Kazuko Ishibashi

A phage-resistant mutant of Lactobacillus casei which permits phage adsorption but not genome injection

A phage-resistant mutant of Lactobacillus casei (strain YIT 9021) was capable of adsorbing both PL-1 and J-1 phages, but did not yield phage-infected cells. The mutant and wild-type strains were identical in morphology and sugar composition of the cell walls. Attempts to induce prophages from strain YIT 9021 were unsuccessful. Electron microscopic examination of negatively stained mixtures of phage PL-1 and YIT 9021 bacteria revealed that the phages were adsorbed to the cells in a tail-first orientation. All the absorbed phages had DNA-filled heads. It was concluded that PL-1 adsorbed normally but was blocked in the injection of the phage genome into the cell.

Adenosine Triphosphate Content in Lactobacillus casei and the Blender-resistant Phage-Cell Complex-forming Ability of Cells on Infection with PL-1 Phage

The intracellular ATP content of Lactobacillus casei ATCC 27092 grown in a glucose-containing medium was almost constant (2 to 3 microgram/mg dry wt. cells) through the early to middle stage of logarithmic phase, but it was lowered to less than 0.1 microgram/mg after cessation of growth owing to the exhaustion of available glucose. All the cells in the early stage of stationary phase were still viable and thus considered to be in a starved state. When such starved cells were infected with PL-1 phages in a tris-maleate buffer of pH 6.0, the process of forming blender-resistant phage-cell complexes signifying the complete injection of phage genomes into the cells was much inhibited. There was a good correlation between the ATP content of cells and the extent of the formation of blender-resistant phage-cell complexes and the correlation coefficient between them was 0.89 + 0.09 at the 95% confidence limit. On the other hand, the process of forming both the phage-adsorbed cells and the anti-phage serum-resistant phage-cell complexes were not affected by the ATP content of cells. Feeding of glucose to such starved cell cultures caused the cells to restore both the ATP content and the ability to form blender-resistant phage-cell complexes. Such restoration was also observed when the starved cells collected by centrifugation were incubated in a glucose-containing medium. The significance of the intracellular level of high energy compounds such as ATP for the mechanism of the injection of phage genomes into the cells is discussed.

Studies on frost hardiness in Chlorella ellipsoidea. V. The role of glucose and related compounds

Abstract Chlorella ellipsoidea cells at an intermediate stage in the ripening phase of the life cycle were hardened at 3°C. d -Glucose added during hardening in the dark induced a tolerance to freezing. Sucrose, d -galactose and d -fructose were also capable of increasing hardiness, but pyruvate, succinate and citrate were not. These results suggest the involvement of the pentose-phosphate cycle in the hardening process. The amounts of monosaccharide and dextrin in Chlorella cells did not change during hardening. Sucrose concentration increased during hardening in both the light and the dark in the presence of glucose. Starch concentration remained unchanged in the dark in the presence of glucose, but increased remarkably in the light. These results suggest that the hardening process involves an accumulation of sucrose, and indicate that there is no direct correlation between starch content and frost hardiness. Glucose 6-phosphate dehydrogenase activity increased greatly during the early phase of hardening; 6-phosphogluconate dehydrogenase activity also increased with hardiness. These results suggest that pentose-phosphate cycle activity occurs at a high level during hardening. Cycloheximide and oligomycin completely inhibited the hardiness increase in the dark in the presence of glucose; chloramphenicol and DCMU had no effect. These results suggest that protein synthesis on cytoplasmic ribosomes and ATP synthesis in mitochondria are involved in the process of hardening in the dark in the presence of glucose, but protein and ATP synthesis in chloroplasts are not. The inhibition of the hardiness increase by sodium dodecyl sulfate was greater in the light than in the dark in the presence of glucose. Triton X-100 inhibited completely the hardiness increase in both the light and the dark in the presence of glucose. It seems likely, therefore, that membrane alterations during hardening in the dark in the presence of glucose may occur; these alterations are different from those which occur in the light.

Reversibility of the adsorption of bacteriophage PL-1 to the cell walls isolated from Lactobacillus casei.

Bacteriophage PL-1 adsorbed specifically to fragments of the isolated cell walls of its host Lactobacillus casei ATCC 27092 and failed to adsorb to cell wall fragments of resistant strains. Soon after mixing, an equilibrium situation of phage adsorption was attained. The equilibrium position was dependent on the cell wall concentration, but was not affected by the incubation temperature. The adsorbed phages were not inactivated by the cell wall fragments, but formed phage-cell wall complexes maintaining original phage infectivity. The infectivity of phage-cell wall complexes was neutralized by antiphage sera in the same manner as free phages. When the phage-cell wall complexes were repeatedly washed by centrifuging and resuspending in a fresh medium, the adsorbed phages were eluted as infective virions, confirming that phage adsorption was reversible. When the reactants concerned were allowed to approach equilibrium from the opposite direction, the same equilibrium state was achieved. The value of the euqilibrium constant (Keq) with respect to reversible adsorption was constant with various phage concentrations under the conditions used here. When a mixture of phages and cell walls at an equilibrium state was diluted, the unadsorbed phages increased in accordance with the decrease in the concentration of the reactants.