INTRODUCTION

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The cell wall of gram-positive bacteria is host to a wide variety of molecules and serves a multitude of functions, most of which are critical to the viability of the cell. Although the primary function of the cell wall is to provide a rigid exoskeleton for protection against both mechanical and osmotic lysis (694, 695) the cell wall of gram-positive bacteria also serves as an attachment site for proteins that interact with the bacterial environment. Over the past decade, it has become apparent that the gram-positive bacteria have evolved a number of unique mechanisms by which they can immobilize proteins on their surface. These mechanisms involve either the covalent attachment of protein to the peptidoglycan or the noncovalent binding of protein to either the peptidoglycan or secondary wall polymers such as teichoic acids.

This review describes our current knowledge about surface proteins of gram-positive bacteria and the mechanisms of their anchoring to the cell wall. Functions performed by the various wall proteins are incredibly diverse. For example, many covalently linked surface proteins of gram-positive pathogens are thought to be important for survival within an infected host (713). Other wall-targeted proteins are responsible for the controlled synthesis and turnover of the peptidoglycan at specific sites (division septa) during cell growth and division (348). It is believed that these enzymes are targeted to the division sites through a noncovalent interaction with specifically localized septal receptors. Still other surface proteins of gram-positive bacteria, including the internalin B molecule of Listeria spp., lysostaphin, and S-layer proteins, are immobilized to the cell surface by binding to secondary wall polymers present throughout the cell wall.

To facilitate this discussion of the mechanisms of protein attachment, we briefly discuss the mechanisms of protein secretion in these bacteria. We also summarize what is known about the structure, assembly, and turnover of the cell wall of gram-positive bacteria. For more detailed treatises on these subjects, we refer the reader to other excellent reviews (252, 707).

Gram-positive bacteria are simple cells. On the basis of morphological criteria three distinct cellular compartments can be distinguished: the cytosol, a single cytoplasmic membrane, and the surrounding cell wall (261). Some gram-positive bacteria synthesize a large polysaccharide capsule, whereas others elaborate a crystalline layer of surface proteins (739); both structures may envelope the entire cell. Spore-forming gram-positive bacteria, such as Bacillus subtilis, generate morphologically distinct daughter cells by a developmental program of asymmetric cell division (501). The cell wall of spores differs from that of mother cells and contains specific sets of proteins (645, 852). Some gram-positive bacteria divide without separating their cell walls and thus continue to grow as strings of cells (streptococci) or as clusters (staphylococci). Figure 1 is a transmission electron micrograph of thin-sectioned Staphylococcus aureus cells, revealing the characteristic morphology of the subcellular compartments of gram-positive bacteria.

View larger version (156K): [in this window] [in a new window]   FIG. 1.   Transmission electron micrograph of S. aureus cells undergoing cell division. Completed division septa are indicated by a single arrowhead, and new division sites are marked by double arrowheads. Newly formed cell wall (W2) is distinguished from preexisting cell wall (W1). Also visible are the bacterial chromosome (Chr) and cytoplasmic membranous bodies (M). This electron micrograph was generated by P. Giesbrecht. Reprinted from reference 260 with permission of the publisher.

The cell wall of gram-positive bacteria is a peptidoglycan macromolecule with attached accessory molecules such as teichoic acids, teichuronic acids, polyphosphates, or carbohydrates (302, 694). The glycan strands of the cell wall consist of the repeating disaccharide N-acetylmuramic acid-(1-4)-N-acetylglucosamine (MurNAc-GlcNAc) (254, 255). Glycan strands vary in length and are estimated to contain 5 to 30 subunits, depending on the bacterial species investigated (270, 331, 749). In most cases, the D-lactyl moiety of each MurNAc is amide linked to the short peptide component of peptidoglycan (256, 564, 792). Wall peptides are cross-linked with other peptides that are attached to a neighboring glycan strand (787-789, 791), thereby generating a three-dimensional molecular network that surrounds the cell and provides the desired exoskeletal function (463, 760) (Fig. 2).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Diagram of peptidoglycan structures from S. aureus, S. pyogenes, and L. monocytogenes. Glycan chains composed of a repeating disaccharide, GlcNAc and MurNAc, are linked to short wall peptides through the lactyl moeity of MurNAc. Adjacent wall peptides can be linked through crossbridge peptides (pentaglycine in S. aureus or dialanine in S. pyogenes). In some cases, such as L. monocytogenes, adjacent wall peptides are not cross-linked via peptide crossbridges. Instead, wall peptides are linked via an amide bond between the -amino group of a meso-diaminopimelic acid (m-Dpm) residue at position 3 of one wall peptide and the carboxy terminus of the D-alanyl residue at position 4 of the adjacent wall peptide.

Cell wall synthesis in gram-positive bacteria can be divided into three separate stages that occur in distinct subcellular compartments, the cytoplasm, the membrane, and, finally, the cell wall itself (759, 761) (Fig. 3). Although we discuss here peptidoglycan synthesis in S. aureus, all bacteria use a nearly identical synthesis pathway. Peptidoglycan synthesis begins by generating UDP-MurNAc from UDP-GlcNAc and phosphoenolpyruvate (287, 758). Five amino acids are linked to UDP-MurNAc in four consecutive steps, L-Ala, D-isoGlu, L-Lys, and, finally, the D-Ala-D-Ala dipeptide (370, 554, 572). Synthesis of D-Ala-D-Ala requires two enzymes. The first, D-Ala racemase, converts L-Ala to D-Ala, while the second, D-Ala ligase, creates a peptide bond between two D-Ala residues (113, 578, 823). Synthesis of each peptide (amide) bond within the wall peptide consumes 1 high-energy phosphate (ATP) (812). The end product of the cytoplasmic segment of the cell wall synthesis pathway, UDP-MurNAc-L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala (UDP-MurNAc-pentapeptide, Park's nucleotide), has been solubilized by acid extraction of gram-positive cells and purified (616, 617). Radiolabeled Park's nucleotide was used as substrate for the in vitro synthesis of peptidoglycan, which provided a powerful assay for the elucidation of this biochemical pathway (116). UDP-MurNAc-pentapeptide is phosphodiester linked to an undecaprenyl-pyrophosphate carrier molecule at the expense of UDP (C₅₅-PP-MurNAc-L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala, or lipid I) (13, 371). UDP-GlcNAc is linked to the muramoyl moiety to generate the disaccharide lipid II precursor [C₅₅-PP-MurNAc(-L-Ala-D-isoGlu-L-Lys(Gly₅)-D-Ala-D-Ala)-1-4-GlcNAc] (335-337). Lipid II is further modified by the addition of amino acids to the -amino of lysine (12, 417). Figure 3 shows these reactions for the biosynthesis of staphylococcal peptidoglycans, which contain five glycine residues linked to L-Lys. Three glycyl-tRNA species are thought to be dedicated to this biosynthetic pathway (281, 672, 673, 755). Finally, the modified lipid II precursor is translocated across the cytoplasmic membrane and serves as the substrate for the assembly of peptidoglycan (Fig. 3).

