Shigeshi Takesue

The Requirement for Calcium in Infection with Lactobacillus Phage

Summary Calcium ions were specifically required for a stage in the formation of phage-infected cells in the growth cycle of PL-1 phage active against Lactobacillus casei atcc 27092. Both the adsorption of phage on to the host cells and the intracellular multiplication of phage were independent of the presence or absence of calcium ions. The optimum pH for successful infection was 5.5 to 6.0 and the optimum temperature was approximately 30 °C. Among the divalent salts tested SrCl2 was as effective as CaCl2 in allowing infection, whereas MgSO4, MnCl2, BaCl2, BeSO4, ZnSO4 and CoSO4 were not. The impairment of infection by lack of calcium ions could not be reversed by the delayed addition of CaCl2. An experiment with host cells infected with [32P]-labelled phage showed that calcium ions were required for the penetration of phage genomes into the host cells. The host cells, which had adsorbed phage but not permitted the penetration of phage genomes owing to the absence of calcium ions, were not killed by the phage particles. PL-1 phage infection was, therefore, abortive in the absence of calcium ions owing to the inhibition of a stage of the formation of phage-infected cells involving penetration.

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.

Energy requirement for the formation of blender-resistant complexes in Lactobacillus phage infection.

Summary The formation of blender-resistant complexes signifying the complete penetration of PL-1 phage genomes into the host cells, Lactobacillus casei atcc27092, was studied in a tris-maleate buffer using the technique of blending described by Hershey & Chase (1952). The process was not affected much by the age of the cells, but it was greatly inhibited when the cells had been previously starved. The process was dependent on the temperature, and therefore it was considered to be an energy-requiring process. Apparent activation energy (µ) was calculated to be about 8.3 kcal. The process which had been first inhibited at 0 °C was recovered at any time when the temperature was raised to 37 °C. Among the metabolic inhibitors tested, 10 mm-arsenate inhibited the process selectively without affecting either the infectivity of phage particles or the viability of the cells. On the contrary, cyanide, azide, arsenite, monofluoroacetate, dinitrophenol, pentachlorophenol, dicumarol, chloramphenicol and gramicidin S did not exhibit such a selective inhibition. The significance of the active metabolism of the cells for the mechanism of phage genome penetration is discussed.

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.

Activate sludge treatment of slacked lime pretreated formalin containing hospital waste.

石灰処理して無毒化した病院ホルマリン廃液のCOD値を活性汚泥法で低下させる実験を行った. (1) 円筒型ガラスビン(容量2l)中で試料1容と活性汚泥2容との混合液(COD 100～400mg/l, MLSS 3000～8400mg/l)を曝気すると,混合液のCOD値は1夜で60mg/l以下に減少した. (2) この曝気処理を1日, 1回, 3分の1回分方式で繰り返すと,経日的に活性汚泥は試料に馴致され, COD値の減少速度はしだいに速くなり,数日でほぼ最高値に達した. (3) 活性汚泥法による既設排水処理場(曝気槽170m3×2)に試料(COD 62,000 mg/l)201lを投入速度約3l/分で投入した場合,曝気槽出口のCOD値は1時間後に最高33mg/lに達したが,以後しだいに減少し,通常の運転時(雑排水のみの対照)と同じ20mg/l以下になった.放流水は無色透明で,そのCOD値は常に20mg/l以下であった. (4) 1日量108lの試料を隔日的に3回連続処理した場合も, COD値, SVI,透視度等は試料を投入しない対照とほとんど同じ良い値を示した. 以上のことから,病院ホルマリン廃液の石灰処理と活性汚泥法を組み合せた2段階式の浄化処理法の有効性が示された.