In vivo analysis of the Escherichia coli ultrastructure by small-angle scattering

Abstract

The flagellated Gram-negative bacterium Escherichia coli is one of the most studied microorganisms. Despite extensive studies as a model prokaryotic cell, the ultrastructure of the cell envelope at the nanometre scale has not been fully elucidated. Here, a detailed structural analysis of the bacterium using a combination of small-angle X-ray and neutron scattering (SAXS and SANS, respectively) and ultra-SAXS (USAXS) methods is presented. A multiscale structural model has been derived by incorporating well established concepts in soft-matter science such as a core-shell colloid for the cell body, a multilayer membrane for the cell wall and self-avoiding polymer chains for the flagella. The structure of the cell envelope was resolved by constraining the model by five different contrasts from SAXS, and SANS at three contrast match points and full contrast. This allowed the determination of the membrane electron-density profile and the inter-membrane distances on a quantitative scale. The combination of USAXS and SAXS covers size scales from micrometres down to nanometres, enabling the structural elucidation of cells from the overall geometry down to organelles, thereby providing a powerful method for a non-invasive investigation of the ultrastructure. This approach may be applied for probing in vivo the effect of detergents, antibiotics and antimicrobial peptides on the bacterial cell wall.

Keywords: Escherichia coli; in vivo analysis; small-angle scattering; ultrastructure.

Figure 1

Top: schematic diagram of the E. coli ultrastructure. The diderm cell envelope is distinguished by the presence of the periplasmic space (PP), which is separated from the cytoplasm (CP) by the inner membrane (IM). In turn, the periplasm is separated from the outside by the outer membrane (OM), which is firmly bound to the peptidoglycan layer (PG) inside via Lpp proteins. Bottom: scheme of the core multiple shell SLD profile used to model the bacterial scattering form factor.

Figure 2

(a) Representative USAXS/SAXS from an E. coli suspension and the corresponding fit with (5). Data are for the sample at 0 wt% D₂O in suspension medium with OD₆₀₀ = 10. The sum of the membrane model 〈P _shell(q)〉, the SAW polymer model P _SAW(q) and the constant value is shown in the inset. (b) SANS data at 11, 42, 65 and 100 wt% D₂O were fitted with (5).

Figure 3

Fitting of B _SAW/n values as a function of the nominal D₂O concentration. The minimum of the parabola is close to the matching point for proteins, which is roughly 36 wt% D₂O (1.9 × 10⁻⁴ nm⁻²).

Figure 4

(a) XSLD profile for the system suspended in PBS buffer. (b) NSLD profiles for five different contrast-match points. The x axis refers to the minor radius of the ellipsoid. The same profile is applied all over the ellipsoid. The smearing of the rectangular SLD profile is only used for better visibility.