-colour and DIC images were acquired sequentially with separate laser lines and merged using the ImageJ software.

View full figure (202 KB)

Reconstruction of surface protein deposits in the staphylococcal envelope

Following labelling of staphylococci with BODIPY-vancomycin and antibodies, serial images in optical planes separated by approximately 60 nm along the z axis were collected by sequential laser scanning confocal microscopy. These series were then used to generate three-dimensional fluorescence reconstructions of surface proteins in the murein sacculus. We first acquired images for large clusters of staphylococci to reveal protein deposits in bacteria where cell wall envelopes had not yet been completely separated. Figure 4A represents a summary image for such a staphylococcal cluster. ClfA staining of staphylococcal clusters revealed the ring-shaped distribution of surface proteins secreted through YSIRK/GS motif signal peptides as well as the perpendicular pattern of protein rings in neighbouring cells (Figure 4A). The three-dimensional reconstruction of the acquired data can be appreciated when the summary image is rotated (Supplementary Movie 1). As a control, ClfA antibody staining of spa, clfAermB mutant staphylococci detected only background fluorescence. Three-dimensional image reconstruction of staphylococcal clusters stained with anti-SasF revealed the discrete, circumscript nature of protein deposits derived from signal peptide precursors without the YSIRK/GS motif (Figure 4A; Supplementary Movie 2). Control experiments with spa, sasFermB mutant staphylococci revealed occasional background fluorescence signals with SasF antibodies.

Figure 4.

Confocal laser microscopy reveals the three-dimensional distribution of ClfA and SasF on the staphylococcal surface. (A) S. aureus strains SEJ1 (spa), ACD1 (spa, clfAermB) or ACD2 (spa, sasFermB) were labelled with BODIPY-vancomycin (green) as well as ClfA- or SasF-specific antibodies followed by secondary goat anti-rabbit conjugated to Alexafluor 647 (red). Confocal microscopy was performed and z-series was acquired with 70-nm increments. Three-dimensional (3D) projections were created from stacks using ImageJ software. For 3D rotation of assembled images, see Supplementary Movies 1 and 2. (B) Representative single cell series for S. aureus SEJ1 labelled with BODIPY-vancomycin (green) and either ClfA- or SasF-specific antibodies (red).

View full figure (589 KB)

A series of confocal images of single staphylococcal cells with ClfA deposits confirmed the hemispherical or ring-like distribution of this surface protein (Figure 4B). These images also identified foci of abundant ClfA deposition within the ring of surface proteins, in agreement with earlier observations generated for protein A (DeDent et al, 2007). Our working model (vide infra) predicts that YSIRK/GS motifs containing surface proteins are deposited at specific sites, the location of which in the murein sacculus may change as bacteria grow and expand their cell wall envelope. Acquisition of similar images for staphylococci stained with SasF-specific antibody identified three discrete envelope deposits for proteins secreted without YSIRK/GS motif signal peptides (Figure 4B). These foci, however, are not connected by ring-like or hemispherical distributions of this surface protein, as is observed with ClfA and protein A (Figure 4B).

Two destinations for staphylococcal surface protein secretion

The distribution patterns of surface proteins with or without YSIRK/GS motif signal peptides in the staphylococcal envelope appear distinctly different. Staining for individual proteins, however, does not establish whether the foci of surface protein deposition are identical for ClfA and SasF. In other words, we sought to determine whether the destiny of surface protein secretion in staphylococci is determined by the presence of specific signal peptides. To address this question, antibodies against ClfA (+YSIRK) were conjugated to Alexafluor 488 (green), whereas antibodies against SasF (-YSIRK) were conjugated to Alexafluor 647 (red). Staphylococci were stained with both antibodies and samples subjected to sequential laser scanning confocal microscopy and optical sections of 120 nm through bacterial samples were acquired. Figure 5A depicts summary images of deconvoluted three-dimensional reconstructions for stacked confocal images. Although abundant foci of ClfA and SasF deposition were detected in the staphylococcal envelope, the vast majority of these fluorescent signals did not colocalize. As a control, double labelling of staphylococci for ClfB and SdrD, two surface proteins with YSIRK/GS signal peptides, revealed their colocalization (Supplementary Figure 1).

Figure 5.

ClfA and SasF do not colocalize in the staphylococcal envelope. (A) ClfA and SasF antibodies were conjugated to Alexafluor 488 (ClfA, green) or Alexafluor 647 (SasF, red) and used to label S. aureus SEJ1 (spa). Confocal microscopy was performed and z-series was acquired with 120-nm increments. Three-dimensional projections were created from stacks using the ImageJ software. (B) Representative single-cell DIC images and confocal microscopy z-series for staphylococci labelled with ClfA (green)- and SasF (red)-specific antibodies.

View full figure (227 KB)

Single staphylococcal cells were also subjected to sequential laser scanning confocal microscopy. Optical sections along the z axis for two exemplary cells are displayed in Figure 5B. The sites of ClfA and SasF deposition in the staphylococcal cell wall envelope were distinct in each case examined. In some cells, deposition sites for ClfA and SasF appear to be positioned adjacent to one another (cell 1), whereas in others (cell 2), they appeared to be juxtaposed or perpendicular to one another (Figure 5B).

