In vivoanalysis of theEscherichia coliultrastructure by small-angle scattering

Abstract

The flagellated Gram-negative bacteriumEscherichia coliis 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 probingin vivothe effect of detergents, antibiotics and antimicrobial peptides on the bacterial cell wall.

Structure of LetB reveals a tunnel for lipid transport across the bacterial envelope

“…Grey cylinders represent transmembrane helices (TM, not present in the structure) at their expected positions. The width depicted between the IM and OM is within a range of 210-240 Å, based on previously measured periplasmic widths, 210 Å 22 and 230 Å 21 , and deduced from the periplasmic spanning region of AcrAB-TolC, 240 Å 59 . sequence specifying one ring in the resulting structure.…”

“…2d). The length of LetB is ~220 Å, comparable to the width of the periplasmic space [19][20][21][22] (Fig. 2e) and to the length of the periplasm spanning region of AcrAB-TolC 23,24 .…”

“…Given that the length of MCE proteins varies with the number of MCE domains, and the length of LetB is approximately the same as the reported width of the periplasm in E. coli [19][20][21][22] , we hypothesised that LetB may physically bridge the gap between the IM and OM, creating a continuous path for lipid transport. If this model is correct, then the length of LetB would be important for its function.…”

Increasing complexity in small-angle X-ray and neutron scattering experiments: from biological membrane mimics to live cells

“…Nevertheless, TEM has been instrumental for constraining the multiscale scattering model of E. coli. 24 The model accounts for all cellular contributions by combining ultra-SAXS (USAXS)/SAXS and contrast-variation SANS. Note that USAXS extends the range of studied length scales to a few mm and a recent upgrade in synchrotron X-ray instrumentation for USAXS 102,103 allowed to fully exploit this technique for bacteria.…”

“…In addition to all these bottom-up approaches of increasing system complexity, several groups have started to work out the scattering contributions of natural systems, starting from the early work on nerve myelin 1,2 and red blood cells 22 (which were still studied under quite dehydrated conditions) to live cells such as bacteria. 23,24 This opens up new avenues to bridge the gap between studies on model membranes and cells using the same techniques, i.e. with comparable experimental windows.…”