Role of plasmid DNAs in the establishment of symbiosis between amoebae and bacteria

The possible roles of plasmid BNAs in the establishment of symbiosis between symbiotic X-bacteria and xD strain of Amoeba proteus were studied by testing the infectivity of X-bacteria after curing with plasmid-curing agents. For the purpose of this test, plasmids were eliminated from X-bacteria in vivo and in vitro. First, plasmid DNAs were cured by culturing xD amoebae in a medium containing 10^-6 M acridine orange or 10^-5 M ethidium bromide for up to 2 weeks. For curing in vitro, isolated X-bacteria were incubated with plasmid-curing agents for up to 12 hours at 4%deg;C. After treatment with plasmid-curing agents, the relationship between the loss of infectivity and the absence of plasmid DNAs was determined. Infection kinetics were studied by poly-L-lysine-induced phagocytosis, and the presence or absence of plasmid DNAs was verified by agarose-gel electrophoresis.

By studying the infection kinetics of the plasmid-cured X-bacteria, it was shown that X-bacteria which had been treated with plasmid-curing agents disappeared completely from the amoeba cytoplasm within a few days after experimental infection and failed to establish new symbiotic bacterial populations. X-Bacteria isolated from xD amoebae cultured with plasmid-curing agents for 7 days and 14 days disappeared within 72 and 48 hours respectively after experimental infection. When X-bacteria had been cured for longer than 9 hours in vitro, no X-bacteria were detected after 96 hours of infection.

Agarose-gel electrophoretic studies of DNAs showed that the loss of infectivity of X-bacteria in the symbiont-free amoebae was caused by the elimination of plasmid DNAs from the symbiotic bacteria. It appeared that the amount of plasmid UNA of X-bacteria, isolated from xD amoebae grown in the medium with plasmid-curing agents, decreased as the length of the culture period increased. No plasmid DNA was detected in X-bacteria isolated from xD amoebae that had been grown in plasmid-curing agents for longer than 7 days. At that time, X-bacteria lost their infectivity due to the subsequent loss of plasmid DNA which they normally harbor.

The results presented in this study support the idea that plasmid DNAs in the symbiotic bacteria play important roles in the establishment of symbiosis between amoebae and bacteria.