Evolution of the analytical scattering model of live Escherichia coli

Abstract

A previously reported multi-scale model for (ultra-)small-angle X-ray (USAXS/SAXS) and (very) small-angle neutron scattering (VSANS/SANS) of live Escherichia coli was revised on the basis of compositional/metabolomic and ultrastructural constraints. The cellular body is modeled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was replaced by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale. Specifically, this approach provides detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. The model was also successfully applied to E. coli K12, used for the authors' original modeling, as well as for two other E. coli strains. Significant differences were detected between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of the approach outlined here to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labeling agents.

Lactoferricins access the cytosol of Escherichia coli within few seconds

“…Here, end-state refers to one hour of incubation of bacteria at a given AMP concentration. We have recently reported initial-state structures of different E. coli strains at different hierarchical length scales—including size of bacteria, distance between inner and outer membranes, and LPS packing density—in terms of a multi-scale analytical model using joint USAXS/SAXS and (very) small-angle neutron scattering (VSANS)/SANS experiments ( Semeraro et al, 2021 ). The same concept was applied here to reveal the end-state structure of E. coli .…”

“…( A ) Mapping the main structural changes in E. coli ATCC 25922 (green symbols) upon 1 h incubation with LF11-324 (red symbols) as observed by USAXS/SAXS and TEM. Scattering data of E. coli ATCC 25922 are from Semeraro et al ( 2021 ). Black lines are the best fits using Equation 6 .…”

“…Further, the increased scattering contributions originating from O-LF11-215 aggregates impeded the detection of OMVs. Notably, SAS is insensitive to the inner cytosolic structure of bacteria ( Semeraro et al, 2021 ). Hence the peptide induced separation of the nucleoid region from the nucleoid-free cytosol ( Figure 1 A) reported also previously from TEM ( Zweytick et al, 2011 ), is not observed in our scattering data.…”

“…In particular, we report ( i ) contrast changes in terms of the scattering length densities (SLDs) of the cytoplasm, ρ CP , periplasm, ρ PP , and peptidoglycan, ρ PG , ( ii ) microscopic to mesoscopic structural size changes of bacteria, approximated by an ellipsoid of inner radius R , and distance between inner and outer membranes, Δ OM , as well as the corresponding distribution of distances between the two membranes, σ OM , and ( iii ) nanoscopic structural changes as observed for the average number of LPS, N OS , per cell. See Semeraro et al ( 2021 ), for a justification of all used parameters. The decrease of ρ CP , along with the increase of ρ PP , signifies leakage of mainly low-weight molecules from the cytoplasm ( Figure 1–Figure Supplement 2 B-C).…”

“…We have recently reported an analytical model for elastic X-ray and neutron scattering from live Escherichia coli without the need to resort to any specific labelling technique ( Semeraro et al, 2021 ). In particular, we combined the different sensitivities of X-rays and neutrons to matter, including H/D contrast variation, with a compositional multi-scale model.…”