Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.

Most bacteria have multiple peptidoglycan hydrolases capable of cleaving covalent bonds in peptidoglycan sacculi or its fragments. An overview of the different classes of peptidoglycan hydrolases and their cleavage sites is provided. The physiological functions of these enzymes include the regulation of cell wall growth, the turnover of peptidoglycan during growth, the separation of daughter cells during cell division and autolysis. Specialized hydrolases enlarge the pores in the peptidoglycan for the assembly of large trans-envelope complexes (pili, flagella, secretion systems), or they specifically cleave peptidoglycan during sporulation or spore germination. Moreover, peptidoglycan hydrolases are involved in lysis phenomena such as fratricide or developmental lysis occurring in bacterial populations. We will also review the current view on the regulation of autolysins and on the role of cytoplasm hydrolases in peptidoglycan recycling and induction of beta-lactamase.

We solved the crystal structure of a secreted protein, EXLX1, encoded by the yoaJ gene of Bacillus subtilis. Its structure is remarkably similar to that of plant ␤-expansins (group 1 grass pollen allergens), consisting of 2 tightly packed domains (D1, D2) with a potential polysaccharide-binding surface spanning the 2 domains. Domain D1 has a double-␤-barrel fold with partial conservation of the catalytic site found in family 45 glycosyl hydrolases and in the MltA family of lytic transglycosylases. Domain D2 has an Ig-like fold similar to group 2/3 grass pollen allergens, with structural features similar to a type A carbohydratebinding domain. EXLX1 bound to plant cell walls, cellulose, and peptidoglycan, but it lacked lytic activity against a variety of plant cell wall polysaccharides and peptidoglycan. EXLX1 promoted plant cell wall extension similar to, but 10 times weaker than, plant ␤-expansins, which synergistically enhanced EXLX1 activity. Deletion of the gene encoding EXLX1 did not affect growth or peptidoglycan composition of B. subtilis in liquid medium, but slowed lysis upon osmotic shock and greatly reduced the ability of the bacterium to colonize maize roots. The presence of EXLX1 homologs in a small but diverse set of plant pathogens further supports a role in plant-bacterial interactions. Because plant expansins have proved difficult to express in active form in heterologous systems, the discovery of a bacterial homolog opens the door for detailed structural studies of expansin function.family 45 endoglucanase ͉ lytic transglycosylase ͉ peptidoglycan ͉ plant cell wall ͉ plant-microbe interactions B acterial and plant cell walls have similar functions but distinctive structures. Bacterial peptidoglycan forms a network of linear polysaccharide strands of alternating Nacetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues cross-linked by short polypeptides. As a giant bag-shaped sacculus, peptidoglycan expands via the action of endopeptidases, amidases, and lytic transglycosylases that cleave covalent bonds and allow insertion of new subunits (1). In contrast, the growing plant cell wall is formed from a scaffold of cellulose microfibrils tethered together by branched glycans such as xyloglucan or arabinoxylan that bind noncovalently to cellulose surfaces. The cellulose-hemicellulose network enlarges via polymer slippage or ''creep,'' mechanically powered by turgorgenerated forces in the cell wall and catalyzed by expansins and other wall-loosening agents (2).Expansins are known principally from plants where they function in cell enlargement and other developmental events requiring cell wall loosening (3). Canonical expansins are small proteins (Ϸ26 kDa, Ϸ225 aa) consisting of 2 compact domains: D1 has a fold similar to that of family 45 glycosyl hydrolases (GH45), and D2 has a ␤-sandwich fold. Expansins facilitate cell wall creep without breakdown of wall polymers (3-5). Plant expansins consist of 2 major families: ␣-expansins, which preferentially loosen the cell walls of dicots compa...

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Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.