Granular Layer in the Periplasmic Space of Gram-Positive Bacteria and Fine Structures ofEnterococcus gallinarumandStreptococcus gordoniiSepta Revealed by Cryo-Electron Microscopy of Vitreous Sections

Abstract

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryoelectron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.Gram-positive bacteria possess a cell envelope composed of a plasma membrane and a thick network of peptidoglycan, secondary acids, and proteins, which maintains cell shape and provides resistance to osmotic pressure (39). Electron microscopy studies performed on fixed (either chemically or by freezing) and dehydrated specimens favored the widely accepted model of the cell envelope consisting of the peptidoglycan network in direct contact with the plasma membrane (4). Nevertheless, the existence of a periplasmic space separated from the cytoplasm and the outer medium by a plasma membrane and a thick cell wall has been suggested by previous biochemical studies (3,14,25). Recent data obtained with cryo-electron microscopy of vitreous sections (CEMOVIS), a method that allows the observation of biological specimens in the closest-to-native state, challenged the classical model (23,24). CEMOVIS consists of cooling the specimen at high pressure so rapidly that water cannot crystallize and instead becomes vitreous. Water stays liquid, but its viscosity dramatically increases (10). It is then possible to make thin sections at low temperatures and observe them using a cryo-electron microscope (1). Consequently, water does not need to be removed from the sample, and molecular aggrega...

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“…This is not unique in biology. It is only recently that cryoelectron microscopy identified a 2-to 4-nm granular layer on the outer face of the cytoplasmic membrane in grampositive bacteria (35) and a periplasmic space also in grampositive organisms (20). The second explanation is that in prior studies, the envelope was removed by the EM preparation procedure, leaving only the network and the crystalline lamellar layer, which would constitute an electron-dense layer that is approximately 4 nm thick.…”

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

“…The inherently low contrast of frozen-hydrated samples, combined with their beam sensitivity, makes it difficult to find regions of interest without destroying them; this severely limits the benefit of an otherwise powerful technique for answering biological questions. Features that have so far been visualized by CEMOVIS/CETOVIS are, not surprisingly, those that are comparatively easy to discern, such as bacteria and their cell surface, mitochondria and microtubules in cross section (Matias et al, 2003;Zuber et al, 2006;Bouchet-Marquis et al, 2007;Marko et al, 2007).…”

Localization and expression of the Bacillus subtilis dl-endopeptidase LytF are influenced by mutations in LTA synthases and glycolipid anchor synthetic enzymes

“…Cryo-electron microscopy of frozen-hydrated sections has indicated that Gram-positive bacteria such as B. subtilis and S. aureus also have a periplasmic space, termed the inner wall zone, between the plasma membrane and thick CW (Matias & Beveridge, 2005;Matias & Beveridge, 2006;Zuber et al, 2006) and that LTA is a major component of the B. subtilis periplasm (Matias & Beveridge, 2008). Moreover, subcellular localization analysis of UgtP fused to GFP showed that the fusion protein was predominantly localized at the septa and that proper localization was abolished in the pgcA and gtaB mutants (Weart et al, 2007).…”