Dynamics of surface protein ring formation

To monitor the deposition of surface protein and formation of ring-like assemblies, staphylococci were treated with trypsin, a protease that removes surface-exposed proteins such as protein A, ClfA and SasF from the bacterial envelope, thereby abolishing antibody binding to the bacterial surface. Protease was quenched with inhibitor and cells were incubated in fresh media at 37°C for growth and anchoring of surface proteins. Deposition of newly synthesized ClfA and SasF in the staphylococcal envelope was monitored by fluorescence microscopy. Immediately following trypsin treatment, no fluorescent signal was detected for ClfA or SasF (data not shown). At 20 min following trypsin treatment, fluorescent signals were detected in the staphylococcal envelope for both ClfA and SasF, revealing localized deposition of newly synthesized surface protein (Figure 6). It is noted that ClfA and SasF were deposited at different sites in the bacterial envelope, as their red and green fluorescent signals were spatially restricted, but not colocalized. SasF appeared at the perimeter of diplococcal cells, and very rarely on single cells. ClfA was found almost exclusively at or near the cross wall of diplococcal cells, and rarely, if at all, on single cells (Supplementary Figure 5). Incubation for 40 min led to increased deposition of ClfA at the cross wall, which in some cases expanded to form ring-like structures surrounding staphylococci. In contrast, areas of SasF deposition remained primarily at the cell periphery (Figure 6). At the 40-min time point, we observed occasional colocalization of ClfA and SasF in the bacterial envelope. Nevertheless, discrete SasF-specific fluorescent signals were distinguishable. Thus, ClfA and SasF appear to be deposited at unique sites in the staphylococcal envelope. In addition, the foci of ClfA, but not those of SasF deposition, are expanded to form ring-like assemblies of surface proteins at the cross wall.

Figure 6.

Deposition of newly synthesized ClfA and SasF in the cell wall envelope following trypsin treatment of staphylococci. (A) Staphylococci were treated with trypsin, which abolished all ClfA- and SasF-specific surface protein labelling in the bacterial envelope. At timed intervals, 20 or 40 min, following dilution of bacteria into fresh growth media, culture aliquots were labelled with ClfA (green, conjugated to Alexafluor 488) or SasF (red, conjugated to Alexafluor 647). DIC images as well as confocal laser microscopy z-series in 120-nm increments were acquired. Three-dimensional projections were created from stacks using the ImageJ software. (B) Representative single-cell DIC images and confocal microscopy z-series for staphylococci labelled with ClfA (green)- and SasF (red)-specific antibodies after 20 or 40 min of growth following trypsin treatment.

View full figure (231 KB)

Signal peptides address proteins to their final destination in the envelope

In agreement with the streptococcal model (Carlsson et al, 2006), we presumed that staphylococcal signal peptides may address surface proteins to their final destination, either a ring-like distribution or a focal assembly in the staphylococcal envelope. To test this, we transformed spa, clfAermB or spa, sasFermB mutant strains with recombinant plasmids expressing either clfA (pClfA) or sasF (pSasF). Immunofluorescence microscopy experiments revealed focal assemblies of SasF in the envelope of spa, sasFermB (pSasF) cells and ring-like or spherical distributions of ClfA on the surface of spa, clfAermB (pCflA) cells (Figure 7A). These results suggest that plasmid complementation with the wild-type gene in trans not only restored the expression defects of sasF or clfA mutants but also promoted trafficking of surface proteins to their proper destination. The signal peptides of SasF and ClfA were exchanged and plasmid-encoded recombinant proteins, SP_SasF-ClfA and SP_ClfA-SasF, were expressed in spa, clfAermB or spa, sasFermB mutant staphylococci. Immunofluorescence microscopy revealed ring-like or spherical distribution of SasF-specific signals in spa, sasFermB (pSP_ClfA-SasF) cells (Figure 7B). In contrast, ClfA deposition was restricted to discrete focal assemblies in the envelope of spa, clfAermB (pSP_SasF-ClfA) cells (Figure 7B). Taken together, these results indicate that the signal peptides of ClfA and SasF are necessary and sufficient for delivery of surface proteins to two distinct locations in the staphylococcal cell wall envelope.

Figure 7.

Signal peptides address staphylococcal surface proteins to their proper location in the cell wall envelope. (A) S. aureus strains ACD1 (spa, clfAermB) and ACD2 (spa, sasFermB) were transformed with plasmids to generate ACD3 (spa, clfAermB, pClfA) and ACD4 (spa, sasFermB, pSasF). Staphylococci were viewed with differential interference contrast (DIC) or fluorescence microscopy using BODIPY-vancomycin (green), ClfA- or SasF-specific antibodies followed by secondary goat anti-rabbit conjugated to Alexafluor 647 (red). (B) S. aureus strains ACD1 and ACD2 were transformed with plasmids to generate ACD5 (spa, clfAermB, pSP_SasF-ClfA) and ACD6 (spa, sasFermB, pSP_ClfA-SasF). Staphylococci were viewed with DIC or fluorescence microscopy using BODIPY-vancomycin (green), ClfA- or SasF-specific antibodies followed by secondary goat anti-rabbit conjugated to Alexafluor 647 (red).

View full figure (260 KB)

Expression of surface protein genes from multi-copy plasmids results in increased abundance of polypeptides in the bacterial envelope (Figure 7). To determine whether surface protein trafficking occurs also with genetic constructs that are inserted in single copy into the staphylococcal genome, we utilized the integration plasmid pCL55 (Lee et al, 1991). Wild-type sasF and a sasF variant with the clfA signal peptide were cloned into the integration plasmid pCL55 (pCL55-SP_ClfA-SasF). Following transformation and plasmid integration, S. aureus cells were treated with trypsin, protease quenched and bacterial growth was initiated by dilution into fresh media at 37°C. Sample aliquots were stained with primary labelled antibody to SdrD (green) or SasF (red) and viewed by fluorescence microscopy 20 and 40 min following trypsin treatment. S. aureus with wild-type sasF and the empty pCL55 vector control (spa (pCL55)) displayed SasF at 20 and 40 min primarily in the periphery of dividing cells, with only rare signals in the proximity of the cell division septum (Figure 8). SdrD, on the other hand, was found primarily at or near the cross wall at 20 min and at both the cross wall and periphery at 40 min (Figure 8). In contrast, staphylococci expressing SasF with the ClfA signal peptide (+YSIRK/GS motif) (spa, sasFermB (pCL55-SP_ClfA-SasF)) generated immunofluorescent signals for both SdrD and SasF that localized to the cross wall of dividing staphylococci at 20- and 40-min incubation intervals (Figure 8). Surface protein colocalization on SP_ClfA-SasF-expressing cells, though not complete, was markedly increased as compared with cells expressing wild-type SasF. Similar to what has been reported for protein F with the M protein signal peptide in group A streptococci (Carlsson et al, 2006), SP_ClfA-SasF was also found in the periphery of cells (Figure 8).

