Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation - PubMed (original) (raw)
Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation
Elitza I Tocheva et al. Cell. 2011.
Abstract
Two hallmarks of the Firmicute phylum, which includes the Bacilli and Clostridia classes, are their ability to form endospores and their "Gram-positive" single-membraned, thick-cell-wall envelope structure. Acetonema longum is part of a lesser-known family (the Veillonellaceae) of Clostridia that form endospores but that are surprisingly "Gram negative," possessing both an inner and outer membrane and a thin cell wall. Here, we present macromolecular resolution, 3D electron cryotomographic images of vegetative, sporulating, and germinating A. longum cells showing that during the sporulation process, the inner membrane of the mother cell is inverted and transformed to become the outer membrane of the germinating cell. Peptidoglycan persists throughout, leading to a revised, "continuous" model of its role in the process. Coupled with genomic analyses, these results point to sporulation as a mechanism by which the bacterial outer membrane may have arisen and A. longum as a potential "missing link" between single- and double-membraned bacteria.
Copyright © 2011 Elsevier Inc. All rights reserved.
Figures
Figure 1. Stages of sporulation in A. longum
(A) A vegetative cell showing typical Gram-negative cell wall architecture. (B) A sporulative septum separates the mother cell (M) from the prespore (S). The septum is formed from the inner membrane (IM) of the mother cell. (C) The diameter of the prespore enlarges before engulfment, and the septum eventually turns into the inner spore membrane (IsM) and the outer spore membrane (OsM) of the prespore. (D) Engulfment begins as the IsM and OsM curve and move along the mother cell wall. Storage granules appear at the leading edges of the engulfing membranes (SG, black bodies). (E) The prespore continues to enlarge and eventually becomes spherical. The number and size of SGs increases as engulfment proceeds. (F) Engulfment is completed and a forespore surrounded by an IsM and an OsM is formed in the middle of the mother cell. Each panel is a 20-nm thick tomographic slice through a 3-D reconstruction of an intact cell. Scale bar 200 nm (note E and F are slightly smaller scale to show the entire cell pole). See also Figure S1 and Figure S2, Table S1 and Movie S1.
Figure 2. Spore maturation, germination and outgrowth
(A) A forespore (S) in the middle of a mother cell (M) is surrounded by a double membrane (IsM and OsM) and a PG layer between them. Multiple layers of coat are deposited on the outside of the OsM. (B) The IsM and OsM are separated apart (40 nm) as cortex is synthesized. (C) The main features of a mature spore: core, storage granules (SG), IsM, inner and outer cortex (ICx, OCx, respectively), OsM, coat, exosporium (Ex, also see D). (D) A germinating spore shows that cortex (Cx) hydrolysis is uneven. The IsM and OsM come closer together as the cortex gets degraded. (E) Cortex degradation nears completion before outgrowth begins. (F) Outgrowth of a bacterium shows the new IM and OM that are derived from an IsM and the OsM. Again each panel is a 20-nm thick tomographic slice through a 3-D reconstruction. Scale bars 200 nm. See also Figure S3 and Figure S4.
Figure 3. Structural details
(A) 2 to 3 periplasmic layers are observed between the IM and OM of vegetative cells. (B) Septa at early stages of sporulation exhibit two septal layers of density between the IsM and OsM. The layers are continuous with the innermost periplasmic layer. A layer of protein density is observed on the mother side of the septum. (C) Leading edge of an engulfing membrane. A layer of septal material is present between the IsM and OsM, a protein layer is connected to the mother side of a septum and appears as regularly spaced densities connected to the OsM, and storage granules appear at the leading edge of the engulfing membranes. (D) Advanced stage of engulfment. The number and size of the SG increases, the septal layer between the IsM and OsM is still observed, a protein layer is also present on the mother side of OsM. (E) Presence of periplasmic layers between the IM and the OM of an outgrowing cell. Insets show the area of a cell that has been magnified. Scale bars 200 nm. (F) Immunofluorescence images of quiescent (top) and outgrowing spores (middle and bottom) showing that LPS appears on the surface A. longum cells immediately upon outgrowth. See also Figure S5 and Figure S6.
Figure 4. Higher order structure of a protein layer on the mother side of the septum
(A) Density projections of a protein layer on the mother side of the OsM. Left column: tomographic slices through the cells showing the shape of the OsM and the protein density connected to it. Right column: projection of all the density within 5–15 nm on the mother cell's side of the OsM, revealing concentric, parallel cables. Control projections of density both further into the mother cell and on the spore side of the IsM appear random (data not shown). (B) Schematic representation of the pattern generated by the observed protein layer (red rings) on the mother side of the OsM.
Figure 5. Peptidoglycan is present between the IsM and OsM during engulfment
(A) Images of negatively-stained, collapsed sacculi of engulfing cells. (B) Tomographic slices through reconstructions of purified sacculi of engulfing cells. Black arrows mark the prespore PG and white arrows mark folds in the mother cell PG caused by the collapse of the sacculi on the EM grid. The inset shows a slice where the prespore PG can be seen merging with the mother cell PG at the tip. (C) Two models for the structure and role of PG during sporulation. The key difference is the presence of PG between the IsM and OsM during engulfment, as highlighted in the red intermediate unique to the “continuous PG” model. See also Figure S7 and Figure S8.
Figure 6. SSU rRNA phylogenetic tree
The tree was constructed using the Fitch Distance method with Kimura 2-parameter correction and was based on 1117 unambiguously aligned nucleotide positions. The A. longum 16S gene sequence was aligned to the database, which incorporated the Silva SSU alignment (
), using parsimony methods. Values at nodes represent percent support for 1000 step bootstrap parsimony analyses of the dataset. Bar represents 10% sequence variation. Symbols: ●documented sporulation, Ø sporulation not observed. Where no symbol is given, spore formation was not classified. ✶ Certain members of the phylum Firmicutes are classified as Gram-negative or Gram-variable. See also Figure S9 and Table S2.
Figure 7. Schematic highlighting key findings
Frames 1–3 were taken from Movie S2 and illustrate the transformation of an IM (IM, green) to an OsM (green) and finally the OM (black) of an outgrowing cell. A yellow star is used to show that the IM also gets inverted in the process. PG is shown in red and is present in the septum throughout engulfment, elaborating to form the cortex of the mature spore and then degrading to restore the thin cell wall of the outgrowing cell. See also Movies S2 and S3.
References
- Ben-Harush K, Maimon T, Patla I, Villa E, Medalia O. Visualizing cellular processes at the molecular level by cryo-electron tomography. J Cell Sci. 2010;123:7–12. - PubMed
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