The molecular architecture of engulfment during Bacillus subtilis sporulation.

Kit Pogliano,Kanika Khanna,Ziyi Zhao,Joseph Sugie,Yuan Yuan,Reika Watanabe,Elizabeth Villa,Javier Lopez-Garrido

doi:10.7554/elife.45257

Abstract

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

Highlights

From an architectural point of view, bacterial cells are among the simplest forms of life on the planet
Their cytoplasm is typically devoid of membrane bound organelles, and their cellular morphology relies upon a semi-rigid peptidoglycan (PG) cell wall that imposes its shape on the malleable cell membrane(s)
We used cryo-FIB-ET to illustrate the role of DNA translocation in inflating the forespore (Lopez-Garrido et al, 2018). These data confirmed the presence of a thin layer of PG between the forespore and the mother cell membranes in the wild type strain, as previously visualized by cryo-electron tomography (cryo-ET) of a slender ponA mutant of B. subtilis (Tocheva et al, 2013)

Summary

Introduction

From an architectural point of view, bacterial cells are among the simplest forms of life on the planet. Microbiology and Infectious Disease Structural Biology and Molecular Biophysics eLife digest Much of what happens in biology occurs at scales so small that the microscopy methods traditionally used by biologists cannot visualize them One such process is bacterial sporulation: in stressful conditions, bacteria like Bacillus subtilis can divide to produce a smaller cell called a forespore, which the larger mother cell engulfs. By combing cryo-ET with another methodology that allowed them to focus in on thin sections of their sample, Khanna et al generated high resolution images, which provided a look at forespore engulfment in unprecedented detail These images revealed that the peptidoglycan wall separating the mother cell from forespore is not completely degraded: a thin layer of peptidoglycan persists. The methodology, images and analyses presented here will provide valuable resources for future studies of spore assembly and other fundamental cell biological processes

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health