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

Enrico F Semeraro,Lionel Porcar,Juliette M Devos,Theyencheri Narayanan,V Trevor Forsyth

doi:10.1107/s2052252517013008

Enrico F Semeraro, Lionel Porcar + Show 3 more

Open Access

https://doi.org/10.1107/s2052252517013008

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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.

Highlights

Escherichia coli is a model prokaryotic cell classified as a Gram-negative bacterium whose morphology has been studied over the last 60 years (Lieb et al, 1955) using different techniques including optical microscopy (Latimer, 1979), light scattering (Wyatt, 1970), atomic force microscopy (Lonergan et al, 2014) and X-ray imaging (Miao et al, 2003)
We present a method for multiscale structural analysis of the E. coli bacterium with the aim of providing a framework for quantitative structural elucidation of any diderm prokaryotic cells
The comparison between SAXS and SANS measurements allowed the mutual exclusion of both X-ray radiation damage and toxic effects owing to the D2O medium

Summary

Introduction

Escherichia coli is a model prokaryotic cell classified as a Gram-negative bacterium whose morphology has been studied over the last 60 years (Lieb et al, 1955) using different techniques including optical microscopy (Latimer, 1979), light scattering (Wyatt, 1970), atomic force microscopy (Lonergan et al, 2014) and X-ray imaging (Miao et al, 2003). Cryo-TEM imaging needs to be performed on thin cell sections obtained after highpressure freezing and cryosectioning (Matias et al, 2003) or other more invasive methods (Hobot et al, 1984) that may introduce artefacts. Progress in these methods has allowed a better understanding of the bacterial ultrastructure (Milne & Subramaniam, 2009), in particular shedding light on the spatial arrangement within the cell envelope.

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