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Diagram of the biosynthetic pathway of cell wall assembly. As discussed in the text, the generation of cell wall precursors begins in the cytoplasm, resulting in the synthesis of UDP-MurNAc-L-Ala-D-iGlu-L-Lys-D-Ala-D-Ala (Park's nucleotide). The peptidoglycan precursor subunit is transferred to a lipid carrier in the membrane to generate lipid I. After further modification, the lipid-anchored peptidoglycan precursor is translocated to the extracellular face of the cytoplasmic membrane. The peptidoglycan precursor is subsequently incorporated into the cell wall by transpeptidation and transglycosylation reactions with the concomitant displacement of the lipid carrier. The terminal D-alanine of the wall pentapeptide is subject to substitution by cross-linking to other wall subunits or can be removed by the action of a D-alanyl-D-alanine carboxypeptidase. PEP, phosphoenolpyruvate; MN-GN, MurNAc-GlcNAc.

Cell wall assembly is catalyzed by penicillin binding proteins (PBPs) (251). The high-molecular-weight PBPs are bifunctional enzymes that have recently been categorized into one of two classes based on sequence similarity (251, 272). Class A PBPs promote both the polymerization of glycan from its disaccharide precursor, i.e., the successive addition of MurNAc(-L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala)-GlcNAc to C₅₅-PP-MurNAc(-L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala)-GlcNAc, and the transpeptidation (cross-linking) of wall peptides (570). The latter reaction results in the proteolytic removal of the D-Ala at the C-terminal end of the pentapeptide and the formation of a new amide bond between the amino group of the crossbridge and the carbonyl group of D-Ala at position 4 (791). The transpeptidation reaction of staphylococcal peptidoglycan is depicted in Fig. 3. This reaction is the target of penicillin and other -lactam antibiotics which mimic the structure of D-alanyl-D-alanine (791). After cleavage, the -lactam ring continues to occupy the active site serine residue of PBPs, thereby inhibiting PBPs (871, 872). Less is known about the class B PBPs; however, they are speculated to be involved in morphogenetic networks (272).

Not all cell wall peptides are cross-linked to their neighboring peptidoglycan strands. Unsubstituted (free) peptides may be preserved by trimming the terminal D-Ala off the pentapeptides (839). This reaction is catalyzed by other, low-molecular-weight PBPs that function as carboxypeptidases (372, 805). The reactions carried out by transpeptidases and carboxypeptidases are similar in nature. Transpeptidases use an amino group as a nucleophile to resolve the acyl-enzyme intermediate between the active-site hydroxyl of serine and the carbonyl of D-alanine, whereas carboxypeptidases use water as a nucleophile (251). Consequently, there are sequence and structural similarities between these enzymes (251). The ratio between transpeptidation and carboxypeptidation is thought to be important in generating a three-dimensional structure of the cell wall (463). For example, different degrees of cross-linking could allow wall synthesis at distinct angles and the generation of curves in otherwise cylindrical cells. However, this assumption has never been tested for the distinctly shaped gram-positive bacteria. Peptidoglycan with a low degree of cross-linking is much more sensitive to degradation by cell wall hydrolases (760). It is conceivable that the degree of cross-linking plays a role in specifying sites on the cell wall that are more prone to either degradation or additional synthesis.

While the repeating disaccharide [MurNAc-(1-4)-GlcNAc] is found in all bacterial peptidoglycans, the wall peptides differ in composition between bacterial species (710). Some organisms, such as Listeria, replace L-Lys with another diamino acid, in this case m-diaminopimelic acid (710). Others add amino acids to the side chain -amino of L-Lys to synthesize extended peptidoglycan cross-bridges (710). Figure 2 provides an example for the peptidoglycan structures of three different gram-positive bacteria. The wall peptides can either be cross-linked with those of another glycan strand such that the -amino of L-Lys or any other amino acid added at this position will be amide linked to the carbonyl of D-Ala in the wall peptide or be trimmed to generate an un-cross-linked wall tetrapeptide (710).

The cell wall of many gram-positive bacteria is modified by O-acetylation of MurNAc at C-6 (790). Although the enzymatic mechanism for this decoration remains unknown, it does confer resistance to cell wall degradation by animal lysozymes (97). Other chemical modifications include additions of teichoic acids, phosphorylation, and attachment of carbohydrates (see below). Recent advances in cell wall structure have been made by analyzing peptidoglycan breakdown products by reverse-phase high-pressure liquid chromatography combined with mass spectrometric analysis (144, 244, 269, 801). These techniques allow the characterization of different degrees of cross-linking as well as the identification of new cross-linking reactions within the cell wall (145).

Gram-positive bacteria synthesize several compounds that decorate their peptidoglycan exoskeleton (694). Based on structural differences, one can distinguish teichoic acids, teichuronic acids, lipoteichoic acids, lipoglycans, and polysaccharide modifications (694). Over the past 30 years, the complete structures of some of these compounds as well as the genes required for their synthesis have been characterized. This research has been reviewed in detail elsewhere, and we provide here a brief summary of these elements, since they may be important for protein targeting mechanisms (17, 205, 643).

All gram-positive bacteria are thought to synthesize anionic polymers that are covalently attached to the peptidoglycan or tethered to a lipid anchor moiety (18, 23). Typically these polymers consists of polyglycerol phosphate (Gro-P), poly-ribitol phosphate (Rit-P), or poly-glucosyl phosphate (Glc-P) all of which may be glucosylated and/or amino acid esterified (205, 643). Figure 4 compares the structure of staphylococcal cell wall teichoic acid with that of two other bacteria. A polymer of 30 to 50 Rit-P subunits is phosphodiester linked to 2 or 3 Gro-P residues which are linked to the disaccharide ManNac(1-4)GlcNAc (16, 183, 184, 700, 701). The GlcNAc moiety is (1-6) phosphodiester linked to MurNAc within the peptidoglycan repeating disaccharide (MurNAc-GluNac) (128, 318, 438). The (Gro-P)_n-ManNac-GlcNAc tether between wall teichoic acids and the peptidoglycan is referred to as the linkage unit (15). Although many gram-positive bacteria are known to synthesize identical linkage units, some species modify its structure with other carbohydrates whereas a few others use entirely different compounds to tether their wall teichoic acids (15).