Figure 8.

YSIRK/GS signal peptides direct cell wall-anchored proteins to the septum and cross wall. (A) S. aureus strain ACD2 (spa, sasFermB) was transformed with the integration plasmid pCL55 encoding wild-type SasF (pCL55-SasF) or SasF bearing the ClfA signal peptide (pCL55-SP_ClfA-SasF) to generate strains ACD8 (spa, sasFermB, pCL55-SasF) and ACD9 (spa, sasFermB, pCL55-SP_ClfA-SasF). Staphylococci were treated with trypsin, which removed all SasF-specific surface protein labelling in the bacterial envelope. At timed intervals, 20 and 40 min, following dilution of bacteria into fresh growth media, aliquots of staphylococcal cultures were labelled with SdrD (green, conjugated to Alexafluor 488) or SasF (red, conjugated to Alexafluor 647). DIC images as well as confocal laser microscopy z-series in 120-nm increments were acquired. Images were deconvoluted using the Huygens™ software. Three-dimensional projections were created from deconvoluted stacks using the ImageJ software. Representative wide-field images are shown. (B) Representative images of diplococci from the experiment described in (A).

View full figure (307 KB)

To examine the contribution of the YSIRK/GS motif in surface protein trafficking, we generated alanine amino-acid substitutions in any one motif residue encoded by the clfA gene inserted through pCL55 into the staphylococcal genome; however, mutant proteins displayed no defect in deposition (ring-like distribution and deposition at the cross wall, data not shown). We therefore generated a variant with a scrambled YSIRK sequence (but leaving the G/S segment intact) and inserted the variant into the staphylococcal genome. Again, we observed no phenotype in immunofluorescence microscopy experiments (Supplementary Figure 2), indicating that the YSIRK/GS signal peptide, but not the YSIRK sequence alone, is responsible for directing ClfA into a ring-like surface distribution and deposition within the envelope at the cross wall.

Contributions of specialized secretion systems to surface protein trafficking

YSIRK/GS motif containing signal peptides harbour two positively charged amino acids, RK or RR, similar to substrates recognized by the twin-arginine translocase (TAT) pathway, which is capable of secreting fully folded polypeptides across the cytoplasmic membrane of bacteria (Berks et al, 2003). To test whether or not the TAT pathway is involved in the secretion or localized deposition of surface proteins, we viewed staphylococci harbouring a bursa aurealis insertion into the tatC gene (spa, tatCermB) by immunofluorescence microscopy. When compared with spa staphylococci, we observed no differences in ClfA deposition in the tatC mutant strain (Supplementary Figure 3).

Similar to Streptococcus gordonii, S. aureus encodes two accessory secretion genes, designated secA2 and secY2 (Bensing and Sullam, 2002; Baba et al, 2007). S. gordonii secY2 and secA2 are required for the secretion and functional display of GspB, a surface protein homologue of S. aureus SasA (Mazmanian et al, 2001; Bensing and Sullam, 2002). However, mutations that affect secA2 and gtfA, encoding a glycosyl transferase that modifies GspB, cause the increased surface display of non-glycosylated GspB, in agreement with a model whereby SecA2 is involved in substrate recognition of a single polypeptide after it has been modified by glycosylation (Bensing and Sullam, 2002; Takamatsu et al, 2004; Bensing et al, 2007). To examine the possibility that the two accessory secretion genes, secA2 and secY2 are required for the secretion of surface proteins, we used bursa aurealis insertions into secY2 (secY2ermB) or in secA2 (secA2ermB). Bursa aurealis insertion into secY2 abolished the surface display of SasA (Figure 9), but did not affect the deposition of other surface proteins, such as SdrD or SasF (Supplementary Figure 4). In contrast, secA2ermB increased the surface display of SasA (Figure 9); however, this mutation also caused no phenotype for the deposition of other surface proteins (data not shown).

Figure 9.

Contribution of secA2 and secY2 towards the surface display of SasA. S. aureus strains SEJ1 (spa), ACD22 (spa, sasAermB), ACD19 (spa, secA2ermB) or ACD20 (spa, secY2ermB) were analysed by immunofluorescence microscopy with SasA antibodies followed by Alexafluor 647-conjugated secondary antibody and staining with BODIPY-conjugated vancomycin and compared with captured DIC images. See text for details and Supplementary Figure 4 for SasF and ClfA staining of secY2 mutant staphylococci.

