C ELL WALL TURNOVER AND RECYCLING IN G RAM - NEGATIVE BACTERIA

The Gram-negative bacterium E. coli has been studied extensively as a model organism for the processes of cell wall turnover and recycling (Park and Uehara, 2008). Cell wall turnover occurs simultaneously with the insertion of cell wall material along the cylindrical surface of the rod-shaped E. coli cell during the cell elongation process, whereas the cell poles remain metabolically inactive and basically do not turn over (Burman et al., 1983; de Pedro et al., 1997). During cell elongation the essentially single-layered murein is continuously degraded by peptidoglycan cleaving enzymes such as endopeptidases and carboxypeptidases, which hydrolyze the peptide crosslinks and release terminal D-Ala residues, respectively, of the peptide portion of the peptidoglycan as well as lytic transglycosylases, which cleave the MurNAc-GlcNAc bonds in the glycan backbone of the peptidoglycan releasing unique β-1,6-anhydro-N-acetylmuramic acid (anhMurNAc)-containing peptidoglycan fragments (anhydromuropeptides). Anhydromuropeptides are trapped in the periplasm, the disaccharide-tetrapeptide GlcNAc-β-1,4-anhMurNAc-^L-Ala-γ-^D-Glu-m-DAP-D-Ala being the major product (Höltje, 1998) (Fig.1). Originally, Goodell determined that about half of the murein was being recycled per generation (Goodell, 1985). Taking into account that the polar caps, which make up 1/3 of the cell wall, are basically inert, it can be calculated that more than 60%

of the murein of the side wall is degraded within one doubling process (Uehara and Park, 2008). Surprisingly, it was reported recently that at least 30% of the newly synthesized septal murein is degraded during cell division, involving lytic transglycosylases in removal of excess peptidoglycan during septation (Uehara and Park, 2008). The periplasmic amidases AmiA, B and C, all of which cleaving MurNAc-L-Ala bonds in the murein, are involved in splitting the septum during daughter cell separation but surprisingly release only minor amounts of peptidoglycan during cell division (Heidrich et al., 2001; Priyadarshini et al., 2006; Uehara and Park, 2008). AmiD, an additional, outer membrane-anchored murein amidase, is not involved in cell separation but was shown to cleave periplasmic anhydromuropeptides (Uehara and Park, 2007) (Fig.1). Murein tripeptides (L-Ala-γ-D-Glu-m-DAP) and tetrapeptides (L-Ala-γ-D-Glu-m-DAP-D-Ala) that are released from the murein or from anhydromuropeptides by the amidases can be taken up from the periplasm into the cytoplasm via the general oligopeptide transporter Opp that recruits the specific binding protein MppA (Park, 1993; Park et al., 1998) (Fig. 13). However, uptake of cell wall-derived peptides is only a minor recycling pathway in E. coli (Park and Uehara, 2008). The major recycling pathway involves the uptake of anhydromuropeptides by the secondary transporter AmpG that is specific for GlcNAc-1,6-anhMurNAc and GlcNAc-1,6-anhMurNAc-peptides (Cheng and

Park, 2002). The anhydromuropeptides are further degraded in the cytoplasm by the N-acetylglucosaminidase NagZ (Cheng et al., 2000; Vötsch and Templin, 2000), the anhMurNAc-L-Ala amidase AmpD (Höltje et al., 1994; Jacobs et al., 1995) and the

L,D-carboxypeptidase LdcA (Templin et al., 1999). The processing finally yields GlcNAc, anhMurNAc, D-Ala and L-Ala-γ-D-Glu-m-DAP tripeptide in the cytoplasm. The tripeptide is directly transferred to UDP-MurNAc by the muropeptide ligase Mpl (Mengin-Lecreulx et al., 1996), generating UDP-MurNAc-L-Ala-γ-D-Glu-m-DAP, thereby entering the murein synthesis pathway. Intriguingly, Mpl can also transfer tetrapeptides, which makes LdcA activity essential. In the absence of LcdA, UDP-MurNAc-tetrapeptide instead of UDP-MurNAc-pentapeptide accumulates, which results in the incorporation of tetrapeptide instead of pentapeptide in the peptidoglycan, leading to abnormal morphology and lysis in the transition to log phase (Templin et al., 1999). In the absence of Mpl, the tripeptide can also be degraded to the single amino acids involving the D-Glu-m-DAP peptidase MpaA and the