View larger version (38K): [in this window] [in a new window]   FIG. 4.   Structure of cell wall teichoic acids from S. aureus, B. subtilis, and L. monocytogenes. Wall teichoic acids are anionic polymers of glycerol-phosphate, ribitol-phosphate, or glucosyl-phosphate and are tethered to the peptidoglycan. The cell wall linkage unit consists of ManNac(1-4)GlcNAc, which is 1-6 1-6phosphodiester linked to MurNAc within the glycan strands of the cell wall envelope. One to three glycerol-phosphate moieties serve as a bridge between the linkage unit and the anionic polymer, which is composed of 15 to 60 subunits. R indicates covalent modification of the hydroxyl side chains of either glycerol-phosphate or ribitol-phosphate and can be either GlcNAc or esterified D-alanine.

Modifications of the repeating subunits differ from one bacterial species to another (17). Both Gro-P and Rit-P repeating units can be decorated with a variety of different sugars and can also be esterified with D-alanine (78-80). Synthesis of the backbone structure is thought to occur in the bacterial cytoplasm (78, 643). Synthesis begins by fusing UDP-GlcNAc to prenol-phosphate (862, 863) (Fig. 5). The resulting GlcNAc-PP-prenol is then mannosylated with UDP-ManNac to yield ManNac-GlcNAc-PP-prenol (873). The lipid-anchored cell wall linkage unit serves as an attachment site for the addition of two or three Gro-P units and is extended by Rib-P, which is added from CDP-ribitol substrates (267, 268, 521, 642). The end product of this synthetic pathway is presumably translocated across the cytoplasmic membrane via an ATP binding cassette transporter system (472). Attachment of wall teichoic acid to MurNAc proceeds during cell wall assembly; however, the enzymatic machinery required for this process is still unknown.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Diagram of the biosynthesis of staphylococcal cell wall teichoic acid. Synthesis begins in the cytoplasm via the addition of UDP-GlcNAc to an undecaprenol carrier molecule (P-C55). UDP-ManNac and 2,3-CDP-glycerol are added to complete the cell wall linkage unit, which is then extended by the polymerization of ribitol-phosphate. This precursor molecule is thought to be translocated across the cytoplasmic membrane and linked to MurNAc within the glycan chains.

Esterification of Gro-P or Rit-P with D-Ala occurs on the surface of the cytoplasmic membrane, i.e., after teichoic acids have been translocated across the membrane (580). Four gene products are required for this process, during which D-Ala is first linked to a diacyl carrier protein and then linked to an undecaprenol phosphate lipid carrier (139, 316, 317, 580, 592, 641). Lipid-linked D-Ala is translocated across the cytoplasmic membrane and serves as a substrate for esterification. This reaction is presumably used for the D-Ala ester modification of cell wall teichoic acids and lipoteichoic acids (579). Cell wall teichoic acids are thought to be uniformly distributed over the entire peptidoglycan exoskeleton (806). Some teichoic acid decorations, for example the esterified D-Ala, are unstable, and S. aureus continuously reesterifies D-Ala residues (431, 433).

Although the structures of cell wall teichoic acids are largely known and some of the genes involved in their synthesis appear to be essential for the growth of gram-positive bacteria, the physiological role of these molecules is still not completely understood (642). It is conceivable that the negatively charged teichoic acids function to capture divalent cations or provide a biophysical barrier to prevent the diffusion of substances (205, 207). However, these claims have been largely speculative and experiments that directly prove or disprove them are difficult to design. Cell wall teichoic acids appear to be the binding sites for some enzymes that cleave the bacterial peptidoglycan (333). For example, the LytA amidase of S. pneumoniae binds to the choline moiety of the cell wall teichoic acids of this organism (351, 352). Conceivably, the affinity for teichoic acids directs murein hydrolases to the cell walls of specific species (discussed below). Thus, teichoic acids may serve as species-specific decorations which allow gram-positive bacteria to synthesize an envelope structure that is chemically distinct from the envelope other organisms that display an otherwise identical peptidoglycan exoskeleton.

Lipoteichoic acids are polyanionic polymers inserted in the outer leaflet of the cytoplasmic membrane via a lipid moiety (205). The polymer extends through the cell wall peptidoglycan onto the surface of gram-positive cells. The precise function of lipoteichoic acids is unknown (205). The cell wall teichoic acids and lipoteichoic acids possess different chemical structures in all gram-positive bacteria except Streptococcus pneumoniae. For example, S. aureus synthesizes poly-Rit-P cell wall teichoic acids, which are modified at the 2' and 4' hydroxyl with GlcNAc and D-Ala (701). Staphylococcal lipoteichoic acids are composed of poly-Gro-P, which can be modified at the 2' hydroxyl of Gro-P with either GlcNAc or esterified D-Ala (165, 208-210). The lipoteichoic acid moiety of S. aureus is attached to Glc(1-4)Glc-(1-3)diacylglycerol (433).

Lipoteichoic acids of other bacteria have different repeating subunits and/or different lipid anchors. Figure 6 compares the structure of staphylococcal lipoteichoic acid with that from S. pneumoniae (205). Lipoglycans are related structures that can be distinguished from lipoteichoic acids by the absence of phosphate from the repeating subunits (205). Synthesis of the lipoteichoic acids is thought to occur on the surface of the cytoplasmic membrane. Thus, the substrates of each lipoteichoic acid constituent must be translocated across the cytoplasmic membrane prior to assembly (205). Indeed, each of the precursor molecules is tethered to a lipophilic moiety: either glycolipid, phosphatidylglycerol, hexosyl-1-phosphoundecaprenol, or undecaprenyl-D-Ala (106, 241) (Fig. 7).

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Structure of lipoteichoic acid from S. aureus and S. pneumoniae. Staphylococcal lipoteichoic acid is composed of polymerized glycerol-phosphate and is linked to a membrane anchor moiety [Glc(1-4)Glc(1-3)diacylglycerol]. The fatty acid side chains of diacylglycerol are indicated by R. The hydroxyl side chains of polymerized glycerolphosphate are modified with GlcNAc or esterified D-alanine. In S. pneumoniae, the repeat unit of lipoteichoic acid, [Glc(1-3)AATGal(14)GalNac(6-Cho-P)(1-3)GalNac(6-Cho-P)(1-1)ribitol-P], is identical to that of cell wall teichoic acids. The choline-modified repeat unit serves as a targeting receptor for several murein hydrolases and surface protein. Modified from reference 205 with permission of the publisher.

View larger version (20K): [in this window] [in a new window]   FIG. 7.   Diagram of the biosynthesis of staphylococcal lipoteichoic acid (LTA). Glycerol-phosphate is acylated with two fatty acids to yield phosphatidic acid. This compound, as well as all other biosynthetic components of lipoteichoic acid, are thought to be located in the outer leaftlet of the cytoplasmic membrane, the presumed site of lipoteichoic acid synthesis. The glycolipid anchor, Glc (1-4)Glc(1-3)diacylglycerol, is linked to the first glycerol-phosphate moiety and then further extended by polymerization with phosphatidyl-glycerol as a substrate. Modified from reference 205 with permission of the publisher.

