Electron microscope studies on the intracellular growth of PL-1 phage of Lactobacillus casei

Ultrathin sections of the cells of Lactobacillus casei infected with or without PL-1 phages were observed by the rapid-freezing and substitution-fixation method. Phage-head-like particles were first observed in the nuclear region. The region was seen more widely dispersed in the cytoplasm than that observed by the conventional chemical fixation method. The features of cells just broken open by the infected phages were observed by the sedimentation method devised by us. The bursting occurred in more than one place in the cells with liberation of progeny phages.     

Preparation and regeneration of bacteriophage PL-1 enzyme-induced Lactobacillus casei protoplasts

Lactobacillus casei ATCC 27092 protoplasts were obtained by treatment with a bacteriolytic enzyme, which was produced in the PL-1 phage lysates of this bacterium, at 37 degrees C for 1.5 h in 50 mM Tris hydrochloride buffer (pH 7.2) containing 20% sucrose and 10 mM MgSO4. The protoplasts lacked the cell wall layer but retained the ability to transport L-[3H]glutamine. The frequency of regeneration was about 1%, whereas about 99% of the cells were osmotically sensitive.     

Preparation and regeneration of bacteriophage PL-1 enzyme-induced Lactobacillus casei protoplasts.

Lactobacillus casei ATCC 27092 protoplasts were obtained by treatment with a bacteriolytic enzyme, which was produced in the PL-1 phage lysates of this bacterium, at 37 degrees C for 1.5 h in 50 mM Tris hydrochloride buffer (pH 7.2) containing 20% sucrose and 10 mM MgSO4. The protoplasts lacked the cell wall layer but retained the ability to transport L-[3H]glutamine. The frequency of regeneration was about 1%, whereas about 99% of the cells were osmotically sensitive.     

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. Received: 11 August 2000 / Accepted: 30 August 2000     

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.     

Calcium Requirement for Protoplast Transfection Mediated by Polyethylene Glycol of Lactobacillus casei by PL-1 Phage DNA

The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca²⁺ , Sr²⁺ increased the transfection rate as well, but Ba²⁺, Mn²⁺, and Mg²⁺ did not. Co²⁺ and Zn²⁺ inhibited transfection. The simultaneous use of Ca²⁺ and Mg²⁺ increased the transfection efficiency. Impairment of transfection caused by lack of Ca²⁺ could not be reversed by the addition of Ca²⁺ later. A decrease in the Ca²⁺ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.     

Effect of Some Lectins on the Adsorption of PL-1 Phage to Lactobacillus casei

The structure of fusariocin C, C₂₇H₂₈O₆ has been established by X-ray diffraction, PMR and IR studies. It is a pseudo dimer structure containing the tropolone skeleton.     

Calcium requirement for protoplast transfection mediated by polyethylene glycol of Lactobacillus casei by PL-1 Phage DNA.

The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca2+. Sr2+ increased the transfection rate as well, but Ba2+, Mn2+, and Mg2+ did not. Co2+ and Zn2+ inhibited transfection. The simultaneous use of Ca2+ and Mg2+ increased the transfection efficiency. Impairment of transfection caused by lack of Ca2+ could not be reversed by the addition of Ca2+ later. A decrease in the Ca2+ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.     

Exposing the Secrets of Two Well-Known Lactobacillus casei Phages,J-1 and PL-1, by Genomic and Structural Analysis

