Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ.

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a "hibernation state." Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction.

Lipoteichoic acid is a major component of the Bacillus subtilis periplasm.

Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.

Cryo-electron microscopy of cell division in Staphylococcus aureus reveals a mid-zone between nascent cross walls.

Cryo-electron microscopy of frozen-hydrated thin sections permits the observation of the real distribution of mass in biological specimens allowing the native structure of bacteria to be seen, including the natural orientation of their surface layers. Here, we use this approach to study the fine ultrastructure of the division site, or septum, of Staphylococcus aureus D(2)C. Frozen-hydrated sections revealed a differentiated cell wall at the septum, showing two high-density regions sandwiched between three low-density zones.

Native cell wall organization shown by cryo-electron microscopy confirms the existence of a periplasmic space in Staphylococcus aureus.

The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e.

Cryo-electron microscopy reveals native polymeric cell wall structure in Bacillus subtilis 168 and the existence of a periplasmic space.

Ultrarapid freezing of bacteria (i.e. vitrification) results in optimal preservation of native structure.

DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential.

Natural membrane vesicles (n-MVs) produced by Pseudomonas aeruginosa PAO1 and PAO1 carrying plasmid pAK1900 (p-MVs) were purified and analysed for DNA content. The MVs were isolated by a procedure designed to ensure no cellular contamination from the parent MV-producing cells. Fluorometry analysis revealed that p-MVs were associated with 7.

Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa.

High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material.