The anatomy of the cell envelope of a marine vibrio examined by freeze-etching

The structure of the cell envelope of the marine vibrio MW^tO was examined by freeze-etching and other techniques. In thin section the organism was similar to other gram-negative bacteria. The cell envelope appeared as two double-track layers. No intermediate dense layer was observed. Purified peptidoglycan was prepared from cells during the exponential phase of growth by treating cells with hot sodium dodecyl sulfate. The peptidoglycan had a typical amino acid and amino sugar composition and did not have a covalently linked lipoprotein. The material appeared fibrous when observed in the electron microscope. Purified 1 ipopolysaccharide (LPS) was extracted by the hot phenolwater procedure. its composition and appearance was typical. The LPS was used to determine the specificity of anti-LPS antiserum. For f reeze-etching, eel 1 s were suspended in a salt solution containing 0.22M NaCl, 0,026M MgCl , and O.OIM KCl. In some cases 20% glycerol was added as a cryoprotective agent. The cell envelope of this organism freeze-fractured in three planes. One fracture split the cytoplasmic membrane along its fiydrophobic center revealing particle-studded fracture faces similar to those seen in other bacteria. The convex face was more densely covered with particles than the concave face. Large particle-free areas were observed on both faces. The appearance of the faces was the same with and without glycerol. A second fracture revealed smooth particle-free concave and convex faces. This fracture occurred primarily without glycerol, but was occasionally seen in glycerol -treated cells. The third fracture produced a rough convex face and a concave face composed of subunits approximately 1 nm in diameter. In areas where the subunit layer was incomplete it was observed that the subunits were globular and were backed by a smooth surface. This fracture occurred in cells freeze-etched with glycerol, and occasionally the globular surface was seen without glycerol. The outer surface of the cell was exposed by etching in preparations without glycerol. The surface appeared smooth or finely granular, but may have been obscured by a thin eutectic layer. Complementary replicas were prepared and it was demonstrated that the three pairs of fracture faces were in fact apposed and were produced by three fractures. Isolated cell envelopes were prepared by lysing cells in a French pressure cell. The appearance of these envelopes when freeze-etched was similar to v/hole cells. It was observed, however, that the globular surface could be exposed by etching alone indicating that it had separated from the rough surface before freezing. Crude outer membrane material was prepared by washing the cells with NaCl and sucrose solutions. This material freeze-fractured producing smooth concave and convex faces. Specific rabbit anti-LPS antiserum was prepared and used to label the LPS on the cell surface. This antibody was then labelled with ferri in-conjugated anti-rabbit immunoglobulin antiserum. In thin sections these cells appeared coated with a band of ferritin along the outer double-track, but separated from it by an electron transparent space. For freeze-etching the cells were suspended in 0.05M MgC1_. The preparations were deep etched and the presence of the ferritin on the etch face proved that the outer surface of the cell was revealed. The smooth convex fracture face was immediately adjacent to this ferritin coated surface. It was concluded that the envelope fractured at three levels. One fracture split the cytoplasmic membrane. A second split the outer membrane along its hydrophobic center revealing smooth fracture faces. The third fracture exposed the rough convex surface of the rigid layer and a globular layer which separated the rigid layer from the outer membrane.