Lipoteichoic acid synthesis begins in the cytoplasm with the synthesis of phosphatidic acid from glycerol, ATP, and two acyl side chains (Fig. 7). Phosphatidic acid is converted to phosphatidyl-glycerophosphate and then to phosphatidylglycerol, which serves as a substrate for the polymerization of Gro-P (266). This synthetic scheme requires large amounts of diacylglycerol, which are recycled to phospatidic acid or used for the biosynthesis of other membrane lipids (433). Glycosylation of lipoteichoic acid requires hexose linked to undecaprenyl, which is presumably synthesized in the cytoplasm and translocated across the membrane (206, 432, 482). It is conceivable that lipoteichoic acids, similarly to the cell wall teichoic acids of S. pneumoniae, also serve as species-specific decorations of the peptidoglycan exoskeleton.

Protein secretion has been studied extensively in Escherichia coli, and the paradigms established through this work are thought to be true for all bacterial and eukaryotic cells (171, 640, 654). Proteins destined for translocation across the cytoplasmic membrane are marked by a signal (leader) peptide (65) that is generally composed of a core of 15 to 20 hydrophobic residues flanked at the N-terminal end by positively charged residues (182, 728). For secreted proteins, signal peptides are proteolytically removed by signal (leader) peptidases upon translocation across the cytoplasmic membrane (136, 153). Signal peptides are necessary and sufficient for protein translocation across membranes if the fused polypeptide substrate can be maintained in an export competent state, a function that can be achieved by one of two separate pathways (142, 808). In one pathway, a signal recognition particle (SRP) can bind to the signal peptides of nascent chains and temporally arrest their ribosomal translation (824-826). The SRP of E. coli is thought to be a ribonucleoprotein particle consisting of Ffh (also known as P48) and 4.5S RNA (46, 648, 678, 762). Translation resumes after the SRP-ribosome complex docks onto its membrane receptor (FtsY), thereby delivering the nascent polypeptide to the Sec translocation channel (551, 808). Alternatively, signal peptide-bearing precursors may be translocated after their synthesis has been completed, i.e., by a posttranslational translocation process. Binding of a secretion chaperone, SecB, can maintain these precursors in an unfolded, translocation-competent state (452, 661). The SecB protein binds to the mature part of signal peptide-bearing precursors but may also interact with the SecA protein, a component of the secretion machinery itself (90, 151, 341, 662). Once initiated into the secretory pathway by the signal peptide, the SecB chaperone dissociates from the precursor, allowing the translocation of the full-length polypeptide across the membrane (197). The pathways converge at the translocation channel, and the choice of pathway that is used may be a function of the hydrophobicity of the signal peptide (807, 808). Recent observations suggest that the SRP pathway may target proteins destined for the inner membrane to the Sec translocase whereas the SecB pathway seems to be used by proteins that are secreted across the membrane (721, 804).

The gene products necessary for the initiation of leader peptide-containing precursors into the secretory pathway were identified as mutants that restored the -galactosidase activity of LacZ fusions to the C terminus of signal peptide bearing proteins (600). These LacZ fusion proteins are substrates for export but arrest at an undefined step of the secretion pathway and block the export of other precursors. The results of the sec screens generally matched those of a suppressor screen (prl, for "protein localization") that scored for the export of a mutant polypeptide harboring a defective signal peptide (181). The functionality of the Sec proteins in membrane translocation of precursor proteins has been elegantly demonstrated in vitro (309, 563). SecYEG is the preprotein translocase channel and requires the SecA ATPase to push polypeptides through a hydrophilic channel (172, 174, 179, 811). The SecDF and YajC proteins function at a later step, perhaps in regulating the activity of SecA (173).

Homologs of SecA, SecD, SecE, SecF, SecY, and YajC have been identified in the sequenced genome of the gram-positive organism B. subtilis; however, no homologs of SecB and SecG were found (454). It is not clear whether SecB and SecG are dispensable for secretion in B. subtilis or whether they have been replaced with other polypeptides that do not display homology to the E. coli gene products. Ffh, 4.5S RNA, and FtsY are conserved between E. coli and B. subtilis (102, 456, 774). Another striking difference between E. coli and B. subtilis is the number of signal peptidases. The gram-positive organism appears to encode seven signal peptidases, whereas E. coli contains only two. Two of the Bacillus genes specify class 2 signal peptidases for the maturation of lipoproteins, whereas the other five signal peptidases are similar to the E. coli lepB gene. Signal peptides of gram-positive bacteria differ from those of their gram-negative counterparts by usually being longer and more hydrophobic and possessing more charge in their N-terminal ends (818, 819). Although these signal peptides of gram-positive organisms generally function in gram-negative bacteria, the same is not always true when signal peptides of gram-negative bacteria are expressed in gram-positive organisms (716). Could it be that gram-positive bacteria require additional components to recognize signal peptides? This has been tested biochemically for both B. subtilis and S. aureus by purifying membranes with bound ribosomes and comparing their protein content with the content of membranes those that did not contain ribosomes (2, 3). Although several different polypeptides could be identified as a secretory (S) complex, analysis of the cloned sequences revealed pyruvate dehydrogenase, an enzyme of intermediary metabolism that is presumably not directly involved in protein secretion (329, 330).

The existence of a periplasmic space in gram-positive bacteria that is analogous to the one found in gram-negative bacteria has been a matter of speculation for many years (276). Morphological inspection has at times revealed a narrow space between the cytoplasmic membrane and the cell wall peptidoglycan (276). Visualization of this space depended on the mode of sample preparation (275). Others have emphasized an operational definition for the periplasm of gram-positive bacteria as a subcellular compartment (544); i.e., it should contain a subset of secreted proteins that are specifically targeted to this compartment (644).

Several proteins that are periplasmic in gram-negative bacteria were observed to be lipid modified in gram-positive bacteria (264). These lipoproteins function to capture specific import substrates, such as carbohydrates, and deliver them to transport machinery embedded within the cytoplasmic membrane. The homologous proteins in gram-negative cells are generally soluble in a periplasmic space that is bounded by both the bacterial inner and outer membranes. Hence, the lipoyl moiety might be a targeting device to retain polypeptides on the membrane surface of gram-positive bacteria (264, 769, 770). For example, -lactamase (Bla) is a soluble periplasmic enzyme that confers resistance to -lactam antibiotics in gram-negative bacteria (444) while the -lactamases of S. aureus and B. licheniformis are lipoproteins (584, 585). -Lactamase precursors in gram-positive bacteria appear to be substrates for both type I and type II signal peptidase, resulting in mixed populations of secreted, soluble Bla as well as glyceride-modified, membrane-anchored Bla (573). Mutant Bla exported solely by a type I signal peptide is secreted into the extracellular medium but fails to protect staphylococci from -lactam antibiotics (573). Thus, tethering of such enzymes to the cytoplasmic membranes is an important factor in both lowering the local concentration of inhibitors such as penicillin and raising the local concentration of necessary nutritional resources.

