Endosymbiotic bacteria in Amoeba proteus strain xD : characterization and comparison of microorganisms associated with symbiotic and non-symbiotic amoebae

A strain of Amoeba 1 proteus (xD) containing bacterial endosymbionts (x-bacteria) was isolated from laboratory cultures of A. proteus strain D, in 1966. The bacteria are Gram-negative rods contained within vesicles in the amoeba cytoplasm and are required for normal functioning of the amoeba nucleus. The bacteria are able to infect nonsymbiotic amoebae and can become required cytoplasmic components in about 200 macrosymbiont generations. Symbiotic amoebae cannot grow at temperatures above 26° C, a characteristic mediated by the endosymbionts.

The purpose of this study was to further characterize x-bacteria, and to ascertain the possible relationships of x-bacteria to more typical, free-living, Gram-negative rods.

Antisera to x-bacteria were prepared and evaluated by agglutination and indirect fluorescent antibody (IFA) tests. Ten strains of bacteria isolated from amoeba cultures were tested by the IFA method to determine possible cross-reactivity with x-bacteria. Attempts to culture x-bacteria in vitro were made out. The numbers and kinds of free-living bacteria present in D and xD cultures were compared by bacteriological techniques. Bacteria with two different electron densities

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within xD amoeba vesicles were compared by statistical methods.

The major findings of the study were:

1) The x-bacteria stimulate antibody production in rabbits, as demonstrated by IFA tests;

2) Antibody to x-bacteria is not produced in rabbits or rats is not detectable by agglutination tests. Immunizing doses were administered which have been shown to result in high antibody titers to antigens prepared from other Gram-negative bacteria;

Bacteria of the genus Xanthomonas were found to be associated only with xD amoebae. While isolates of Xanthomonas are not immunologically crossreactive with x-bacteria, the two organisms do seem to have some characteristics in common.

It was concluded that x-bacteria are less antigenic than other Gram-negative rods. Some unique feature of the x-bacteria cell wall may be responsible for this phenomenon, and could explain the ability of x-bacteria to avoid digestion by amoebae as well.

The x-bacteria could not be cultured in vitro. However, based on studies of organisms present in amoeba culture dishes, the bacterial species ancestral to x-bacteria is believed to be an organism very much like Xanthomonas.

Statistical comparison of the mean lengths of dark and light bacteria seen within xD vesicles suggests that two distinct types of bacteria are present in the amoeba cytoplasm. . The implications of this finding are discussed in terms of what is known about bacterial endosymbionts of Paramecium aurelia.

A possible mechanism for the establishment of the endosymbiosis between x-bacteria and xD amoebae is proposed.