Thickness and Elasticity of Gram-Negative Murein Sacculi Measured by Atomic Force Microscopy

Abstract

Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi from Escherichia coliK-12 and Pseudomonas aeruginosa PAO1. Air-dried sacculi from E. coli had a thickness of 3.0 nm, whereas those fromP. aeruginosa were 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. When E. colisacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 × 107 N/m2 was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 × 108 to 4 × 108N/m2 and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intactE. coli sacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927–953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial Physiol. 24:301–366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.

Peptidoglycan structure and architecture

“…Under normal growth conditions results suggest that E. coli has about two times as much peptidoglycan as would be strictly required to preserve physical integrity, normal shape and growth parameters (Prats & de Pedro, 1989;Caparros et al, 1992). It is interesting to note that recent measurements of cell wall thickness in E. coli and P. aeruginosa indicate that the former is twice as thick as the latter (Yao et al, 1999;Matias et al, 2003). If indeed E. coli does have an 'excess' of peptidoglycan relative to other species as P. aeruginosa, then Gram-negative bacteria should have to be considered a more structurally heterogeneous group than formerly thought.…”

“…However, this somewhat simplistic vision seems to be far from reality and the bacterial sacculus is proving itself to be a particularly intractable subject for structural studies. Application of even the more powerful tools in structural analysis, as X-ray diffraction, EM, AFM, low angle neutron scattering, and others have provided only limited information (Formanek et al, 1976;Labischinski et al, 1979Labischinski et al, , 1985Labischinski et al, , 1991Yao et al, 1999;Matias et al, 2003). Perhaps the most important concept to derive from these studies is that the distribution of subunits in the sacculus is far from the highly regular 'quasi crystalline' schemes proposed in the past and so often used to represent the cell wall.…”

“…The more controversial aspect related to the structure of the sacculus is the orientation and distribution of the glycan strands (Formanek et al, 1976;Verwer et al, 1978;Höltje, 1998;Koch, 1998aKoch, , b, 2000aDmitriev et al, 1999Dmitriev et al, , 2003Yao et al, 1999;Pink et al, 2000;Vollmer & Höltje, 2004;Meroueh et al, 2006). Most available evidence favoured models postulating glycan strands oriented parallel to the cell surface, and in most cases with the glycan backbones transversal to the cell longitudinal axis.…”

Cell wall synthesis is necessary for membrane dynamics during sporulation of Bacillus subtilis

“…Finally, since cell wall peptidoglycan has a Young's modulus of 1.7-25 MPa (Yao et al, 1999;Francius et al, 2008), it is up to 10 4 times stiffer than actin, so formation of mature peptidoglycan would produce mechanical work.…”

Enabling cell–cell communication via nanopore formation: structure, function and localization of the unique cell wall amidase AmiC2 of Nostoc punctiforme

“…Nevertheless, the PGN of most cyanobacterial strains is thicker than in typical Gram-negative bacteria. Its thickness is estimated at approximately 12 nm versus approximately 6 nm in E. coli [9, [37][38][39][40][41]. The higher activity of AmiC2 may therefore reflect the need for efficient processing of the thicker, more complex PGN in cyanobacteria.…”