Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes, however, the lytic transglycosylases are not hydrolases but instead cleave the glycosidic linkage between N-actetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramoyl product. They are ubiquitous in bacteria which produce a compliment of different forms that are responsible for creating space within the peptidoglycan sacculus for its biosynthesis and recycling, cell division, and the insertion of cell-envelope spanning structures, such as flagella and secretion systems. As well, the lytic transglyosylases may have a role in pathogenesis of some bacterial species. Given their important role in bacterial cell wall metabolism, the lytic transglycosylases may present an attractive new target for the development of broad-spectrum antibiotics.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Among its primary mechanisms of resistance is expression of a chromosomally encoded AmpC ␤-lactamase that inactivates ␤-lactams. The mechanisms leading to AmpC expression in P. aeruginosa remain incompletely understood but are intricately linked to cell wall metabolism. To better understand the roles of peptidoglycan-active enzymes in AmpC expression-and consequent ␤-lactam resistance-a phenotypic screen of P. aeruginosa mutants lacking such enzymes was performed. Mutants lacking one of four lytic transglycosylases (LTs) or the nonessential penicillin-binding protein PBP4 (dacB) had altered ␤-lactam resistance. mltF and slt mutants with reduced ␤-lactam resistance were designated WIMPs (wall-impaired mutant phenotypes), while highly resistant dacB, sltB1, and mltB mutants were designated HARMs (high-level AmpC resistant mutants). Double mutants lacking dacB and sltB1 had extreme piperacillin resistance (>256 g/ml) compared to either of the single knockouts (64 g/ml for a dacB mutant and 12 g/ml for an sltB1 mutant). Inactivation of ampC reverted these mutants to wild-type susceptibility, confirming that AmpC expression underlies resistance. dacB mutants had constitutively elevated AmpC expression, but the LT mutants had wild-type levels of AmpC in the absence of antibiotic exposure. These data suggest that there are at least two different pathways leading to AmpC expression in P. aeruginosa and that their simultaneous activation leads to extreme ␤-lactam resistance.

The Pseudomonas aeruginosa inner membrane protein FimV is among several proteins of unknown function required for type IV pilus-mediated twitching motility, arising from extension and retraction of pili from their site of assembly in the inner membrane. The pili transit the periplasm and peptidoglycan (PG) layer, ultimately exiting the cell through the PilQ secretin. Although fimV mutants are nonmotile, they are susceptible to killing by pilus-specific bacteriophage, a hallmark of retractable surface pili. Here we show that levels of recoverable surface pili were markedly decreased in fimV pilT retraction-deficient mutants compared with levels in the pilT control, demonstrating that FimV acts at the level of pilus assembly. Levels of inner membrane assembly subcomplex proteins PilM/N/O/P were decreased in fimV mutants, but supplementation of these components in trans did not restore pilus assembly or motility. Loss of FimV dramatically reduced the levels of the PilQ secretin multimer through which pili exit the cell, in part due to decreased levels of PilQ monomers, while PilF pilotin levels were unchanged. Expression of pilQ in trans in the wild type or fimV mutants increased total PilQ monomer levels but did not alter secretin multimer levels or motility. PG pulldown assays showed that the N terminus of FimV bound PG in a LysM motif-dependent manner, and a mutant with an in-frame chromosomal deletion of the LysM motif had reduced motility, secretin levels, and surface piliation. Together, our data show that FimV's role in pilus assembly is to promote secretin formation and that this function depends upon its PG-binding domain.Type IV pili (T4P) are thin, long, flexible, and retractable protein filaments. The broad distribution of T4P among bacterial genera correlates with their ability to mediate a wide range of functions, including attachment to surfaces, DNA uptake, and twitching motility. During twitching motility, pili extend from the cell and attach to a surface, and pilus retraction occurs, moving the bacteria forward toward the point of adhesion (41,51). T4P have been classified into two distinct subtypes, T4aP and T4bP, based on differences in structure of the major pilin subunit and in architecture of the assembly systems (3,16,43). T4aP have been characterized extensively in species such as Pseudomonas aeruginosa, Neisseria spp., and Myxococcus xanthus (10,22,23,39,40,46,49,52). T4bP are found predominately in enteric pathogens, where they promote adhesion (37,58) and bacterial aggregation (6).In P. aeruginosa, a large number of genes involved in the regulation, assembly, and dynamics of T4aP function have been identified. These include the major pilin subunit PilA, as well as a conserved set of minor pilin-like proteins (21); the N-methyltransferase/peptidase PilD, essential for processing prepilins into their mature form (53); an inner membrane assembly subcomplex composed of PilM/N/O/P (4, 47); an outer membrane PilQ secretin pore oligomerized with the assistance of the lipoprotein PilF (7, 29); a...

Peptidoglycan plays a vital role in bacterial physiology, maintaining cell shape and resisting cellular lysis from high internal turgor pressures. Its integrity is carefully maintained by controlled remodeling during growth and division by the coordinated activities of penicillin-binding proteins, lytic transglycosylases, and N-acetylmuramyl-l-alanine amidases. However, its small pore size (∼2 nm) and covalently closed structure make it a formidable barrier to the assembly of large macromolecular cell-envelope-spanning complexes involved in motility and secretion. Here, we review the strategies used by Gram-negative bacteria to assemble such macromolecular complexes across the peptidoglycan layer, while preserving its essential structural role. In addition, we discuss evidence that suggests that peptidoglycan can be integrated into cell-envelope-spanning complexes as a structural and functional extension of their architecture.