SUMMARYCalcium ions were specifically required for a stage in the formation of phageinfected cells in the growth cycle of PL-I 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 3 ° °C. Among the divalent salts tested SrC12 was as effective as CaC12 in allowing infection, whereas MgSO4, MnC12, BaCI~, BeSO4, ZnSO4 and COSO4 were not. The impairment of infection by lack of calcium ions could not be reversed by the delayed addition of CaCI~.An experiment with host cells infected with [3~P]-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-I 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.

SUMMARYL-rhamnose has been found to be useful to investigate the process of irreversible adsorption of PL-I phage to its host bacterium, Lactobacillus casei ATCC27092. Lrhamnose inhibited phage adsorption to cells without inactivating free phages. Adsorption inhibition was correlated with the concentrations of L-rhamnose. The inhibitory effect of L-rhamnose on phage adsorption was of a competitive nature against host ceils. Among other saccharides tested, L-fucose, L-mannose and o-ribose showed a slight degree of adsorption-inhibiting activity.In early stages of phage adsorption in a tris-maleate buffer, where the binding of phages to cells was still reversible, addition of L-rhamnose resulted in the partial desorption of phages from the cells to which they had adsorbed. However, the number of infective phages desorbed by L-rhamnose treatment gradually fell off as incubation continued, showing that the phages became firmly bound to the cells. Therefore, it is possible to determine the number of phages irreversibly adsorbed to cells by using this desorption technique with L-rhamnose. The process of irreversible phage adsorption, that is, the formation of phage-cell complexes from which no more infective phages could be desorbed, was dependent on temperature and strongly inhibited at o °C.

The polysaccharide (PS) located outside the peptidoglycan layer in Lactobacillus casei ATCC 27092 was found to inhibit the adsorption of PL-1 phage to cell wall preparations without inactivating free phage. Electron microscopic examination of adsorption mixtures showed that the phage were adsorbed to fragments of PS material in a tail-first orientation. Phage did not adsorb to isolated peptidoglycan. The PS was composed of L-rhamnose, D-glucose, Dglucosamine and D-galactosamine, with the hexosamines possibly in N-acetylated form. Prior treatment of L. casei with Streptom-vces haemagglutinin, an anti-human blood group B agglutinin that binds specifically to L-rhamnose, resulted in a concentration-dependent inhibition of phage adsorption. Phage adsorption was inhibited partially by lectins specific for D-glucose and N-acetyl-Dglucosamine, but not by lectins specific for N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. The inhibition of phage adsorption occurred immediately upon the addition of the effective lectins, and was reversed by the addition of the respective lectin inhibitors, a-phenyl galactoside or a-methyl glucoside. The results indicate that L-rhamnosyl residues are the main determinants of the PL-1 phage receptor sites, while D-glucosyl residues may be involved more indirectly.

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