L-Ala-D/L-Glu epimerase AeeP (Park and Uehara, 2008). The sugar portion of the murein is also made available for recovery. It involves the phosphorylation of the two amino sugars GlcNAc and anhMurNAc by the specific kinases NagK (Uehara and Park, 2004) and AnmK (Uehara et al., 2005), respectively, and the conversion of the MurNAc-6-phosphate to GlcNAc-6-phosphate by the MurNAc-6-phosphate etherase MurQ accompanied by the release of ^D-lactate (Jaeger et al., 2005; Uehara et al., 2006) (Fig. 13). Hence, the amino sugars of the cell wall are both converted to GlcNAc-6-phosphate. The latter is then deacetylated by NagA, yielding GlcN-6-phosphate (White and Pasternak, 1967), which subsequently enters either the pathway for the synthesis of the cell wall precursor UDP-GlcNAc, upon isomerization by GlmM (Mengin-Lecreulx and van Heijenoort, 1996) and activation by GlmU (Mengin-Lecreulx and van Heijenoort, 1993, 1994), or the catabolic pathway of glycolysis via deamination by NagB yielding fructose-6-phosphate (White, 1968).

Alternatively, GlcNAc and MurNAc can be taken up into the cell by the phosphotransferase system (PTS) transporters NagE (Plumbridge, 2009) and MurP (Dahl et al., 2004), respectively, yielding, intracellularly, the respective 6-phosphate sugars. Recently, Plumbridge found that NagE is responsible for about 50% of GlcNAc-6-phosphate that accumulates in a nagA mutant whereas the other 50% come from the AmpG/MurQ-dependent peptidoglycan recycling pathway (Plumbridge, 2009). The MurNAc-6-phosphate etherase MurQ was shown to be required in E. coli for both, the recycling of anhMurNAc as well as for utilization of exogenic MurNAc (Uehara et al., 2006). It is induced in the presence of MurNAc-6-phosphate by derepression of the transcriptional regulator MurR as well as by

activation via cAMP-catabolite activator protein (CAP) (Jaeger et al., 2005; Jaeger and Mayer, 2008b).

Fig. 13. The peptidoglycan recycling pathways of E. coli. Anhydromuropeptides like GlcNAc-anhMurNAc-tetrapeptide, the major turnover product, are generated in the periplasm by the action of lytic transglycosylases and endopeptidases. They can be taken up by the AmpG transporter and are further processed in the cytoplasm (for details, see text). Alternatively, cell wall-derived peptides are taken up by the oligopeptide transporter Opp that uses a specific binding protein (MppA) for this purpose. Cell wall-derived amino sugars are taken up by specific phosphotransferase systems (PTS; NagE, MurP) that require the general PTS components EI, HPr, and EIIA-glucose (only MurP).

In some Gram-negative Enterobacteriaceae but not E. coli cell wall recycling is closely connected with cell wall signaling and induction of chromosomal AmpC-type beta-lactamases (Jacobs et al., 1994). The names AmpG (permease) and AmpD (amidase) originate from their initial discovery as positive and negative regulators, respectively, of ampC expression (Park and Uehara, 2008). An ampD-defective mutant accumulates large amounts of anhMurNAc-tripeptide, which apparently stimulate AmpR, the activator of ampC expression (Jacobs et al., 1994). On the other hand, ampC is not expressed in an ampG deletion mutant, which is unable to take up muropeptides. A similar connection of cell wall turnover products and induction of antibiotic resistance has not been identified in Gram-positive bacteria so far.

3.4. Differences in the cell wall structure between Gram-negative and Gram-positive