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.     

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.     

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.     

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     

An N-acetylmuramidase induced by PL-1 phage infection of Lactobacillus casei

A lytic enzyme was isolated and purified from PL-1 phage-induced lysates of the host Lactobacillus casei ATCC 27092. The molecular weight of the enzyme was about 30000. Maximum activity on the lysis of the host cell walls occurred at pH 6.0-6.5 and at 45 degrees C. The enzyme activity was inhibited by heavy metal ions, SH- and serine-enzyme inhibitors and o-phenanthroline. The reducing end of the enzymic digest was muramic acid and the enzyme was considered to be an endo-N-acetylmuramidase. However, the enzyme differed from the other known N-acetylmuramidases including hen's egg-white lysozyme in several enzymic properties.     

Growth Inhibition of Lactobacillus casei Phage J1 by Dehydroacetate

The fine structures of amylopectin and intermediate material characteristic of amylomaize starch were investigated by chemical and enzymatic means. In comparison with waxy-maize amylopectin, that of amylomaize starch was found to possess a img/ approximately 10 glucose units longer. Unit-chain profiles of waxy and amylomaize amylopectins revealed that the clear difference lay simply in the relative amounts of two unit-chain fractions. By fractionations of debranched β-limit dextrins, it was demonstrated that the img/ of the internal chains in amylomaize amylopectin was 9 glucose units longer than that in waxy-maize amylopectin. In addition, the proportion of maltose and maltotriose fractions in the debranched dextrin for amylomaize amylopectin was noticeably smaller than found for waxy-maize amylopectin. These data suggest a lesser branching frequency of outer branches in amylomaize amylopectin, confirming the previous proposal that this amylopectin has longer inner and outer branches than those of normal amylopectin.

As for amylomaize intermediate material, the average degree of polymerization was estimated to be 250 to 300 glucose units per molecule. It was also indicated that there were 5 or 6 glucose residues corresponding to the non-reducing end in the molecule. The unit-chain profile of the intermediate material implied that this molecule was mainly composed of branches with img/ around 50. Moreover, the presence of only small amounts of maltose and maltotriose fractions was demonstrated by the unit-chain distribution of this β-limit dextrin. These findings indicate that amylomaize intermediate material is totally consistent with a branched glucan having a low molecular weight, proposing that this anomalous glucan has such a fine structure that four or five branches with img/ around 50 are linked to a main linear chain of 100 to 150 glucose units.     

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.     

in vitro Phage-inactivating Action of d-Glucosamine on Lactobacillus Phage PL-1

A raw-starch-digesting amylase, Dabiase K-27, was immobilized covalently on an enteric coating polymer (hydroxypropyl methylcellulose acetate succinate: AS) as a carrier which is autoprecipitating in an insoluble state below pH 4 as well as reversibly soluble-insoluble depending on pH. Dabiase immobilized on AS (d-AS) showed a sharp response of solubility to slight changes of pH without decrease in enzymatic activity. Moreover, d-AS in an insoluble state had good properties of sedimentation and a large portion of d-AS spontaneously precipitated after lOmin at pH 4.

D-AS was used successively for repeated ethanol production from raw starch, in which d-AS and flocculating yeast cells were separated simultaneously from a product solution by sedimentation in a reactor with a conical bottom. In the five batches of 10% raw starch, the total amount of ethanol produced from 150g of raw starch was 61 g, a value of which corresponds to the average ethanol productivity of 0.85 g/l/hr. The repeated ethanol production by a combination of d-AS and flocculating yeast cells is a promising procedure for effectively using the enzyme and recovering the product solution economically in a heterogeneous culture system containing a solid substrate.     

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.     

Temperature-sensitive Growth of Wild-type Phage J1 of Lactobacillus casei

Wild-type phage J1 of Lactobacillus casei was found to be temperature-sensitive; the phage failed to grow at 40°C, though the host bacteria grew normally at that temperature.

An analysis of phage growth at 40°C revealed the following. (i) Free phage particle was thermostable, (ii) The adsorption of phage led to the penetration of phage DNA. (iii) No lysis of infected cells occurred, but this was not a major block, (iv) No mature phage particle was formed in the infected cells, (v) No phage-related protein was formed in the cells, (vi) The temperature-shift experiment and the radiobiological study indicated that an early stage of intracellular phage growth was blocked.     

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.     

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.     

Prophage Origin of a Virulent Phage Appearing on Fermentations of Lactobacillus casei S-1

For protection from the abnormal fermentation of Lactobacillus casei S-1 caused by contamination of a virulent phage, FSV, the origin of this phage was studied. Morphologies, viral structural proteins, and DNA structures of three independent isolates of FSV were compared with those of FSW, which is lysogenized in strain S-1. The results showed (i) that the morphology of FSV phages is indistinguishable from that of FSW and (ii) that all viral structural components found in FSW are present in the particles of FSV's. In addition, restriction endonuclease analyses of viral DNA showed that the HindIII-digested fragments of FSW DNA, the sum of which covered at least 94.7% of this phage genome, were conserved in the FSV DNA digests. Results of Southern filter hybridization of the S-1 and prophage-cured cell (C239) DNAs with FSV DNA as a probe revealed that C239 had lost most of the FSV DNA sequence, whereas S-1 had about one copy of the FSV DNA sequence. These results indicate that virulent phage FSV is derived from the lysogenized phage FSW. Therefore, the appearance of FSV can be eliminated by using the prophage-cured derivative of S-1.     

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.     

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.