C ELL WALL GROWTH AND TURNOVER

The task of maintaining and remodeling the cell wall during growth and division by inserting new glycan strands into the preexisting structure is accomplished by cell wall modifying enzymes, which include a number of hydrolases.

Specific hydrolases are essential for cell growth and exist for most connecting building blocks in the peptidoglycan. These enzymes can be classified into three types depending on the specific bond cleaved by the enzyme (Fig. 6). There are glycan strand-hydrolyzing enzymes such as muramidases (lysozyme-related and/or lytic transglycosylases) and glucosaminidases. Peptide bonds within the stem peptides of the peptidoglycan structure are hydrolyzed by endopeptidases and carboxypeptidases. Furthermore, (anhydro-)N-acetylmuramic acid-L-Ala amidases cleave the junction between the glycan strands and the linked stem peptides. Besides their important role in the growth of the cell wall, peptidoglycan hydrolases are also proposed to be involved in other cellular processes such as cell separation, turnover and recycling of cell wall components or sporulation (Scheffers and Pinho, 2005).

Several models for the growth of the peptidoglycan sacculus have been proposed for Gram-negative and Gram-positive bacteria, since the former have a thick multilayered cell wall compared to the predominantly monolayered structure of the latter. A safe enlargement of the sacculus can only be ensured during growth, when newly synthesized material is incorporated and cross-linked before the existing stress-bearing peptidoglycan is cleaved. This process is called the “make-before-break” strategy (Koch and Doyle, 1985). For Gram-positive bacteria, it has been shown that labeled peptidoglycan precursors are incorporated close to the inner membrane and are gradually displaced within the cell wall to the outside according to an “inside-to-outside” growth mechanism (Koch and Doyle, 1985). Therefore, new material is inserted in an unextended state, randomly along the cell cylinder, and underneath the stress-bearing peptidoglycan layers. This is followed by dissolution of the outermost layers, which are assumed to be more susceptible to the activity of peptidoglycan hydrolases at the cell surface, and their release into the growth medium. As a result of continuous peptidoglycan synthesis, newly synthesized material would be pushed outward and becomes stretched due to surface tension caused by the turgor pressure. In this model, the

cell wall is always steady and strong during growth as long as the covalently closed layers of the peptidoglycan are formed before the older and outer ones are degraded to prevent rupture of the cell wall.

Fig. 6. Peptidoglycan hydrolyzing enzymes in E. coli or B. subtilis. The arrows indicate the cleavage sites of the different hydrolases. Glycosidic bonds in the peptidoglycan are hydrolyzed by glucosaminidases and muramidases, yielding GlcNAc (grey circle) and disaccharides containing among MurNAc (grey square) or 1,6-anhydroMurNAc (grey square with cap), respectively. Amidases, (1,6-anhydro-)N-acetylmuramic acid-L-Ala amidases, hydrolyze the amide bonds between the lactyl group of (1,6-anhydro-)MurNAc and the N-terminal L-alanine of the stem peptides, thereby releasing oligopeptides. In contrast to endopeptidases, which cleave specific peptide bonds within the stem peptides, carboxypeptidases remove only C-terminal D- or L-amino acids.

The stem peptides L-Ala-γ-D-Glu-m-DAP-D-Ala-D-Ala are abbreviated with Ala-Glu-DAP-Ala-Ala. Figure was adapted from (Mayer, 2012).

Based on the “inside-to-outside” growth mechanism of Gram-positive bacteria, even the predominantly monolayered peptidoglycan sacculus of Gram-negative bacteria follows the

“make-before-brake” strategy. Accordingly, the “three-for-one” model has been proposed for the peptidoglycan synthesis in E. coli (Höltje, 1998) (Fig. 7A). This model suggests that three new glycan strands are synthesized and cross-linked to each neighbor, whereby a triplet is formed. This is achieved by transglycosylation and transpeptidation catalyzed by a dimer of a bifunctional PBP (PBP1A or PBP1B) as well as a monofunctional PBP (PBP1C) (Bertsche et al., 2005; Born et al., 2006; Schiffer and Höltje, 1999). Now, the triplet is covalently attached in a relaxed conformation below an existing glycan strand, the so-called docking strand, in the sacculus. A dimer of a transpeptidase (PBP2 or PBP3) cross-links the glycan strands on the outside of the triplet to both sides of the docking strand (Spratt, 1975). Upon simultaneous removal of the docking strand by degradation to monomeric disaccharide peptide subunits by

the concerted action of a dimer of an endopeptidase (PBP4 or PBP7) (Korat et al., 1991;

Romeis and Höltje, 1994a) and a lytic transglycosylase, the triplet of glycan strands is pulled into the stress-bearing sacculus by the force of the internal pressure of the cell.