The high incidence of streptococcal and staphylococcal human diseases at the beginning of this century sparked the interest of many medical investigators in these microbes. Initial efforts concentrated on the characterization of the predominant antigens during infection by gram-positive bacteria with the rationale of developing protective vaccines (285, 466). Although vaccines for Streptococcus pyogenes and Staphylococcus aureus are still not commercially available, this research rapidly identified and characterized protein and carbohydrate structures on bacterial surfaces. Lancefield and colleagues classified streptococci on the basis of carbohydrate antigens. The group A streptococcus (GAS), S. pyogenes, is the causative agent of pharyngitis, purulent skin lesions, and postinfectious sequelae. GAS strains display M protein on their surface and can be typed with antisera raised against the N-terminal portion of this -helical coiled-coil molecule (467). More than 100 types are known, and it was recognized early that these type-specific antibodies conferred immunity to infection of strains carrying one specific M type by promoting the phagocytic killing of GAS (461, 467). Hence, much effort was directed at characterizing the M protein molecule as an antiphagocytic antigen (212).

In 1958, Jensen observed that several S. aureus strains could precipitate immunoglobulins (Ig) from both human and preimmune animal serum in gel precipitation assays (380). The nonimmune precipitation of antibodies is due to the binding of protein A on the staphylococcal surface to the Fc portion of Ig (222). However, antibodies directed specifically at protein A are not protective during animal infections, and strains deficient in protein A do not display a significant reduction in their pathogenic potential in an animal experimental system (403). Although we do not know the precise role of protein A during S. aureus infections, this molecule has served as a model system for immunological, structural, and microbiological studies (588).

It is widely assumed that surface proteins of Gram-positive bacteria might interact with eukaryotic proteins as a means of establishing residence at unique locations or evading the immune system. Although these assumptions have not always been rigorously tested, they have increased research interest in this field. The advent of molecular cloning techniques yielded a rapid accumulation of DNA sequence and allowed structural comparison of surface proteins. The current completion of several microbial genome sequences will soon provide us with a comprehensive view of all sequences. Therefore, we assume that future work will concentrate on the physiological and biochemical characterization of surface proteins in gram-positive bacteria.

Initial experiments to characterize surface proteins focused on solubilizing and purifying streptococcal M protein and staphylococcal protein A from the bacteria. Lancefield used acid extraction of streptococci to release M proteins from the cell surface (467). This treatment caused partial hydrolysis of polypeptides but not of the bacterial cell wall, and released peptide fragments were recovered in the supernatant after centrifugation of acid extracts. Treatment with detergents, bases, or proteases or by boiling also released some M protein; however, all of these preparations yielded either only peptide fragments or minute amounts of material, indicating that the solubilization technique was insufficient (212). This problem was overcome by the use of cell wall-hydrolytic enzymes, which enabled the efficient solubilization of full-length surface proteins (212, 340). The biochemical analysis of protein A was facilitated by its ability to be purified by affinity chromatography on matrices containing linked immunoglobulins after its enzymatic solubilization from the staphylococcal cell wall (340). When purified S. aureus cell walls were digested with lysostaphin, a bacteriolytic enzyme secreted by Staphylococcus simulans bv. staphylolyticus, protein A was released as a homogeneous population (737). Egg white lysozyme, an N-acetylmuramidase, does not effectively degrade the staphylococcal cell wall. Nevertheless, Sjöquist et al. were able to purify small amounts of lysozyme-released protein A and to show that it migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than did the lysostaphin-released counterpart, suggesting an increase in molecular mass (738). Acid hydrolysis of the lysozyme-released species yielded the amino sugars GlcNAc and MurNAc, which were not observed for lysostaphin-released protein A (738). On the basis of these observations, Sjöquist et al. concluded that protein A must be linked to the staphylococcal cell wall (738).

Cloning and sequencing of the emm (streptococcal M protein) and spa (staphylococcal protein A) genes revealed open reading frames that specified polypeptides harboring an N-terminal signal peptide and a C-terminal hydrophobic domain (346, 803). Because hydrophobicity is a universal signal for membrane anchoring of polypeptides (138), it seemed plausible that surface proteins could be tethered to the cytoplasmic membrane of gram-positive bacteria rather than linked to the cell wall. Comparison of several additional surface protein gene sequences allowed a more detailed analysis of their primary structures and revealed a striking C-terminal homology, namely, the presence of an LPXTG sequence motif, where X is any amino acid, followed by a C-terminal hydrophobic domain and a tail of mostly positively charged residues (216). The remarkable conservation of the LPXTG motif in surface proteins of gram-positive bacteria suggested that this element must be involved in the anchor mechanism of surface proteins in gram-positive bacteria.

To characterize the C-terminal part of M proteins, Pancholi and Fischetti digested the surface exposed portion of this molecule with trypsin (613). Streptococci were washed to remove trypsin as well as cleaved peptide fragments and subsequently digested with phage lysin amidase, which resulted in a spectrum of released M-protein fragments with increasing mass when analyzed by SDS-PAGE (613). The fastest-migrating species was further purified and characterized by Edman degradation as well as amino acid composition. The data indicated that the C-terminal end of M protein, beginning at residue 302, is uniformly protease protected (613). Amino acid analysis revealed that the C-terminal peptide did not contain phenylalanine and suggested that the phenylalanine-containing hydrophobic domain might be absent from mature M protein (612). In a subsequent study of the possible membrane anchor cleavage activity, this data was interpreted as indicating a cleavage between the proline and the serine of the LPSTG motif (612), an estimate that was close to the later-identified cleavage site between the threonine and the glycine of the LPXTG motif within protein A (see below). Phage lysin released anchor fragments do not contain amino sugars (396). This can be explained by the amidase activity of this enzyme, which removes the glycan component of the cell wall from its crosslinked peptide backbone (613). Thus, although this has never been demonstrated, the data corroborate a model in which the C-terminal end of M proteins may also be covalently linked to the peptidoglycan.

Chemical analysis of the peptidoglycan of several other bacterial species including S. pneumoniae (476), S. mutans, S. sanguis (64, 680), Lactobacillus fermenti (822), and Mycobacterium tuberculosis (853) revealed the presence of significant amounts of nonpeptidoglycan amino acids. Analysis of the cell walls of S. mutans led to the conclusion that it contained covalently attached proteins, and it was suggested that this may be a feature common to the cell walls of all gram-positive species (577, 689).

