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 beta-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-alpha-Aminoadipic acid, a glutamate receptor-operated Ca2+-channel blocker, had no effect. Propranolol, which blocks beta-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 beta-adrenergic receptor found in mammalian cells.