Masters Theses

Date of Award

3-1983

Degree Type

Thesis

Degree Name

Master of Science

Major

Zoology

Major Professor

Kwang W. Jeon

Committee Members

G. L. Whitson, L. Etkin

Abstract

The symbiotic X-bacteria that hove established an obligatory symbiosis with the xD strain of Amoeba proteus contain two plasmid DNAs, pHJII and pHJ12. In this study, the effect of high temperature on the two plasmid DMAs of X-bacteria was studied by electron microscopy and agarose gel electrophoresis.

By examining the growth rotes of symbiotic and nonsymbiotic amoebae in single cell cultures at different temperatures and the average number of symbionts per amoeba, it is shown that symbiotic amoebae cultured at 27°C grow at the same rate as those grown at 22°C during the first 8 days, after which they cease to divide, duplicating only three divisions on the overage, and then all cytolyze by the 14th day. At 27°C, the average number of symbionts per amoeba declines rapidly, and the symbionts disappear completely from amoebae after 10 days. The depletion of symbionts appears to be the cause of death of xD amoebae which closely follows the disappearance of symbionts.

The growth characteristics of amoebae in mass cultures at 27°C are somewhat different. They survive longer, i.e., over a month, and the average number of symbionts per amoeba in the mass culture at 27°C decreases slower than those in single-cell cultures. The reasons for these differences ore not clear, but it may be that the high density of amoebae and of symbionts per unit area and unit volume of medium causes somehow favorable (compensation) effect on them so that they can alleviate the adverse effect of the higher temperature.

Agarose gel electrophoretic studies of DNAs show that the decrease of plasmids of X-bacteria can be detected from the 8th day of culture at 27°C, whereas X-bacteria still remain healthy. No plasmids were found in X-bacteria after 14 days of culture at 27°C. At that time X-bacteria started to show the symptom of digestion by amoebae as examined by electron microscopy; that is, lysosomes fused with symbiotic vesicles which contain X-bacteria. The Symbiotic bacteria appear to be digested by amoebae after the symbionts have lost the plasmids which they normally harbor. From this study, the role of plasmids in the symbiotic relationship between amoebae and X-bacteria can be speculated; the protection of X-bacteria from digestion by amoebae. It is possible that the piosmids found in X-bacteria encode some polypeptides which ore somehow inserted into the bacterial outer membrane and/or the symbiotic vesicle membrane, so that X-bacteria and symbiotic vesicles are protected from digestion by amoebae. Thus, X-bacteria can succeed in the establishment of endosymbiosis by protecting themselves during initial stages of infection when the X-bacteria are directly exposed to the amoeba cytoplasm and X-bacteria can maintain successful symbiotic relationship with amoebae by prevention of lysosomol fusion with symbiotic vesicle membranes during sustained multiplication of both host and symbiont.

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