View full figure (218 KB)

Cell wall-anchored surface proteins with YSIRK/GS motif signal peptides have been reported in streptococci (S. pyogenes, S. agalactiae, S. faecalis and S. pneumoniae) and staphylococci (S. aureus and S. epidermidis), but not in Listeria monocytogenes, bacilli (Bacillus subtilis, B. anthracis and B. cereus), clostridia (Clostridium tetani and C. perfringens) or nocardia (Corynebacterium diphtheriae, Actinomyces naeslundii, Streptomyces coelicolor) (Rosenstein and Götz, 2000; Tettelin et al, 2001; Bae and Schneewind, 2003). When comparing the morphology of these microbes, we noticed that the majority of YSIRK/GS motif signal peptides occur in Gram-positive bacteria with round spherical cell shapes (cocci). This report, as well as earlier work from Gunnar Lindahl's laboratory, examined the function of YSIRK/GS motif signal peptides in the delivery of surface proteins to unique locations in the cell wall envelope of cocci (Carlsson et al, 2006). For streptococci, M protein (carrying the YSRIK/GS motif signal peptide) is secreted at the cell division site (Hahn and Cole, 1963), whereas protein F (signal peptide without motif) is preferentially secreted at the cell pole (Carlsson et al, 2006). In staphylococci, five polypeptides with YSIRK/GS motif signal peptides (protein A, ClfA, SdrC, SdrD and FnbpB) are distributed in a ring-like manner in the vicinity of the cell division site. Four surface proteins without the YSIRK/GS motif (SasA, SasD, SasF and SasK) are deposited at discrete focal assemblies in the envelope that, however, do not overlap with the ring-like distribution of proteins secreted with the YSIRK/GS motif.

Pioneering studies on staphylococcal morphology used electron microscopy and revealed the cross wall, a thick layer of newly synthesized peptidoglycan separating adjacent daughter cells (Giesbrecht et al, 1976; Touhami et al, 2004). Cross wall synthesis is preceded by FtsZ ring formation and cytokinesis, that is, the segregation of chromosomes into daughter cells, which is also accompanied by FtsZ constriction and cell separation (Bi and Lutkenhaus, 1991; Lutkenhaus, 1993). Unlike the elongated growth of a rod-shaped microbe along its cylindrical axis (de Boer et al, 1990), the overall size of staphylococcal cells does not increase prior to cross wall separation (Touhami et al, 2004). These findings imply that newly replicated chromosomes and cross wall, in short, two newly formed daughter cells, are all accommodated within a murein sacculus that has not expanded following the last round of cell separation. Once the cross wall has been cleaved by lytic enzymes (autolysin, Atl) (Oshida et al, 1995; Yamada et al, 1996), the separating daughter cells immediately assume similar size and shape as older cells already engaged in cell division (Touhami et al, 2004).

These observations provide an important base for our working model on protein targeting to the cell wall envelope (Figure 10). Once formed, murein sacculi of staphylococcal cells cannot expand unless the cross wall of dividing daughter cells is cleaved. We and others did not observe half-cells, hemispherical staphylococci with one flat surface (Giesbrecht et al, 1998). It therefore seems reasonable to assume that the bacterial osmotic pressure expands the shape of dividing daughter cells immediately to form round spheres (Touhami et al, 2004). Bulk de novo cell wall synthesis occurs mostly at the cross wall, which represents about half of the murein sacculus of a future daughter cell (Briles and Tomasz, 1970; Pinho and Errington, 2003). Proteins deposited in the cross wall initially cannot be displayed on the bacterial surface; the cross wall compartment, including the space between two daughter cells, is secluded from the environment by murein sacculi. If so, protein traffic to the cross wall is probably difficult to measure with immunofluorescence microscopy, as antibodies may be unable to penetrate murein sacculi.

Figure 10.

Model for surface protein trafficking in staphylococci. Newly synthesized peptidoglycan is assembled in the cross wall and, once daughter cells have been separated, this compartment is exposed as staphylococcal envelope. YSIRK/GS motif signal peptides address surface proteins to the cross wall of staphylococci, whereas conventional signal peptides direct proteins to peripheral secretion sites at the cell pole. Protein secretion sites accessed by conventional signal peptides by default lead to protein transport into extracellular milieu. Sortase-mediated anchoring of proteins with conventional signal peptides is thought to immobilize proteins in the immediate vicinity of staphylococcal secretion sites.

View full figure (135 KB)

Considering the unique attributes of cell division in staphylococci, we propose that the destiny of surface protein traffic through YSIRK/GS motif signal peptides resides in the vicinity of future cell division sites and within the cross wall. Massive synthesis of peptidoglycan within the cross wall may ensure that assembly sites for surface protein deposition are mobile within the peptidoglycan layer as the cross wall expands, first within the division plane and then following separation of daughter cells in spherical staphylococci. Such presumed protein traffic to the cross wall would be unable to promote protein secretion into the extracellular medium, unless daughter cells have already separated. In agreement with this conjecture, the cross wall pathway appears to be engaged by YSIRK/GS motif signal peptides and may be largely restricted to surface proteins. The alternative secretion pathway, travelled by substrates with conventional signal peptides, by default leads across the murein sacculus into the extracellular medium. This pathway is engaged by surface proteins that get deposited in the cell wall as discrete foci surrounding these secretion sites.

Carlsson et al (2006) generated substitutions in the YSIRK/GS motif of M protein and measured polypeptide targeting to the cell wall envelope by immune-electron microscopy. Signal peptide variants with YAARK/GS or YSAAK/GS motifs continued to direct proteins to the cell division site, similar to wild-type M protein (Carlsson et al, 2006). We have also examined the amino-acid requirement of the YSIRK/GS motif for cross wall secretion. Single amino-acid substitutions of each conserved residue of the YSIRK/GS motif (data not shown) or a scrambled YSIRK sequence, all failed to abolish surface protein localization. Thus, although YSIRK/GS signal peptides are clearly sufficient to address surface proteins to the cross wall, the YSIRK sequence may not be required for this mechanism and another, discrete feature of these signal peptides must provide for surface protein traffic.

