Thickness and elasticity of gram-negative murein sacculi measured by atomic force microscopy

Atomic force microscopy was used to measure the thickness of air-dried, col lapsed murein sacculi from Escherichia coil K-12 and Pseudomonas aeruginosa PAO1. Air-dried sacculi from E. coli had a thickness of 3.0 nn, whereas th ose from P. aeruginosa were 1.5 nm thick. When rehydrated, the sacculi of b oth bacteria swelled to double their anhydrous thickness. Computer simulati on 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 resu lts. When E. coil sacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope u sed to depress and stretch the peptidoglycan, an elastic modulus of 2.5 x 1 0(7) N/m(2) was determined for hydrated sacculi; they were perfectly elasti c, springing back to their original position when the tip was removed. Drie d sacculi were more rigid,vith a modulus of 3 x 10(8) to 4 x 10(8) N/m(2) a nd at times could be broken by the atomic force microscope tip. Sacculi ali gned over the groove with their long axis at right angles to the channel ax is were more deformable than those with their long axis parallel to the gro ove 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 elastic ity of intact E. coli sacculi is such that, if the peptidoglycan strands ar e aligned in unison, the interstrand spacing should increase by 12%,vith ev ery 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated in terstrand spacing of 1.3 nm (R. E. Purge, A. G. Fowler, and D. A. Reaveley, J. Mel. 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, 1 983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clea rly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must s omehow be disrupted.