SORTING: COVALENT LINKAGE OF SURFACE PROTEINS TO THE CELL WALL

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Cell Wall Anchoring of Staphylococcal Protein A

Staphylococcal protein A has been used as a model system to study the anchoring of surface proteins in gram-positive bacteria (716). The cytoplasmic protein A precursor is exported and processed to generate the mature anchored species within 1 min of its synthesis. As described above, the anchored mature species is accessible to protease on the bacterial surface and requires enzymatic release from the staphylococcal cell wall for solubility. Lysostaphin cleaves at the pentaglycine crossbridge of the staphylococcal cell wall and solubilizes protein A as a uniform species on SDS-PAGE (716). In contrast, muramidase cleaves the glycan strands of the cell wall and muramidase-released protein A appears as a spectrum of bands on SDS-PAGE, all of which migrate more slowly than the lysostaphin-released counterpart (715). The notion that these mass differences must be the result of linked peptidoglycan was confirmed by an experiment in which muramidase-released protein A is digested with lysostaphin, which converts all material to the same uniform mobility as that observed for protein A directly solubilized by lysostaphin (715). Because of the uniform migration of lysostaphin-released fragments on SDS-PAGE, the anchoring point of surface proteins must be more proximal to the pentaglycine crossbridge, i.e., the cleavage site of lysostaphin, than to the glycan chains where muramidase cuts.

Protein A lacking a C-terminal sorting signal, i.e., the LPXTG motif, hydrophobic domain, and charged tail, is secreted into the medium and thus does not require the enzymatic digestion of the cell wall in order to be soluble when cells are boiled in SDS (716) (Fig. 8). Mutant protein A with the charged tail truncated behaves similarly. Lysostaphin-solubilized protein A migrates faster on SDS-PAGE than the secreted, unanchored species does, suggesting that the polypeptide chain is proteolytically cleaved during the anchoring process (716). To confirm that the secreted species comprised the complete (unprocessed) polypeptide chain, a protein A mutant that contains a single cysteine at the C terminus has been generated. Metabolic labeling of this cysteine revealed that the secreted protein A mutant is uncleaved and that some form of processing must be required for the anchoring of surface proteins (716).

View larger version (17K): [in this window] [in a new window]   FIG. 8.   Phenotypes of protein A cell wall sorting signal deletion mutants. Full-length protein A is found to be quantitatively anchored to the cell wall. Deletions of the charged tail, charged tail and hydrophobic domain, or the entire sorting signal result in the complete secretion of the polypeptide into the extracellular milieu. Deletion of the LPXTG motif of the C terminal abolishes the anchoring of the polypeptide to the cell wall. In the case of the protein A sorting signal, these mutant proteins are loosely associated with the cytoplasmic membrane.

Mutants with mutations within the LPXTG motif have a different phenotype. These polypeptides are not secreted into the medium but remain cell associated. Compared to the wild type, the LPXTG mutants do not reveal the characteristic size differences of lysostaphin- and muramidase-released species, indicating that these substances are not covalently linked to the cell wall (715). Furthermore, the mutant polypeptides also migrate more slowly on SDS-PAGE than their anchored counterparts do, suggesting that processing, i.e., proteolytic cleavage and cell wall linkage, is disrupted. Thus, the two different mutant phenotypes reveal that surface protein anchoring is arrested at distinct steps. The hydrophobic domain and charged tail cause protein A to be retained from the secretory pathway, whereas the LPXTG motif is needed for cleavage and linkage to the cell wall.

To determine the signal sufficient for cell wall anchoring of proteins normally secreted into the medium or lipid anchored to the cytoplasmic membrane, C-terminal protein A sequences have been fused to staphylococcal enterotoxin B (Seb), -lactamase (BlaZ), or E. coli alkaline phosphatase (PhoA) exported by the protein A signal peptide (715, 716). The C-terminal 35 residues of protein A, comprising the LPXTG motif, hydrophobic domain, and charged tail, are sufficient for cell wall anchoring of each of these fusion proteins. Homologous sequence elements of other surface proteins also function to signal cell wall anchoring (715). When fused to the C terminus of secreted reporter proteins, some of these signals function in a manner indistinguishable from that of the protein A signal. Others fail to signal anchoring but can be mutated to gain this function. In all cases examined, these mutations alter the spacing between the LPXTG motif and the charged tail. The absolute number of residues between the LPXTG motif and the charged tail does not appear to be critical. It has been proposed that the folding of the hydrophobic domain determines the correct spacing between the flanking elements required for proper recognition of the sorting signal (715).

Retention is signaled by the positively charged residues within the charged tail of the sorting signal. Although a single arginine is most effective in signaling retention, this residue can be replaced by lysine when other positively charged residues are present. Increasing the spacing between the LPXTG motif and the charged tail further does not interfere with cell wall sorting but proves to be toxic for staphylococci. The reason for this phenomenon is not known (715).

The LPXTG motif is conserved within the sorting signals of all known wall-anchored surface proteins of gram-positive bacteria (Table 1). The threonine (T) displays some variation in that either alanine or serine can be found at this position. A threonine-to-alanine substitution was tested in the sorting signal of staphylococcal protein A, and this mutation does not affect anchoring, which suggests that the identified sequences are indeed functional sorting signals. Other mutations in the LPXTG motif, for example a proline (P)-to-asparagine (N) mutation, do abolish sorting. Nevertheless, a rigorous mutagenesis of this sequence element has never been performed, and the basis for the sequence conservation is thus unknown. The simplest explanation for the conservation of the LPXTG motif may be the preservation of a proteolytic cleavage site. Sortase, the presumed enzymatic activity that cleaves at this point and catalyzes the transpeptidation reaction, may require a specific sequence or fold for substrate recognition. It is surprising that other transpeptidation mechanisms, for example that of glycosylphosphatidylinositol (GPI) anchoring, seemingly have much less stringent substrate requirements (see below).

The hydrophobic domain and charged tail together are thought to retain the polypeptide from the secretory pathway and provide an opportunity for the proteolytic cleavage of surface proteins. One way in which this might be achieved is to anchor the polypeptide in the cytoplasmic membrane. If so, fusion of the C-terminal hydrophobic domain to other secreted proteins such as PhoA or Seb should insert the hybrids in the membrane by acting as a stable transmembrane domain. This, however, is not the case, and the mutant proteins are instead found to be peripherally associated with the membrane (715). Furthermore, mutation or truncations of the charged tail, even at positions that cannot play a role in membrane insertion, lead to the secretion of the polypeptide into the surrounding medium. Perhaps the C-terminal hydrophobic domain and charged tail function to arrest translocation of the polypeptide within the secretion channel but without permitting the actual insertion into the membrane. This arrest in translocation could suffice to allow cleavage of the polypeptide and concomitant cell wall anchoring. A simple test of this hypothesis might be to engineer sorting signals with increased hydrophobicity to allow membrane anchoring. Once inserted into the membrane, the surface protein could diffuse away from the site of cell wall sorting without permitting a transpeptidation reaction at the LPXTG motif.