In summary, we report that staphylococci address surface proteins to discrete locations in the bacterial envelope by mechanisms requiring recognition of specific signal peptides. Protein traffic to discrete sites in the cell wall envelope of staphylococci seems a fascinating problem, the future focus of which is the unravelling of genes and mechanisms responsible for directing surface proteins to alternate envelope sites. Conservation of the YSIRK/GS motif in signal peptides of several bacterial species suggests that these mechanisms could apply to different microbes.

Bacterial strains, media and growth conditions

S. aureus strains were grown in tryptic soya broth (TSB) at 37°C to mid-log phase (optical density at 600 nm (OD₆₀₀) of 0.5), unless otherwise stated. Escherichia coli DH5 was gown in Luria–Bertani broth at 37°C (Hanahan, 1983). The spa mutant strains SEJ1, a derivative of RN4220 (Kreiswirth et al, 1983), and SEJ2, a variant of strain Newman (Duthie and Lorenz, 1952), have been described earlier (Stranger-Jones et al, 2006; Gründling and Schneewind, 2007). Surface protein mutants harbouring the bursa aurealis mariner transposon insertions were obtained from the Phoenix (N) library (Bae et al, 2004) (Supplementary Table 1). Mutations were transduced with bacteriophage 85 into SEJ1 or SEJ2 and grown in TSB supplemented with 10 g ml^-1 erythromycin. Following plasmid transformation, E. coli DH5 transformants were selected in the presence of 100 g ml^-1 ampicillin. Following transformation of relevant S. aureus strains, transformants were selected by growth on 10 g ml^-1 chloramphenicol.

Phage transduction, plasmids and mutagenesis

85 transductants were confirmed by inverse PCR and DNA sequencing as described (Bae et al, 2004). For plasmid complementation studies, the open reading frame of surface protein genes, for example, clfA or sasF, and 1000 bp of upstream sequence was PCR amplified from S. aureus Newman chromosomal DNA with primers AAAGAATTCGTTGTAATGCATTTTTCTATAAGATA
GAACTAAAAGGAG and AAAGCTAGCAATAATTAACTGTTGATGAATTAATT
ATTAATTATATTCAAACAA for clfA or AAAGAATTCTATGAAACGATGCTCAATATAG
CAGAC and AAAGCTAGCTTCATTATAACGTTAAAATGATGGAC
AATCTATTCA for sasF. Resulting PCR products were digested with the restriction enzymes NheI and BamHI and ligated into pOS1 (Schneewind et al, 1993) cut with the same enzymes. Restriction sites flanking coding sequence for N-terminal signal peptides were cloned by the QuikChange method (Stratagene). Specifically, an XhoI site was inserted 30 nucleotides downstream of the predicted cleavage site with primers GTTACGCAATCTGATAGCCTCGAGGCAAGTAACGA
AAGCAAAAG and CTTTTGCTTTCGTTACTTGCCTCGAGGCTATCAGA
TTGCGTAAC (clfA) or GGATACTGAAATTTCAAAACTCGAGATATTATCTA
AGCAAG and CTTGCTTAGATAATATCTCGAGTTTTGAAATTTCA
GTATCC (sasF). An AvrII site was generated following mutation of nucleotides 9 bp upstream from the annotated open reading frame start site with primers TAGTTAATATAAAATGACTTTTTACCTAGGGGGAA
TAAAATGAATATGAAGAAA and TTTCTTCATATTCATTTTATTCCCCCTAGGTAAAA
AGTCATTTTATATTAACTA (clfA) or TAATTCTTTGAAGGAAGTAAGTGACCTAGGAGTAT
GTTGATG GCTAAATATCGA and TCGATATTTAGCCATCAACATACTCCTAGGTCACT
TACTTCCTTCAAAGAATTA (sasF). Following confirmation by restriction digest and DNA sequencing analysis, the resulting plasmids, pClfA or pSasF, or empty vector were transformed by electroporation into the S. aureus strains spa, clfAermB or spa, sasFermB, respectively. For construction of signal peptide exchange hybrid proteins, pClfA or pSasF was digested with the restriction enzymes AvrII and XhoI. The resulting fragments were ligated together (clfA signal peptide insert ligated into the sasF vector fragment and sasF signal peptide insert ligated into the clfA vector fragment), generating pSP_ClfA-SasF and pSP_SasF-ClfA, respectively. Plasmid signal peptide exchanges were screened by colony PCR and confirmed by DNA sequencing analysis and pSP_ClfA-SasF and pSP_SasF-ClfA were transformed into S. aureus. The plasmid pClfA_{YSIRKscramble} was cloned from pClfA using QuikChange mutagenesis (Stratagene) with the primers AAAAAAGAAAAAATTAAACACGCATCAAAATCGAT
TCGGGTGGCTGGCGTGCTTGTAGGT and ACCTACAAGCACGCCAGCCACCCGAATCGATTTTG
ATGCGTGTTTAATTTTTTCTTTTTT. The integration plasmids pCL55-SP_ClfA-SasF and pCL55-ClfA_{YSIRKscramble} were generated by subcloning the SP_ClfA-SasF or ClfA_{YSIRKscramble} fragment into SmaI-digested pCL55 by blunt end ligation following digestion with EcoRI and NheI and generation of blunt ends with Klenow enzyme. pCL55-SasF was generated following PCR amplification of SasF using the same primers described for pSasF. The resulting PCR product was ligated together with SmaI-digested pCL55. pCL55-SP_ClfA-SasF, pClfA_{YSIRKscramble} and pCL55-SasF were transformed by electroporation into the S. aureus strains.

