Coupling of polymerase and carrier lipid phosphatase prevents product inhibition in peptidoglycan synthesis.

Peptidoglycan (PG) is an essential component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG precursor lipid II is assembled at the inner leaflet of the cytoplasmic membrane, translocated to the periplasmic side, and polymerized to glycan chains by membrane anchored PG synthases, such as the class A Penicillin-binding proteins (PBPs). Polymerization of PG releases the diphosphate form of the carrier lipid, undecaprenyl pyrophosphate (C55-PP), which is converted to the monophosphate form by membrane-embedded pyrophosphatases, generating C55-P for a new round of PG precursor synthesis. Here we report that deletion of the C55-PP pyrophosphatase gene pgpB in E. coli increases the susceptibility to cefsulodin, a ß-lactam specific for PBP1A, indicating that the cellular function of PBP1B is impaired in the absence of PgpB. Purified PBP1B interacted with PgpB and another C55-PP pyrophosphatase, BacA and both, PgpB and BacA stimulated the glycosyltransferase activity of PBP1B. C55-PP was found to be a potent inhibitor of PBP1B. Our data suggest that the stimulation of PBP1B by PgpB is due to the faster removal and processing of C55-PP, and that PBP1B interacts with C55-PP phosphatases during PG synthesis to couple PG polymerization with the recycling of the carrier lipid and prevent product inhibition by C55-PP.

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus.

Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to ß-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of ß-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to ß-lactams in S. aureus.

AmiD Is a Novel Peptidoglycan Amidase in Wolbachia Endosymbionts of Drosophila melanogaster.

Wolbachia endobacteria are obligate intracellular bacteria with a highly reduced genome infecting many arthropod and filarial species, in which they manipulate arthropod reproduction to increase their transmission and are essential for nematode development and survival. The Wolbachia genome encodes all enzymes required for the synthesis of the cell wall building block lipid II, although a peptidoglycan-like structure has not been detected. Despite the ability to synthesize lipid II, Wolbachia from arthropods and nematodes have only a subset of genes encoding enzymes involved in the periplasmic processing of lipid II and peptidoglycan recycling, with arthropods having two more than nematodes. We functionally analyzed the activity of the putative cell wall hydrolase AmiD from the Wolbachia endosymbiont of Drosophila melanogaster, an enzyme not encoded by the nematode endobacteria. Wolbachia AmiD has Zn2+-dependent amidase activity and cleaves intact peptidoglycan, monomeric lipid II and anhydromuropeptides, substrates that are generated during bacterial growth. AmiD may have been maintained in arthropod Wolbachia to avoid host immune recognition by degrading cell wall fragments in the periplasm. This is the first description of a wolbachial lipid II processing enzyme putatively expressed in the periplasm.