The proteolytic cleavage site during the anchoring of surface proteins has been determined by purifying and sequencing the C-terminal cleavage fragment (574). This has been done by using a hybrid protein in which the mature part of maltose binding protein is fused to the C-terminal end of the sorting signal. The hybrid protein is exported by its N-terminal signal peptide and cleaved, and the N-terminal portion is linked to the bacterial cell wall. In contrast, the C-terminal cleavage fragment with the fused maltose binding protein domain remains within the cytoplasm and can be purified by affinity chromatography (574). Edman degradation reveals that the cleavage site is located between the threonine and the glycine of the LPXTG motif (Fig. 9). Deletion of the LPXTG motif abrogates cleavage as well as anchoring, indicating that the two events are linked. Proteolytic cleavage at the LPXTG motif absolutely requires protein export by the Sec machinery. When cells are poisoned with sodium azide, which at low concentrations is an inhibitor of the SecA motor of preprotein translocase (223, 601), no secretion or cleavage of the fusion protein occurs. Similarly, protein A mutants harboring a defective signal peptide are not cleaved at the LPXTG motif. This result suggests that the site of proteolytic cleavage may be either in the membrane, i.e., during secretion of protein A, or on its extracytoplasmic side, which coincides with the locus for cell wall assembly.

View larger version (22K): [in this window] [in a new window]   FIG. 9.   Proposed model for the cell wall sorting reaction. The model can be divided into four distinct steps. 1, the full-length precursor is exported from the cytoplasm via an N-terminal leader peptide. 2, the protein is prevented from release into the extracellular milieu by the charged tail and hydrophobic domain. 3, the protein is cleaved between the threonyl and glycyl residues of the LPXTG motif. 4, the newly liberated carboxy terminus of threonine is transferred via an amide bond exchange to an amino group found at the end of the wall crossbridge. In this proposed model, steps 3 and 4 are coupled in a two-step reaction. Overall, the reaction bears similarity to the amide bond exchange mechanisms by which proteins are attached to GPI moieties and by which transpeptidation occurs during cell wall synthesis.

To date, the only cell wall anchor structure to be solved in molecular detail is that of staphylococcal protein A (575, 713, 796, 797). The structure was determined through the use of specifically designed hybrid proteins that enabled the purification of large amounts of surface protein after solubilization from the cell wall with muralytic enzymes. The C-terminal anchor structure was removed and isolated from the full-length hybrid surface protein by proteolysis or cyanogen bromide cleavage at sites specifically engineered into the hybrid protein. The resulting C-terminal anchor peptides allowed detailed studies of associated cell wall structures by chemical analysis, Edman degradation, and mass spectrometry.

In an early study, a maltose binding protein harboring the C-terminal sorting signal of protein A was solubilized with lysostaphin, purified, and subjected to limited proteolysis to identify C-terminal anchor peptides (713). The C-terminal threonine of the LPXTG motif was found to be amide linked to a triglycine peptide. Because the pentaglycine crossbridge of the staphylococcal cell wall is also the site of lysostaphin cleavage, it was proposed that surface proteins are amide linked to the free amino group of the wall crossbridges. Because both the LPXTG motif and the free amino group of wall crossbridges are conserved features in gram-positive bacteria, this mechanism might be universal for all organisms expressing surface proteins via a C-terminal sorting signal (574, 713).

In later studies, a hybrid protein containing the C-terminal sorting signal of protein A fused to Seb was used in similar experiments to analyze the C-terminal anchor structure of surface protein solubilized with several other muralytic enzymes (575, 796, 797). In these studies, six histidyl residues placed at the fusion junction between Seb and the sorting signal facilitated the purification of the full-length protein by chromatography on nickel resin. C-terminal anchor peptides were obtained as follows. The purified surface protein was first cleaved with cyanogen bromide at a methionyl immediately adjacent to the six histidyl. The anchor peptides were then purified by another round of affinity chromatography on nickel resin. As mentioned above, surface protein solubilized from staphylococcal peptidoglycan with muramidases migrate as a ladder of bands on SDS-PAGE (Fig. 10). Muralytic amidases, i.e., enzymes that cut at the amide linkage between the D-lactyl of MurNAc and the L-Ala of the wall peptide, also solubilize surface protein as a ladder of bands (575). Mass-spectrometric analysis of C-terminal anchor peptides from the hybrid surface protein solubilized with these enzymes confirmed that the distinct pattern observed on SDS-PAGE is due to the attachment of a variable number of linked peptidoglycan subunits (575). Amidase-solubilized surface proteins were found to be linked to one or more peptidoglycan subunits of the structure [NH₂-L-Ala-D-iGlu-L-Lys(Gly₅)-D-Ala-D-Ala], whereas the muramidase-solubilized surface proteins were linked to subunits of the structure MurNAc[-L-Ala-D-iGlu-L-Lys(Gly₅)-D-Ala-D-Ala]-GlcNAc. The attached peptidoglycan subunits were found to be linked to one another through their pentaglycine crossbridges. As would be expected, redigestion of amidase- and muramidase-solubilized surface protein with lysostaphin removed the linked peptidoglycan subunits (575). The structure of a surface protein linked to a peptidoglycan dimer is shown in Fig. 11.

View larger version (83K): [in this window] [in a new window]   FIG. 10.   SDS-PAGE of surface protein solubilized with five different muralytic enzymes. Shown is a Coomassie blue-stained SDS-PAGE gel of a hybrid surface protein containing the cell wall sorting signal of protein A after solubilization from the staphylococcal peptidoglycan with lysostaphin (L), mutanolysin (M), the staphylococcal phage 11 murein hydrolase (), the amidase portion of the major staphylococcal autolysin (A, Atl), or the lytic amidase from B. subtilis (A, CwlA). Proteins solubilized with these enzymes display distinct mobilities on SDS-PAGE due to the presence of covalently linked cell wall subunits (see the text). See Fig. 17 for a diagram of the enzymatic activities of these enzymes. A species of 28 kDa is apparent in the amidase-, phage hydrolase-, and mutanolysin-digested samples, which migrates faster than the lysostaphin-solubilized protein. The generation of this species is apparently due to degradation during the prolonged incubation necessary for complete solubilization of the cell wall with these enzymes. Reprinted from reference 575 with permission of the publisher.

View larger version (23K): [in this window] [in a new window]   FIG. 11.   Structure of a surface protein linked to the peptidoglycan of S. aureus. As shown here, the protein is linked to a wall subunit that has been incorporated into the peptidoglycan network by both transpeptidation and transglycosylation. The neighboring wall subunit exists as a pentapeptide and is therefore not substituted. Adapted from reference 575 with permission of the publisher.