Immunofluorescence microscopy

Overnight cultures of S. aureus cells were diluted 1:100 into fresh growth media and grown to mid-log phase (OD₆₆₀ of 0.6) and equivalent of 4–10 cell divisions as surface proteins are regulated by agr (Novick, 2003). The culture (6 ml) was centrifuged for 5 min at 8000 g. Bacterial cell pellets were washed twice with 10 ml phosphate-buffered saline (PBS, 10 mM sodium phosphate, pH 7.0). Cells were suspended in 500 l of PBS, sonicated for 15 s and fixed with 2.5% paraformaldehyde, 0.006% glutaraldehyde in 30 mM PBS (pH 7.4) for 20 min at room temperature. Cells were washed three times with 1 ml PBS, suspended in a final volume of 250 l of 3% bovine serum albumin (BSA) in PBS as a blocking reagent for nonspecific antibody binding, and incubated for 1 h at room temperature or overnight at 4°C with shaking. Primary antibodies were adsorbed prior to incubation with fixed staphylococci by pre-incubation of immune sera with acetone precipitates of transduced N library mutant staphylococci lacking the gene to which the serum was raised. Following blocking, staphylococci were sedimented by centrifugation at 8000 g for 3 min and suspended in 250 l of adsorbed primary antibody (1:500 in 3% BSA–PBS) and incubated for 1 h at room temperature with rotation. Primary incubation was followed by incubation with Alexafluor 647-IgG secondary, and BODIPY-vancomycin (Invitrogen Molecular Probes). Cells were washed five times with 1 ml PBS containing 0.025% Tween 20 (PBS-T) followed by 1 h incubation in the dark with 250 l of 1:250 goat anti-rabbit Alexafluor 647-conjugated secondary antibody in 3% BSA–PBS. Staphylococci were sedimented and incubated with 200 l of 1 g ml^-1 BODIPY-vancomycin conjugate for 5 min. Cells were again washed five times with 1 ml PBS and suspended in 100 l. Cell suspensions were applied to L-poly-lysine-coated coverslips for 3 min followed by three washes with 300 l PBS. Slides were mounted with N-propylgallate, sealed, viewed and images were collected with a Leica SP5 AOBS spectral two-photon confocal microscope (DeDent et al, 2007). Captured images and Z-stacks were processed with ImageJ software and plugins (Abramoff et al, 2004).

Double labelling and trypsin treatment experiments

To prevent cross-reactivity of secondary antibodies for labelling of two surface proteins, primary antibodies were either labelled using the Zenon Alexafluor rabbit IgG labelling kit (conjugated Fab fragments; Invitrogen) or directly conjugated to Alexafluor 488 or 647 (Invitrogen Molecular Probes). Prior to conjugation, ClfA- or SasF-specific rabbit serum was incubated with acetone-precipitated cells of each mutant, supernatant was applied to a protein A column (Pierce), eluted and dialysed overnight with PBS. Following incubation of cells with 3% BSA–PBS, cells were incubated for 1 h at room temperature with both ClfA-Alexafluor 488 and SasF-Alexafluor 647 (1:25 in 3% BSA–PBS). Cells were washed and prepared for microscopy as described. For trypsin treatment, staphylococci were treated for 1 h with 0.2 mg ml^-1 trypsin in PBS at 37°C. Cells were washed twice with 10 ml PBS and suspended in 1 ml TSB containing 1 mM phenylmethylsulphonyl fluoride (PMSF) (Figure 6) or soyabean trypsin inhibitor (Figure 8; Supplementary Figure 5) and grown at 37°C. Aliquots of cells were removed at 0, 20 or 40 min following addition of PMSF and growth at 37°C. Cells were sonicated for 15 s, fixed, labelled with primary-conjugated antibodies, and slides were prepared as described. Images were collected with a Leica SP5 AOBS spectral two-photon confocal microscope and z-series scans were sampled in 120-nm increments.

We thank Dr Vytas Bindokas, Director of the University of Chicago BSD Light Microscopy Core Facility, for assistance with microscopy experiments, Juliane Bubeck-Wardenburg, Gabriella Garufi, Luciano Marraffini, Alice Cheng, Hwan Keun Kim and Brandon Wojcik for critical reading of the paper, and members of our laboratory for discussion. This study was supported by a grant from the National Institute of Allergy and Infectious Diseases, Infectious Disease Branch (AI38897) to OS. AD acknowledges support from the Molecular Cell Biology Training Grant T32GM007183 at the University of Chicago.

Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11: 36–42

Armstrong JJ, Baddiley J, Buchanan JG, Davison AL, Kelemen MV, Neuhaus FC (1959) Composition of teichoic acids from a number of bacterial cell walls. Nature 184: 247–249 | Article | PubMed | ISI | ChemPort |

Baba T, Bae T, Schneewind O, Takeuchi F, Hiramatsu K (2007) Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes. J Bacteriol 190: 300–310 | Article | PubMed | ChemPort |

Baba T, Schneewind O (1998) Targeting of muralytic enzymes to the cell division site of Gram-positive bacteria: repeat domains direct autolysin to the equatorial surface ring of Staphylococcus aureus. EMBO J 17: 4639–4646 | Article | PubMed | ChemPort |

Bae T, Banger AK, Wallace A, Glass EM, Aslund F, Schneewind O, Missiakas DM (2004) Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proc Natl Acad Sci USA 101: 12312–12317 | Article | PubMed | ChemPort |

Bae T, Schneewind O (2003) The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing. J Bacteriol 185: 2910–2919 | Article | PubMed | ChemPort |

Bensing BA, Siboo IR, Sullam PM (2007) Glycine residues in the hydrophobic core of the GspB signal sequence route export toward the accessory Sec pathway. J Bacteriol 189: 3846–3854 | Article | PubMed | ChemPort |

Bensing BA, Sullam PM (2002) An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets. Mol Microbiol 44: 1081–1094 | Article | PubMed | ChemPort |

Berks BC, Palmer T, Sargent F (2003) The Tat translocation pathway and its role in microbial physiology. Adv Microb Physiol 47: 187–254 | Article | PubMed | ISI | ChemPort |

