The lytic transglycosylases cleave the bacterial cell wall heteropolymer peptidoglycan with the same specificity as the muramidases (lysozymes), between the N-acetylmuramic acid and N-acetylglucosamine residues, with the concomitant formation of a 1,6-anhydromuramoyl residue. The putative catalytic residue in the family 3 lytic transglycosylase from Pseudomonas aeruginosa, Glu162 as identified by sequence alignment to the homologous enzyme from Escherichia coli, was replaced with both Ala and Asp by site-directed mutagenesis. Neither mutant enzyme differed structurally from the wild-type enzyme, as judged by CD spectroscopy, but both were enzymatically inactive confirming the essential role of Glu162 in the mechanism of action of this lytic transglycosylase. The bhexosaminidase inhibitor NAG-thiazoline was shown to inhibit the activity of lytic transglycosylase activity, thus providing the first direct evidence that the formation of the 1,6-anhydromuramoyl residue may proceed through an oxazolinium ion intermediate involving anchimeric assistance. Using surface plasmon resonance and difference absorbance spectroscopy, K d values of 1.8 and 1.4 mM, respectively, were determined for NAG thiazoline, while its parent compound N-acetylglucosamine neither inhibited nor appeared to bind the lytic transglycosylase with any significant affinity.

Objectives: To investigate the function of penicillin-binding protein 2 (PBP 2) in Pseudomonas aeruginosa PAO1. Methods:The growth and morphology of P. aeruginosa cultured in the absence and presence of mecillinam was assessed. The gene encoding PBP 2, pbpA, was identified in the genome of P. aeruginosa PAO1 and both its full-length and an engineered truncated form were cloned and expressed in Escherichia coli. Site-directed mutagenesis was used to confirm Ser-327 as the catalytic nucleophile of its transpeptidase domain. Allelic exchange was used to construct a chromosomal mutant of pbpA in strain PAO1.Results: PAO1 grew with a spherical morphology in the presence of mecillinam at concentrations as high as 2000 mg/L. Both wild-type and truncated, soluble forms of PBP 2 were shown to bind penicillins and a competition assay demonstrated their specificity for mecillinam. The PAO1 DpbpA insertional mutant also grew as spheres, and complementation with a plasmid encoding active pbpA, but not with an inactive Ser-327 ! Ala derivative, restored rod-shape morphology. MIC values of a variety of b-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 DpbpA analysed by HPLC/MALDI TOF MS indicated wild-type levels of cross-linking despite the loss of PBP 2 transpeptidase activity.Conclusions: PBP 2 in P. aeruginosa is responsible for the rod-shape morphology of the cells and contributes significantly to b-lactam resistance. The viability of cells lacking an active PBP 2 suggests that the organization of the peptidoglycan biosynthetic machinery is different in this pathogen compared with E. coli.

Soluble lytic transglycosylase B1 from Pseudomonas aeruginosa was coupled to Sepharose and used to immobilize interaction partners from membrane protein extracts. Penicillin-binding protein 2 (PBP2) was identified as a binding partner, suggesting that the two proteins function together in the biosynthesis of peptidoglycan. By use of an engineered truncated derivative, the N-terminal module of PBP2 was found to confer the binding properties.Enlargement and growth of the peptidoglycan (PG) sacculus is effected by the coordination of both synthetic and lytic enzymes. The penicillin-binding proteins (PBPs) are biosynthetic enzymes that catalyze transglycosylation and/or transpeptidation reactions for the incorporation of new material into the PG layer (11). The lytic transglycosylases (LTs), on the other hand, cleave the ␤-1,4-glycosidic bond between MurNAc and GlcNAc and are understood to function as space-makers for the incorporation of new material (reviewed in reference 29). These conflicting enzymatic activities are thought to be controlled by the association of the respective enzymes in multienzyme complexes (14, 29). Support for this hypothesis is confined to only a few studies involving a limited number of bacterial species. With Escherichia coli, a protein-protein interaction network between high-molecular-weight (HMW) PBPs, HMW LTs, and low-molecular-weight PBPs (specifically, D,D-endopeptidases) has been demonstrated by affinity chromatography experiments (3,26,34,35). Affinity chromatography was also used to indicate that membrane-bound LT A (MltA) interacts with PBP2 in

Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their Nterminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

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