Fig. 7. Proposed model for the peptidoglycan synthesis in Gram-negative bacteria. (A) The “three-for-one”

growth model. A triplet of three newly synthesized, cross-linked glycan strands is covalently attached in an unextended state to the free amino groups of m-DAP (A2pm) present in the stem peptides on both sides of an

“old” docking strand. Specific cleavage of the preexisting cross-linkages results in the removal of the docking strand followed by the insertion of the triplet into the stress-bearing peptidoglycan sacculus due to surface tension. (B) According to the “three-for-one” model, a multienzyme complex consisting of peptidoglycan synthases and hydrolases slides along the docking strand to synthesize the peptidoglycan triplet. While a monofunctional transglycosylase (TG) polymerizes the middle strand, two bifunctional transpeptidase/transglycosylases (TP/TG) polymerize the two outer strands and cross-link them to the middle strand. After its attachment to the existing peptidoglycan by two monofunctional transpeptidases (TP), the docking strand is removed by a lytic transglycosylase (TG) and two endopeptidases (EP) and/or two amidases (AM). Figure (A) was taken from (Vollmer and Höltje, 2001) and (B) from (Höltje, 1998).

Interestingly, the localization of the enzymes reflects the “inside-to-outside” growth mechanism of the “three-for-one” model in E. coli. The polymerizing enzymes (PBP1A, 1B, 1C, 2, 3) that form the new peptidoglycan layers are bound to the cytoplasmic membrane, whereas most lytic transglycosylases that act on the outer layers are lipoproteins anchored to the outer membrane. In this regard, the “three-for-one” model predicts that an interaction of peptidoglycan synthases (i.e., transpeptidases and transglycosylases) and peptidoglycan hydrolases with each other must be coordinated by the formation of a multienzyme complex to keep the activities of both in a balanced ratio (Höltje, 1996, 1998) (Fig. 7B). In agreement with the “make-before-break” strategy, peptidoglycan hydrolases in the multienzyme complex should be arranged behind the peptidoglycan synthases, thus ensuring that the insertion of new material precedes the hydrolysis of preexisting peptidoglycan. This allows a safe enlargement of the stress-bearing peptidoglycan sacculus. An evidence for the existence of this multienzyme complex in E. coli was presented by the detection of specific protein-protein

interactions between different PBPs, peptidoglycan hydrolases and PBPs, as well as PPBs and structural proteins (Bhavsar and Brown, 2006; Romeis and Höltje, 1994b; Vollmer and Bertsche, 2008; Vollmer et al., 1999; von Rechenberg et al., 1996). The results of these studies suggest the presence of two multienzyme complexes, which were predicted to be involved in cell elongation and cell division, respectively. So far, a clear evidence for a multienzyme complex in Gram-positive bacteria has not been demonstrated.

Peptidoglycan synthesis might be directed in most rod-shaped bacteria, including E. coli and B. subtilis, by interaction of the intracellular cytoskeleton acting as a scaffold that recruits and assembles the multienzyme complex required for cell wall growth. During cell elongation, the actin-like cytoskeletal elements MreB in Gram-negative bacteria and its paralogs in Gram-positive bacteria are essential for peptidoglycan synthesis along the cylindrical sidewall to maintain the rod shape of the cell. They appear to form helical filaments on the inner side of the cytoplasmic membrane extending from one cell pole to the other (Jones et al., 2001). As visualized by fluorescence microscopy in B. subtilis, peptidoglycan insertion into the existing sacculus was observed to occur in a pattern similar to that formed by Mbl (Daniel and Errington, 2003). In contrast, total internal reflection fluorescence microscopy (TIRFM) is a more sensitive method for imaging events near the cell surface. Studies that based on this technique revealed that during exponential growth, MreB isoforms in B. subtilis (MreB, Mbl and MreBH) did not form helical structures but assembled into discrete patches that moved bidirectionally along peripheral tracks perpendicular to the cell axis (Domínguez-Escobar et al., 2011). In the absence of Mbl, peptidoglycan synthesis is localized at mid-cell. Accompanied by the accumulation of the bacterial tubulin-homolog FtsZ (Bi and Lutkenhaus, 1991), a contractile ring-like structure is polymerized that presumably redirects the multienzyme complex to the division site. This switch from cell elongation to cell division depends on remodeling of the multienzyme complex as illustrated for E. coli (Vollmer and Bertsche, 2008) (Fig. 8).

