Yukari Kakita

Mechanism of the photocatalytic inactivation of Lactobacillus casei phage PL-1 by titania thin film

The mechanism of the inactivation of Lactobacillus casei phage PL-1 suspended in a phosphate buffer by black-light (BL) -catalytic titanium dioxide (TiO2) thin film was studied. Generation of both superoxide anions (O2−) and hydroxyl radicals ( · OH) was confirmed in the aqueous medium in which TiO2 film was settled with BL irradiation under gentle shaking. With BL-irradiation alone without TiO2 film, only O2− was generated to some extent. The genome DNA inside the phage particles was found to be fragmented by the treatment of PL-1 phages with BL-catalytic TiO2 film. The phage inactivation by BL-catalytic TiO2 film was inhibited by the addition of albumin in a concentration-dependent manner. BL-catalytic TiO2 film was considered to cause primarily the damage to the capsid protein through the generation of active oxygen species such as · OH, followed by damage to the genome DNA inside the phage particles.

Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL‐1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first‐order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage‐inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.

Effect of ionic strength on the inactivation of micro-organisms by microwave irradiation.

Microwave irradiation at 2450 MHz inactivated the cells of Escherichia coli, Staphylococcus aureus and Candida albicans suspended in a phosphate buffer. The rate of cell inactivation was proportional to that of the increase in temperature accompanied by microwave irradiation. The inactivation rates of E. coli and C. albicans were affected by addition of NaCl and KCl, but not by sucrose. The maximal inactivation effect was exerted at concentrations of 0·5–1·0 mol l−1, and the end‐point temperature was the highest at the same salt concentrations. Correlation of both the electroconductivity and di‐electric loss of ionic solutions with the heating by microwave irradiation was discussed.

The possible involvement of protein synthesis in the injection of PL-1 phage genome into its host, Lactobacillus casei

The process of genome DNA injection, after adsorption, by phage PL-1 into host cells of Lactobacillus casei was monitored by using the electron microscope. Injection of DNA was inhibited by the protein-synthesis inhibitors chloramphenicol and erythromycin at concentrations where the colony-forming ability of cells not infected by phage was unaffected. The results suggest that protein synthesis may be involved in some way in the process of genome injection.

Electron microscope studies on the host cell energy requirement for injection of PL-1 phage DNA intoLactobacillus casei

The process of genome DNA injection, after adsorption, by phage PL-1 intoLactobacillus casei ATCC 27092 was monitored by electron microscopy. The DNA injection depended on the incubation temperature, and the apparent activation energy was about 11 kcal. It was inhibited when the cells had been previously starved, where their intracellular ATP contents was lowered less than one-hundredth that of the unstarved cells. There was a good correlation between the ATP contents of cells and the extent of the phage DNA injection. Dicyclohexyl carbodiimide inhibited the process with little effect both on the viability of cells and the infectivity of phages. These results agreed with the view that a high energy level of the host cells would be required for the formation of blender-resistant phage-cell complexes to complete injection of phage DNA into host cells.

A temperate phage with cohesive ends induced by mitomycin C treatment of Lactobacillus casei

SummaryA temperate phage, named PL-2, was induced from Lactobacillus casei ATCC 27092 by mitomycin C treatment of the cells at exponential growth phase. The phage had an isometric head of 45 nm in diameter and a flexible, non-contractile tail, 150 nm long and 10 nm wide, with a sharp tip. Along the tail axis, about 40 regularly spaced striae were seen. The phage DNA had complementary cohesive ends. The restriction enzyme map of the DNA was constructed by using 13 different restriction endonucleases. The size of the DNA was 35.2 kb, 83% in size of that of phage PL-1 lytic for the same Lb. casei strain.

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25μF, a transformation efficiency as high as 1.4×107 transformants per μg of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.

Effects of Some Calcium-Related Agents on the Protoplast Transfection of Lactobacillus casei with Phage PL-1 DNA

Abstract. To clarify the mechanism of Ca2+involvement in the DNA transfer through cell membrane, we studied the effects of Ca2+-chelator, Ca2+-ionophore, and Ca2+-channel blocker on the protoplast transfection of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in the presence of Ca2+. Ca2+-chelators, citrate, EDTA, and dipicolinic acid, inhibited the transfection probably by compensating the effect of Ca2+. Ca2+-ionophores, A23187 and N,N,N′,N′-tetracyclohexyl-3-oxapentanediamide, which were expected to accelerate transfection by introducing Ca2+ into cells, inhibited the transfection. This fact indicated the absence of correlation between the entry of Ca2+ and the transport of DNA into protoplasts. Verapamil, which blocks voltage-dependent Ca2+-channel besides β-adrenergic receptor, inhibited the transfection with little effect on the survival of the protoplasts. Both flunarizine and vinpocetine, voltage-dependent Ca2+-channel blockers, did not show the selective toxicity. D-α-Aminoadipic acid, a glutamate receptor-operated Ca2+-channel blocker, had no effect. Propranolol, which blocks β-adrenergic receptor as does verapamil, inhibited the transfection without severely damaging the protoplasts. These results suggested that a kind of receptor-operated Ca2+-channel was involved in the transport of PL-1 phage DNA into the cells and that the cell membrane might have a receptor structure somewhat similar to the β-adrenergic receptor found in mammalian cells.

Involvement of host cell energy in the transfection of Lactobacillus casei protoplasts with phage PL-1 DNA

Transfection of Lactobacillus casei ATCC 27092 protoplasts with phage PL-1 DNA was studied under various conditions. The process of transfection was dependent on the incubation temperature, and the apparent activation energy was calculated to be about 11 kcal/mol. Transfection was inhibited by treating the cells before protoplasting either with monoiodoacetate, N,N′-dicyclohexylcarbodiimide (DCCD), or NaN3, without affecting both the viability of uninfected cells and protoplasting. The addition of DCCD after mixing protoplasts and DNA had no influence on transfection efficiencies. The transfection of L. casei protoplasts with phage PL-1 DNA was considered to require cell energy such as proton-motive force, probably in the initial stages, although the direct involvement of cell energy in the transfer of DNA across the cell membrane is still unclear.