Role of Peptidoglycan Amidases in the Development and Morphology of the Division Septum in Escherichia coli

Abstract

Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.Most eubacteria produce multiple hydrolases that cleave different bonds within the peptidoglycan (PG or murein) cell wall: amidases remove peptide side chains from the carbohydrate polymer, endopeptidases cleave cross-linked peptides that connect the glycan chains, and lytic transglycosylases hydrolyze the glycan backbone (24, 47). These PG-specific hydrolases were once thought to be essential for inserting new material into the wall during bacterial growth (24, 25), a view based on the reasonable hypothesis that cross-links between the glycan chains had to be broken so that new PG strands could be incorporated into the existing wall (31). However, bacterial growth continues perfectly well in the absence of these dedicated hydrolases, including almost all of the amidases, endopeptidases, and lytic transglycosylases (14,22,23), meaning that any bond-breaking during normal cell wall elongation must be performed by other enzymes. So why, then, does Escherichia coli carry so many murein-specific hydrolases?In the gram-positive bacteria, PG hydrolases split the septum that separates daughter cells at the end of cell division. For example, in Streptococcus pneumoniae, LytA (an amidase) and LytB (a glucosaminidase) localize to the equatorial and polar regions, respectively, and mutants lacking either hydrolase grow in long chains, with double mutants being especially deficient in cell separation (13,46). In Bacillus subtilis, the DL-endopeptidases ...

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

“…When immobilized sacculi were labeled with anti-PG antibody, they appeared more uniformly fluorescent (Fig. S1B), as reported previously (10). We conclude that septal enrichment of GFP-SPOR constructs reflects binding to a septal PG structure rather than simply a greater abundance of PG in division septa than in the cell cylinder.…”

“…In the model Gram-negative bacterium Escherichia coli, daughter-cell separation is driven primarily by three cell-wall amidases (8)(9)(10). These enzymes cleave the amide bond that joins the lactyl group of the MurNAc to the α-amino group of the first amino acid (L-Ala) of the stem peptide, releasing as products free oligopeptides and "denuded" glycan strands (Fig.…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…AmiA, AmiB and AmiC have a high sequence homology and assist in cell separation during division [133]. Loss of these three amidases affects septal formation resulting in a chaining phenotype with aggregates of three to six cells [83,130]. The activity of these three enzymes is regulated spatiotemporally by proteins with LytM domains, which have conserved sequences and are collectively known as dLytM factors [134].…”

“…The periplasmic amidases remove the amino acid chain from the muramyl moieties of the sacculus as well as from the recycling by-products such as GlcNAc-anhMurNAc peptides [127][128][129][130][131]. E. coli expresses four periplasmic enzymes AmiA, AmiB, AmiC and AmiD that have Nacetylmuramyl-L-alanine amidase activity [129,132,133].…”

Overview of cell shape: cytoskeletons shape bacterial cells

“…Transglycosylases are part of the general PG synthetic machinery and are not specific for cell division. The Z ring also recruits PG hydrolases necessary for progression of septation and for the separation of the daughter cells after division [10][11][12]. Importantly, mutations in ftsZ that markedly reduce the GTPase activity produce a spiral FtsZ resulting in a spiral septum indicating that the morphology of the septal FtsZ structure dictates the pattern of PG growth [13,14].…”