On the basis of sequence homology and limited biochemical analysis, the murein hydrolase of staphylococcal bacteriophage 11 was predicted to be an amidase (835). Hybrid surface proteins solubilized from the staphylococcal cell wall with the 11 hydrolase migrate as a doublet of bands on SDS-PAGE, in marked contrast to the ladder pattern observed when the protein is solubilized with other well-characterized amidases (796) (Fig. 10). Analysis of 11 hydrolase-solubilized C-terminal anchor peptides revealed that surface proteins were linked to a single peptidoglycan subunit, of which approximately 50% lacked the GlcNAc-MurNAc disaccharide, indicating that cross-linked peptidoglycan subunits had been removed (796). Recent results reveal that the 11 hydrolase displays two activities, the expected amidase as well as a D-Ala-Gly peptidase activity (575, 576).

Genetic analysis of staphylococcal methicillin resistance has provided new insights into the synthesis of the peptidoglycan crossbridge (43). Staphylococcal strains expressing PBP2a (PBP2') are resistant to most -lactam antibiotics including methicillin (310, 520). Genetic screens designed to identify other elements necessary for methicillin resistance yielded mutations in at least 10 different genes (44, 45, 146). Some of these genes are involved in the synthesis of the pentaglycine crossbridge (145, 331, 513, 756) or the amidation of D-isoglutamyl within the wall peptide (292, 394). Staphylococcal strains harboring mutations in the femA, femB, or femX gene synthesize altered cell wall crossbridges with either three glycyl (femB), one glycyl (femA), or a combination of no or one glycyl. The last phenotype has been reported for a mutant with a combination of a femA mutation and a second one leading to a partially nonfunctional FemX protein (442). Although this has not yet been demonstrated directly, it seems likely that FemA, FemB, and presumably also FemX catalyze the addition of glycyl to the -amino group of L-lysyl in the wall peptide portion of lipid II. Ton-That et al. tested whether the sorting reaction could proceed in staphylococcal strains carrying mutations in any of the three genes femA, femB, and femX (797). Although sorting was slowed in all fem mutant strains, surface protein was found anchored to peptidoglycan-bearing crossbridges with only three or one glycyl residues as well as crossbridges containing seryl moieties. However, species anchored directly to the -amino of L-lysyl in the wall peptide were not observed, suggesting that the sorting reaction has restricted substrate specificity for the amino group acceptor of this amide bond exchange mechanism (797).

Although it is now clear that protein A and other staphylococcal surface proteins are linked to the pentaglycine cell wall crossbridge, the specific peptidoglycan substrate of the sorting reaction has thus far not been identified. If the sorting reaction requires mature, assembled peptidoglycan as a substrate, staphylococcal protoplasts in which the peptidoglycan had been removed enzymatically should be unable to catalyze the cleavage of sorting precursors. Movitz studied the synthesis of protein A in staphylococcal protoplasts and reported that most of this polypeptide was released into the extracellular medium (561). A similar result was observed for the secretion of M protein by streptococcal protoplasts (809). Staphylococcal protoplasts generated by lysostaphin digestion and osmotically stabilized with sucrose cleaved sorting precursors similarly to intact cells, suggesting that the mature, assembled cell wall is not required for the sorting reaction (797a). Protein A molecules that were cleaved at the LPXTG motif were released into the extracellular medium. Nevertheless, it is not clear whether these released protein A molecules contain linked peptidoglycan fragments.

On the other hand, if the sorting reaction uses the peptidoglycan synthesis precursor, lipid II, as a substrate, inhibition of peptidoglycan synthesis with known antibiotics might also interfere with the sorting reaction. This question has also been addressed by Movitz (562). Although the anchoring mechanism was unknown at that time, the author suspected that protein A was linked to peptidoglycan and measured the amount of pulse-labeled protein A in the cell walls of staphylococci that had been treated with vancomycin. The results showed that vancomycin inhibited cell wall synthesis but did not interfere with the anchoring of protein A. Movitz concluded that protein A may be linked to mature, assembled cell wall. Vancomycin binds to D-alanyl-D-alanine within the wall peptide, and treatment of staphylococci with this antibiotic leads to the accumulation of lipid II molecules (789). If lipid II served as substrate for the sorting reaction, Movitz's experiments could not have distinguished between protein A molecules that were linked to lipid II and others that were tethered to the assembled cell wall. We think that the identification of the substrate for the sorting reaction will require biochemical studies with purified sortase enzyme as well as the rigorous characterization of presumed sorting intermediates.

The sorting of surface proteins to the cell wall of gram-positive bacteria begins in the cytoplasm with the initiation of precursor molecules into the protein export (Sec) pathway via their N-terminal leader peptides (Fig. 9). By default, this pathway leads to the secretion of polypeptides into the surrounding medium of staphylococci; however, the C-terminal hydrophobic domain and charged tail function to retain protein A along the pathway, perhaps within the preprotein translocase. This retention allows recognition of the LPXTG motif and a proteolytic cleavage between the threonine and the glycine of the LPXTG motif. The carbonyl of threonine may be acylated to an active-site thiol or hydroxyl of sortase, and a subsequent nucleophilic attack of the free amino at the end of the pentaglycine cell wall crossbridge results in the transpeptidation of surface protein to peptidoglycan precursor. The sorting intermediate may subsequently be incorporated into the cell wall. Proteolytic degradation of anchored proteins as well as the physiological turnover of peptidoglycan will finally remove the anchored polypeptides.

Braun's murein lipoprotein (LPP) serves as a paradigm for the synthesis and structure of cell wall-linked proteins in gram-negative bacteria (85) (Fig. 12). The N-terminal signal peptide of LPP directs the polypeptide into the secretory pathway (367). A cysteine residue within the signal peptide is glyceride modified via a thioester linkage prior to proteolytic cleavage at its amino group (305). The amino group of the N-terminal cysteine is acylated prior to the insertion of LPP into the outer membrane (485). The 70-residue polypeptide of mature LPP trimerizes in the periplasmic space (84, 87). About one-third of all lipoprotein is covalently linked to the peptidoglycan layer, thereby tethering the outer membrane to the peptidoglycan skeleton of E. coli (86). Lipid modification of murein lipoprotein first requires membrane-embedded glyceryl transferase and O-acyl transferase activities (240, 289, 783). The glycerol-modified LPP precursor is then cleaved by signal peptidase (type II), and the liberated amino group of cysteine is modified by an N-acyl transferase (365). The -amino of the C-terminal lysine is amide linked to the carboxyl group at the D center of meso-diaminopimelic acid within the E. coli cell wall peptides (88, 89). Neither the chemical reaction nor the enzymes responsible for this linkage have been characterized. It is conceivable that each trimeric murein lipoprotein unit is covalently linked to the cell wall. It also seems plausible that the amide bond between LPP and cell wall is generated at the expense of another amide bon