Bi EF, Lutkenhaus J (1991) FtsZ ring structure associated with division in Escherichia coli. Nature 354: 161–164 | Article | PubMed | ISI | ChemPort |

Briles EB, Tomasz A (1970) Radioautographic evidence for equatorial wall growth in a Gram-positive bacterium. Segregation of choline-3H-labeled teichoic acid. J Cell Biol 47: 786 | Article | PubMed | ISI | ChemPort |

Carlsson F, Stålhammar-Carlemalm M, Flärdh K, Sandin C, Carlemalm E, Lindahl G (2006) Signal sequence directs localized secretion of bacterial surface proteins. Nature 442: 943–946 | Article | PubMed | ChemPort |

Cole RM, Hahn JJ (1962) Cell wall replication in Streptococcus pyogenes. Science 135: 722–724 | Article | PubMed | ISI | ChemPort |

de Boer PAJ, Cook WR, Rothfield LI (1990) Bacterial cell division. Annu Rev Genet 24: 249–274 | Article | PubMed | ChemPort |

DeDent AC, McAdow M, Schneewind O (2007) Distribution of protein A on the surface of Staphylococcus aureus. J Bacteriol 189: 4473–4484 | Article | PubMed | ChemPort |

Duthie ES, Lorenz LL (1952) Staphylococcal coagulase: mode of action and antigenicity. J Gen Microbiol 6: 95–107 | PubMed | ISI | ChemPort |

Emr SD, Schwartz M, Silhavy TJ (1978) Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein. Proc Natl Acad Sci USA 75: 5802–5806 | Article | PubMed | ChemPort |

Giesbrecht P, Kersten T, Maidhof H, Wecke J (1998) Staphylococcal cell wall: morphogenesis and fatal variations in the presence of penicillin. Microbiol Mol Biol Rev 62: 1371–1414 | PubMed | ISI | ChemPort |

Giesbrecht P, Wecke J, Reinicke B (1976) On the morphogenesis of the cell wall of staphylococci. Int Rev Cytol 44: 225–318 | PubMed | ISI | ChemPort |

Gründling A, Schneewind O (2007) Genes required for glycolipid synthesis and lipoteichoic acid anchoring in Staphylococcus aureus. J Bacteriol 189: 2521–2530 | Article | PubMed | ChemPort |

Hahn JJ, Cole RM (1963) Streptococcal M antigen location and synthesis, studied by immunofluorescence. J Exp Med 118: 659–666 | Article | PubMed | ChemPort |

Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557–572 | Article | PubMed | ISI | ChemPort |

Hanski E, Caparon M (1992) Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Proc Natl Acad Sci USA 89: 6172–6176 | Article | PubMed | ChemPort |

Higashi Y, Strominger JL, Sweeley CC (1967) Structure of a lipid intermediate in cell wall peptidoglycan synthesis: a derivative of a C55 isoprenoid alcohol. Proc Natl Acad Sci USA 55: 1878–1884 | Article

Jensen K (1958) A normally occurring Staphylococcus antibody in human serum. Acta Pathol Microbiol Scand 44: 421–428

Kreiswirth BN, Lofdahl S, Betley MJ, O'Reilly M, Schlievert PM, Bergdoll MS, Novick RP (1983) The toxic shock syndrome exotoxin structural gene is not detectably transmitted by prophage. Nature 305: 709–712 | Article | PubMed | ISI | ChemPort |

Lancefield RC (1928) The antigenic complex of Streptococcus hemolyticus. I. Demonstration of a type-specific substance in extracts of Streptococcus hemolyticus. J Exp Med 47: 91–103 | Article | ChemPort |

Lee CY, Buranen SL, Ye Z-H (1991) Construction of single-copy integration vectors for Staphylococcus aureus. Gene 103: 101–105 | Article | PubMed | ChemPort |

Lutkenhaus J (1993) FtsZ ring in bacterial cytokinesis. Mol Microbiol 9: 403–409 | Article | PubMed | ISI | ChemPort |

Marraffini LA, DeDent AC, Schneewind O (2006) Sortases and the art of anchoring proteins to the envelopes of Gram-positive bacteria. Microbiol Mol Biol Rev 70: 192–221 | Article | PubMed | ChemPort |

Matsuhashi M, Dietrich CP, Strominger JL (1965) Incorporation of glycine into the cell wall glycopeptide in Staphylococcus aureus: Role of sRNA and lipid intermediates. Proc Natl Acad Sci USA 54: 587–594 | Article | PubMed | ChemPort |

Mazmanian SK, Liu G, Ton-That H, Schneewind O (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285: 760–763 | Article | PubMed | ISI | ChemPort |

Mazmanian SK, Ton-That H, Schneewind O (2001) Sortase-catalyzed anchoring of surface proteins to the cell wall of Staphylococcus aureus. Mol Microbiol 40: 1049–1057 | Article | PubMed | ISI | ChemPort |

Munoz E, Ghuysen J-M, Heymann H (1967) Cell walls of Streptococcus pyogenes type 14. C polysaccharide-peptidoglycan and G polysaccharide–peptidoglycan complexes. Biochemistry 6: 3659–3670 | Article | PubMed | ISI | ChemPort |

Navarre WW, Schneewind O (1994) Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in Gram-positive bacteria. Mol Microbiol 14: 115–121 | Article | PubMed | ChemPort |

Navarre WW, Schneewind O (1999) Surface proteins of Gram-positive bacteria and the mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev 63: 174–229 | PubMed | ISI | ChemPort |

Navarre WW, Ton-That H, Faull KF, Schneewind O (1998) Anchor structure of staphylococcal surface proteins. II. COOH-terminal structure of muramidase and amidase-solubilized surface protein. J Biol Chem 273: 29135–29142 | Article | PubMed | ChemPort |

