Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron 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.

Transmission electron microscopy of the bacterial nucleoid.

Water-containing biological material cannot withstand the vacuum of the transmission electron microscope. The classical solution to this problem has been to dehydrate chemically fixed biological samples and then embed them in resin. During such treatment, the bacterial nucleoid is especially prone to aggregation, which affects its global shape and fine structure. Initial attempts to deal with aggregation by optimizing chemical fixation yielded contradictory results. Two decades ago, the situation improved with the introduction of freeze-substitution. This method is based on dehydration of unfixed cryo-immobilized samples at low temperature, which substantially reduces aggregation. As a result, the global shape of the nucleoid can be fairly well defined. Overall, in actively growing bacteria, the nucleoids are dispersed and "coralline" but become more confined when growth ceases. However, it is usually impossible to determine the molecular arrangement of DNA in the nucleoids of freeze-substituted bacteria because crystallization and the subsequent removal of water during substitution result in unavoidable distortions at the ultrastructural level. Recently, cryo-electron microscopy of vitreous sections has enabled the fully hydrated bacterial nucleoid to be studied close to the native state. Such studies have revealed aspects of bacterial nucleoid organization that are not preserved by freeze-substitution, including locally parallel or twisted bundles of DNA filaments, which are more frequently observed once bacterial growth has stopped, whereas in actively growing bacteria, the DNA is seen to be in a mostly disordered pattern.