Isolation and characterization of two plasmid DNAs from endosymbiotic bacteria from Amoeba proteus

Two plasmid DNAs have been detected, isolated, and characterized from endosymbiotic bacteria that have established an obligatory symbiosis with a strain of Amoeba proteus. The two plasmid DNAs were detected by electrophoresis in O.7% agarose gels after removing chromosomal DNA either by density-equilibrium centrifugation in a cesium chloride-ethidium bromide medium or by the sodium dodecyl sulfate-sodium chloride cleared lysate method.

The two plasmid DNAs, designated as pHJll and pHJl2, exist as covalently-closed circles. These plasmid DNAs have molecular weights of 39 x 10⁶ and 14 x 10 ⁶ respectively, as determined by their relative mobilities in agarose gels compared with those of known molecular weight plasmids, and by measuring their contour lengths under the electron microscope. The two plasmid DNAs were not amplifiable by the treatment with chloramphenicol and were curable by either ethidium bromide or acridine orange. pHjll molecule was cleaved into several fragments by the restriction endonuclease Bam HI and pHJl2 was cleaved into several fragments by Eco RI.

The roles played by these plasmid DNAs in the symbiotic xbacteria within amoebae are not understood, but indirect evidence suggests that they are involved in the establishment of endosymbiosis during the early stage of infection of the x-bacteria into the amoeba. The presence of plasmid DNAs within the x-bacteria together with other known properties such as inability of x-bacteria to grow in vitro and at temperatures above 27° C might render these symbionts useful as host for recombinant DNA studies.