Novick RP (2003) Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol 48: 1429–1449 | Article | PubMed | ISI | ChemPort |

Oshida T, Sugai M, Komatsuzawa H, Hong YM, Suginaka H, Tomasz A (1995) A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc Natl Acad Sci USA 92: 285–289 | Article | PubMed | ChemPort |

Perry AM, Ton-That H, Mazmanian SK, Schneewind O (2002) Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring. J Biol Chem 277: 16241–16248 | Article | PubMed | ChemPort |

Pinho MG, Errington J (2003) Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery. Mol Microbiol 50: 871–881 | Article | PubMed | ISI | ChemPort |

Rosenstein R, Götz F (2000) Staphylococcal lipases: biochemical and molecular characterization. Biochimie 82: 1005–1014 | Article | PubMed | ChemPort |

Salton MRJ (1952) Cell wall of Micrococcus lysodeikticus as the substrate of lysozyme. Nature 170: 746–747 | Article | PubMed | ISI | ChemPort |

Sanderson AR, Strominger JL, Nathenson SG (1962) Chemical structure of teichoic acid from Staphylococcus aureus, strain Copenhagen. J Biol Chem 237: 3603–3613 | PubMed | ISI | ChemPort |

Schneewind O, Fowler A, Faull KF (1995) Structure of the cell wall anchor of surface proteins in Staphylococcus aureus. Science 268: 103–106 | Article | PubMed | ISI | ChemPort |

Schneewind O, Mihaylova-Petkov D, Model P (1993) Cell wall sorting signals in surface protein of Gram-positive bacteria. EMBO J 12: 4803–4811 | PubMed | ISI | ChemPort |

Schneewind O, Model P, Fischetti VA (1992) Sorting of protein A to the staphylococcal cell wall. Cell 70: 267–281 | Article | PubMed | ISI | ChemPort |

Schuhardt VT, Huber TW, Pope LM (1969) Electron microscopy and viability of lysostaphin-induced staphylococcal spheroplasts, protoplast-like bodies, and protoplasts. J Bacteriol 97: 396–401 | PubMed | ChemPort |

Stranger-Jones YK, Bae T, Schneewind O (2006) Vaccine assembly from surface proteins of Staphylococcus aureus. Proc Natl Acad Sci USA 103: 16942–16947 | Article | PubMed | ChemPort |

Strominger JL (1968) Penicillin-sensitive enzymatic reactions in bacterial cell wall synthesis. Harvey Lect 64: 179–213 | PubMed | ChemPort |

Swanson J, McCarty M (1969) Electron microscopic studies on opaque colony variants of group A streptococci. J Bacteriol 100: 505–511 | PubMed | ChemPort |

Takamatsu D, Bensing BA, Sullam PM (2004) Four proteins encoded in the gspB-secY2A2 operon of Streptococcus gordonii mediate the intracellular glycosylation of the platelet binding protein GspB. J Bacteriol 186: 7100–7111 | Article | PubMed | ChemPort |

Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy RT, Haft DH, Dodson RJ, Durkin AS, Gwinn M, Kolonay JF, Nelson WC, Peterson JD, Umayam LA, White O, Salzberg SL, Lewis MR, Radune D et al (2001) Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293: 498–506 | Article | PubMed | ISI | ChemPort |

Ton-That H, Labischinski H, Berger-Bächi B, Schneewind O (1998) Anchor structure of staphylococcal surface proteins. III. Role of the FemA, FemB, and FemX factors in anchoring surface proteins to the bacterial cell wall. J Biol Chem 273: 29143–29149 | Article | PubMed | ChemPort |

Ton-That H, Mazmanian H, Faull KF, Schneewind O (2000) Anchoring of surface proteins to the cell wall of Staphylococcus aureus. I. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH₂-Gly₃ substrates. J Biol Chem 275: 9876–9881 | Article | PubMed | ChemPort |

Ton-That H, Schneewind O (1999) Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction. J Biol Chem 274: 24316–24320 | Article | PubMed | ChemPort |

Touhami A, Jericho MH, Beveridge TJ (2004) Atomic force microscopy of cell growth and division in Staphylococcus aureus. J Bacteriol 186: 3286–3295 | Article | PubMed | ChemPort |

Tzagoloff H, Novick R (1977) Geometry of cell division in Staphylococcus aureus. J Bacteriol 129: 343–350 | PubMed | ISI | ChemPort |

von Heijne G (1992) Membrane protein structure prediction: hydrophobic analysis and the positive inside rule. J Mol Biol 225: 487–494 | Article | PubMed | ChemPort |

Walsh CT (1993) Vancomycin resistance: decoding the molecular logic. Science 261: 308–309 | Article | PubMed | ChemPort |

Yamada S, Sugai M, Komatsuzawa H, Nakashima S, Oshida T, Matsumoto A, Suginaka H (1996) An autolysin ring associated with cell separation of Staphylococcus aureus. J Bacteriol 178: 1565–1571 | PubMed | ISI | ChemPort |

Main navigation

• Search EMBO Journal

  Search

• Advanced search

• The EMBO Journal

  • Home
  • Aims and scope
  • Current issue
  • Advance online publication
  • Web focuses
  • Archive:
  • Browse by issue
  • Browse by subject
  • Browse by category
  • Free online sample issue
  • Press releases

• Authors and Referees

  • Editorial process
  • Guide for authors
  • Submit an article
  • Guide for referees
  • Editors and Editorial Board
  • Contact editorial office

• Customer Service

  • Subscribe
  • Order sample copy
  • Purchase articles
  • Reprints and permissions
  • Contact NPG
  • Advertising