Bacteriophage J-1 was isolated in 1965 from an abnormal fermentation of Yakult using Lactobacillus casei strain Shirota, and a related phage, PL-1, was subsequently recovered from a strain resistant to J-1. Complete genome sequencing shows that J-1 and PL-1 are almost identical, but PL-1 has a deletion of 1.9 kbp relative to J-1, resulting in the loss of four predicted gene products involved in immunity regulation. The structural proteins were identified by mass spectrometry analysis. Similarly to phage A2, two capsid proteins are generated by a translational frameshift and undergo proteolytic processing. The structure of gene product 16 (gp16), a putative tail protein, was modeled based on the crystal structure of baseplate distal tail proteins (Dit) that form the baseplate hub in other Siphoviridae. However, two regions of the C terminus of gp16 could not be modeled using this template. The first region accounts for the differences between J-1 and PL-1 gp16 and showed sequence similarity to carbohydrate-binding modules (CBMs). J-1 and PL-1 GFP-gp16 fusions bind specifically to Lactobacillus casei/paracasei cells, and the addition of l-rhamnose inhibits binding. J-1 gp16 exhibited a higher affinity than PL-1 gp16 for cell walls of L. casei ATCC 27139 in phage adsorption inhibition assays, in agreement with differential adsorption kinetics observed for both phages in this strain. The data presented here provide insights into how Lactobacillus phages interact with their hosts at the first steps of infection.     

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 phenolic compounds, ethyl alcohol, and sodium metabisulphite on the lytic activity of phage PL-1 on a Lactobacillus casei S strain

The effect of phenolic compounds, ethyl alcohol, and sodium metabisulphite on the lytic activity of virulent bacteriophage PL-1 on a Lactobacillus casei S strain isolated from a lactic acid beverage fermentation was investigated. Catechin, caffeic, and gallic acids, commercially produced red, white, and champagne tannins, ethyl alcohol, and sodium metabisulphite inhibited plaque formation. Catechin, caffeic, and gallic acids were the most effective inhibitors of plaque formation. Commercially supplied oenocyanin was not effective.     

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

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. Received: 6 May 1996 / Accepted: 10 June 1996     

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.     

Enhancement of host resistance against Listeria infection by Lactobacillus casei: efficacy of cell wall preparation of Lactobacillus casei

Cell wall, cytoplasm, polysaccharide, and peptidoglycan fractions prepared from Lactobacillus casei, L. plantarum, and L. acidophilus were examined for their efficacies to enhance resistance of host mice against Listeria monocytogenes infection. Intraperitoneal injections of those cellular fractions of L. casei led to elicitation of inflammatory cells in the peritoneal cavity and the efficacy was highest in the case of peptidoglycan. Macrophage ratio in the resultant peritoneal exudate cells was also highest in mice given peptidoglycan. Macrophages induced with cell wall fraction of L. casei showed the most potent phorbol myristate acetate (PMA)-triggered respiratory burst (chemiluminescence and O2- production determined on the basis of nitroblue tetrazolium reduction) followed by those elicited with peptidoglycan. All the macrophages induced with cell wall of L. casei (two strains) and L. acidophilus enhanced O2- production in response to PMA but L. plantarum did not enhance O2(-)-producing ability in such a manner. The L. casei-cell wall also enhanced in vitro listericidal activity of mouse peritoneal macrophages, but such an activity was not noted in the case of L. acidophilus-cell wall. When mice were intravenously given the cellular fractions 7 or 13 days before L. monocytogenes infection, cell wall fractions of L. casei caused the most potent protective activity. A weak protective activity was also found in peptidoglycan of L. casei. Therefore, the protective action of L. casei against L. monocytogenes infection in host mice may be attributed to cell wall compounds and partially to the peptidoglycan moiety.     

Effect of N-Acetyl Substitution at the Amino Sugar Residues on PL-1 Muramidase-catalyzed Hydrolysis of Cell Wall Peptidoglycan of Lactobacillus casei

Recombinant human BSF-2 (B cell stimulatory factor 2/Interleukin 6; IL-6) proteins were purified from CHO and NIH3T3 cell cultures and characterized. The lectin binding patterns suggested that the proteins have N-linked oligosaccharide(s) with tri-antennary structure of bisecting GlcNAc. Their N-termini were highly heterogeneous; at least five closely related N-termini were detercted. This N-terminal heterogeneity was not generated in the cell culture because no processing activity was found in the culture medium.