Molecular organization of Gram-negative peptidoglycan

Abstract

The stress-bearing component of the bacterial cell wall-a multigigadalton bag-like molecule called the sacculus-is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: ''Layered'' and ''Scaffold'', in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozenhydrated sacculi through electron cryotomography. In the Gramnegative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.cell wall ͉ Cryo-EM ͉ sacculus ͉ tomography ͉ cell shape T he organization of peptidoglycan within the sacculus is a longstanding question in microbiology. Although the sacculus completely envelopes the cell, protecting it from osmotic and mechanical lysis, it is also porous, and thus sieves nutrients and small proteins. Sacculi are constantly remodeled, but they are robust enough that they can be purified from cells and still retain their original shape. Early electron microscope images of purified sacculi were difficult to interpret because the images were 2-D projections through heavy metal-stained sacculi (1, 2). Nevertheless the slender appearance of peptidoglycan in thinsectioned cells (3) and the gaps perpendicular to the polar axis produced by a peptidoglycan-specific endopeptidase (4) lead to the (Circumferential) Layered model (Fig. 1A ) in which the glycan strands lie in the plane of the cell wall and wrap around the cell. The alternative Scaffold model (5) (Fig. 1B) was proposed to fit biochemical data such as pore size and crosslinkage. Various arguments have been made in favor of (6) and against (7) the Scaffold model, but the debate intensified after a recent NMR structure of a synthetic peptidoglycan subunit dimer was interpreted to support the Scaffold model (8). To gain further insight into this issue, we imaged unstained sacculi in 3-D by electron cryotomography (9). Results and DiscussionElectron Cryotomography of Purified Sacculi. Sacculi isolated from C. crescentus strain CB15N and E. coli strains MG1655 and XL-10 were vitrified across holey carbon grids in thin (Ͻ 100 nm) ice. Whereas the gossamer sacculi were barely discernable in the low-dose, 2-D projection images, they were clearly visible in the 3-D tomograms and resembled flattened cells [ Fig. 2 and supporting information (SI) Movie S1 and Movie S2]. In stark contrast to intact cells, the sacculi were flexible and in some positions had radii of curvature less than 20 nm, highlighting how cell shape is not determined by the ''rigidity'' of the sacculus, but rather the cytosolic turgor (osmotic pressure) pushing the cytoplasmic membrane outward against the sacc...

Processivity of peptidoglycan synthesis provides a built-in mechanism for the robustness of straight-rod cell morphology

“…For simplicity and clarity we analyze the effect of processivity on cell straightening independently from the possible contributions of the other mechanisms mentioned above. Here we assume that the PG grows by insertion of new glycan strands parallel to existing ones in a direction that on average is perpendicular to the main cell axis (16)(17)(18). There may be some tilt in the orientation of the glycan strands based on the observation that the insertion happens in helical bands (32).…”

“…The prevalent view proposes that cytoskeletal filaments such as tubulin-like FtsZ and actin-like MreB spatially regulate PG insertion (13-15). Experimental evidence suggests that the cell wall exhibits partial order with glycan strands roughly oriented perpendicular (possibly with some tilt) to the long axis of a rod-shaped cell (16)(17)(18). This orientation may be guided in part by MreB (13), which plays a critical role in maintaining the rod shape, because inactivation of MreB cables causes cells to grow spherically (19,20).…”

Getting into shape: How do rod-like bacteria control their geometry?

“…The rigid PG meshwork counteracts the high turgor pressure set by the difference in osmotic potentials between the cell and its environment . In Gram-negative bacteria electron cryotomography images of isolated cell-wall sacculi suggest that the PG forms a monolayer with glycan strands running in a near-circumferential direction around (Gan et al 2008) ( see Fig. 2).…”

Peptidoglycan architecture can specify division planes in Staphylococcus aureus

“…Th e glycan chain lengths in S. aureus are suffi ciently short to permit either of these confi gurations 16 . However, the planar orientation is adopted in the rod-shaped Gram-negative organisms E. coli and Caulobacter crescentus 17 and the rod shaped Gram-positive Bacillus subtilis has a more complex architecture with apparent helices of very long glycan strands 18 .…”