Phospholipid metabolism in Pseudomonas BAL-31 infected with lipid-containing bacteriophage PM2.

Infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2 resulted in no detectable change in the rate of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) biosynthesis. An increase in the PG content of infected cultures was not seen until the cultures began to lyse, and this increase was in fact only a relative increase resulting from the extensive turnover of PE at the onset of culture lysis. Turnover studies revealed that the glycerol, phosphorus fatty acid, and ethanolamine moieties of PE turned over simultaneously at the time of lysis, and therefore made it unlikely that there was a PE to PG conversion during the latent period of the phage. The lipid found in the bacteriophage did not reflect a preferential selection for lipid synthesized before or after infection, but in fact reflected the composition of the host membrane at the time the phage were assembled. The use of a modified medium that allowed the cultivation of Pseudomonas BAL-31 as a prototroph and resulted in reliable lysis times of infected cultures led us to the conclusion that PM2 infection effects little change in host phospholipid metabolism, and that there is sufficient PG in the host cytoplasmic membrane to account for a full burst of phage. As a result of the reliable lysis times that we have achieved, we concluded that certain metabolic events, i.e., PE turnover, are lytic phenomena and must not be confused with events relevant to the biosynthesis and maturation of the phage.

Heterogeneity in lipid composition of the outer membrane and cytoplasmic membrane and cytoplasmic membrane of Pseudomonas BAL-31.

The outer membranes and cytoplasmic membranes of the marine bacterium Pseudomonas BAL-31 were separated by washing the cells three times in 0.5 M NaCl and twice in 0.5 M sucrose. Electron microscopy during the removal of membranes revealed that the outer membranes fragmented in a regular manner to give rise to fairly uniform vesicles measuring approximately 140 nm in diameter. Isolated outer membranes had a buoyant density in sucrose of 1.230 g per cm(3), whereas the cytoplasmic membranes had a density of 1.194 g per cm(3). The removal of the outer membrane during the application of this procedure was monitored by measuring the release of 2-keto-3-deoxyoctulosonic acid and phospholipid. The cells lost 85.5% of their 2-keto-3-deoxyoctulosonic acid and 47.3% of their phospholipid during this treatment. Complete recovery of outer membrane material could be achieved. The removal of 25.5% of the 2-keto-3-deoxyoctulosonic acid and 0.9% of the phospholipid rendered the cells sensitive to lysis with Triton X-100. The phospholipid composition of the outer membrane was calculated to be 78.9% phosphatidylethanolamine and 16.1% phosphatidylglycerol. The phospholipid composition of the cytoplasmic membrane proved to be 71.5% phosphatidylethanolamine and 23.5% phosphatidylglycerol. The fatty acid composition was also found to be quantitatively heterogeneous between the two membranes.

Mesosome structure in Chromobacterium violaceum.

Exponentially growing cells of the gram-negative bacterium Chromobacterium violaceum demonstrate invaginations of the cytoplasmic membrane with a high frequency. These invaginations conform to the ultrastructural appearance of mesosomes of gram-positive bacteria. As many as four mesosomes are observed per cell, each of which may increase the total membrane surface of the cell by 30%. Washing of cells in dilute tris(hydroxymethyl)aminomethane buffer effects a distension of the mesosome "neck" and/or cytoplasmic membrane clarifying the association of the mesosome to the cytoplasmic membrane. Plasmolysis effects an eversion of the mesosome into the plasmolysis vacuole.

Organization of bacteriophage tail-like particles in cells of Chromobacterium violaceum.

A strain of Chromobacterium violaceum has been isolated which produces bacteriophage tail-like particles in high numbers. The extracellular morphology and the intracellular arrangement of these particles are described.

Microflora of soil as viewed by transmission electron microscopy.

Several procedures were evaluated for separating and concentrating indigenous microorganisms from soil without the occurrence of growth. Electron microscopy of nontangential, thin sections through these cells revealed that all of the cells examined were less than 0.9 mum in diameter, and up to 72% were "dwarf" cells less than 0.3 mum in diameter. Some were small enough that they should not be resolved with the light microscope. Approximately 27% had a fine structure bearing some resemblance to that of a bacterial cyst or microcyst, but this value may be low because cells having their outer layers partially stripped off were not included in the count. Approximately 25% showed a distinct periplasmic space, which often contained stainable material. Other fine structure features are presented together with frequencies of occurrence for the populations examined.

Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme spheroplasts of Escherichia coli.

Spheroplast production by lysozyme and ethylenediaminetetraacetate (EDTA) was examined as a means of obtaining osmotically sensitive cells for studies of enzyme localization. Physiologically young cells plasmolyzed with 0.5 m sucrose in 0.01 m tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7, 8, or 9) were quantitatively converted to plasmolyzed osmotically sensitive rods after lysozyme treatment. Although such cells were osmotically sensitive, a 1:1 dilution in Tris buffer was necessary for conversion of rods into spheroplasts. Addition of EDTA resulted in a rapid conversion of the plasmolyzed spheroplasts into spherical structures devoid of a plasmolysis vacuole. These structures, which we call EDTA-lysozyme spheroplasts, contained a number of attached membranes. We believe that this conversion results from a weakening of the outer trilaminar component of the cell wall by EDTA, resulting in the collapse of the plasmolysis vacuole. Dilution of sucrose below 0.15 m also resulted in the collapse of the plasmolysis vacuole. Both the lysozyme spheroplasts and the EDTA-lysozyme spheroplasts were osmotically sensitive. Thin sections of the EDTA-lysozyme spheroplasts demonstrated that the outer trilaminar component of the cell wall was broken, exposing large areas of the cytoplasmic membrane to the environment.