Cell growth and division of bacteria is accompanied by a massive release of peptidoglycan fragments, so-called muropeptides, from the stressed, outermost layers of the cell wall. This process that is termed cell wall turnover appears as an intrinsic consequence of a growth mechanism, which couples the degradation of the cell wall with the simultaneous insertion of newly synthesized material. Therefore, cell wall turnover occurs as a result of the need for the cell to expand its surface. It has been studied extensively in Gram-positive bacteria (Doyle et al., 1988).

Fig. 8. Model for the peptidoglycan synthesis complexes controlled by the cytoskeletal elements MreB and FtsZ in E. coli. Indicated are the specific composition and cellular localization of the subcomplexes. During cell elongation, synthesis (MraY and MurG) of peptidoglycan precursors is followed by their incorporation (PBPs) at the filament of MreB. Amidases (AMI), lytic transglycosylases (LT) and endopeptidases (EP) are responsible for the hydrolysis of older peptidoglycan layers. Formation of the FtsZ-ring at mid-cell initiates the assembly of the multienzyme complexes for cell division to generate a septum that matures to two new cell poles. Figure was taken from (Vollmer and Bertsche, 2008).

During vegetative growth, B. subtilis and other Gram-positive species turn over up to 50% of the existing cell wall material in one generation (Boothby et al., 1973; Chaloupka et al., 1962a; Chaloupka et al., 1962b; Mauck et al., 1971; Mauck and Glaser, 1970; Rogers, 1967).

The liberated degradation products are released into the growth medium. It has also been observed that the process of cell wall turnover is not restricted solely to peptidoglycan but also refers to the loss of peptidoglycan-attached polymers, such as teichoic acids and capsular polysaccharides at the cell surface (Hancock and Cox, 1991; Mauck et al., 1971; Wong et al., 1974). Peptidoglycan turnover in B. subtilis proceeds rapidly along the cell cylinder, since the cell poles are metabolically inert (Mobley et al., 1984).

Lysis of the peptidoglycan proceeds by the induction of the autolytic system with its hydrolyzing enzymes (autolysins), which contribute to the cleavage of the covalent bonds in the intact sacculus. For B. subtilis, more than 35 candidate peptidoglycan hydrolases have been identified on the basis of amino acid sequence similarities, and classified as N-acetylglucosaminidases (digesting GlcNAc-MurNAc linkage), N-acetylmuramic acid-L-Ala amidases (digesting MurNAc-L-Ala linkage), D,L-endopeptidases (digesting D-Glu-m-DAP

linkage) and ^L,^D-endopeptidases (digesting ^L-Ala-^D-Glu linkage) (Kunst et al., 1997; Smith et al., 2000). Among the best characterized autolysins, the amidase LytC (Kuroda and Sekiguchi, 1991) and the glucosaminidases LytD (Margot et al., 1994; Rashid et al., 1995) and LytG, an exo-N-acetylglucosaminidase, (Horsburgh et al., 2003) are assumed to be responsible for more than 95% of the autolytic activity in B. subtilis (Blackman et al., 1998).

Recently, two bifunctional autolysins, B. subtilis CwlT (YddH) (Fukushima et al., 2008) and CwlP (YomI) (Sudiarta et al., 2010a), have been associated with cell wall turnover. The C-terminal domain of both enzymes exhibits D,L- and D,D-endopeptidase activity, respectively, whereas the N-terminal domain is an N-acetylmuramidase that hydrolyzes the linkage of MurNAc-GlcNAc, thereby releasing tetra-, hexa- and octasaccharides from purified glycan strands of B. subtilis peptidoglycan. Glycan strand-cleaving enzymes such as muramidases are thought to be responsible for turnover of peptidoglycan in Gram-positive bacteria (Atrih et al., 1999). Lytic transglycosylases act like muramidases because they also target the β-1,4-glycosidic linkage between the MurNAc and GlcNAc residue of peptidoglycan, but they catalyze an intramolecular transglycosylation reaction by formation of a 1,6-anhydro ring at the terminal MurNAc residue, thereby producing anhydromuropeptides (Höltje et al., 1975; Scheurwater et al., 2008) (Fig. 9). Analysis of the peptidoglycan structure of B. subtilis revealed that anhydromuropeptides represent only 0.4% of the total cell wall degradation products, in contrast to the large amounts in E. coli (80%) (Kitano et al., 1986), and thus lytic transglycosylases are thought to have only a minor impact on the peptidoglycan turnover in Gram-positive bacteria (Atrih et al., 1999). Therefore, CwlQ (YjbJ) was identified as another bifunctional cell wall hydrolase, but the first one in B. subtilis that exhibits a strong lytic transglycosylase activity besides muramidase activity (Sudiarta et al., 2010b).

Cell wall turnover in the Gram-negative bacterium E. coli has been underestimated for a long time, since only a minor portion of about 8% of released muropeptides was isolated from the culture supernatants (Goodell and Schwarz, 1985). But E. coli also degrades almost half of its peptidoglycan in each generation. The majority of the turnover products have been found to accumulate in the periplasm as a result of turnover, from where they are continuously transported into the cytoplasm and reutilized (i.e., recycled) efficiently (Goodell, 1985). The main turnover products are anhydromuropeptides such as GlcNAc-anhydroMurNAc-L

-Ala-γ-D-Glu-m-DAP(-D-Ala) (Höltje, 1998). Their monomeric structure indicates that they are in principle products of lytic transglycosylases and endopeptidases (PBP4, 7 and MepA) (Keck et al., 1990; Korat et al., 1991; Romeis and Höltje, 1994a), released as a result of the vegetative growth process. Lytic transglycosylases represent the major class of muramidases

in Gram-negative bacteria. At least one soluble lytic transglycosylase (Slt70) and seven membrane-bound lytic transglycosylases (MltA, B, C, D, E (EmtA), F), which are lipoproteins anchored to the inner leaflet of the outer membrane, have been described in E. coli (Ehlert et al., 1995; Höltje et al., 1975; Kraft et al., 1998; Lommatzsch et al., 1997;

Vollmer and Bertsche, 2008; Vollmer et al., 2008b). Most lytic transglycosylases appear to act as exo-acting enzymes (Slt70 and MltA, B), removing disaccharide-peptide repeats from the 1,6-anhydroMurNAc glycan strand end (Vollmer and Bertsche, 2008). The only known exception in E. coli is MltE (EmtA), which is proposed to be an endo-specific lytic transglycosylase (Kraft et al., 1998). However, Slt70 found in the periplasm of E. coli is predicted to be the principle lytic transglycosylase responsible for the turnover of peptidoglycan.

Fig. 9. Cleavage of the β-1,4-glycosidic linkage in glycan strands of peptidoglycan by glucosaminidases (1) or muramidases such as lysozyme-like transglycosylases (2) or lytic transglycosylases (3). Reaction of the lytic transglycosylases is accompanied by an intramolecular transglycosylation at the MurNAc residue, resulting in the formation of a 1,6-anhydro bond. R, stem peptide attached to the lactyl residue of MurNAc. Figure was taken from (Vollmer et al., 2008b).

Furthermore, E. coli possesses five MurNAc-L-Ala amidases, i.e., three periplasmic enzymes (AmiA, B, C) (Heidrich et al., 2001), one outer membrane-anchored enzyme (AmiD) (Uehara and Park, 2007), and one cytoplasmic enzyme (AmpD) (Höltje et al., 1994). All amidases hydrolyze the amide bonds between the glycan strands and the stem peptides with concomitant turnover of the peptide moiety in form of tetra- or tripeptides besides the anhydrodisaccharides from the sacculus. As a result of the combined lytic activities, more than 80 different types of muropeptides were identified in peptidoglycan preparations from E. coli (Glauner et al., 1988).