Prokaryotic Symbionts of Amoebae and Flagellates

Abstract

Amoebae and flagellates have long been known to be associated with both extracellular and intracellular symbionts (Hall, 1969; Kirby, 1941a; Lee et al., 1985). The presence of prokaryotic symbionts on and in flagellates and in some amoebae, as observed by light microscopy, was reported by several authors during the late 1800s and the early part of this century, as was comprehensively reviewed by Kirby (1941a). Symbiont-bearing flagellates were chiefly found in termite guts, and only a few free-living flagellates were found to have adhering symbionts. Hall (1969) extensively reviewed the literature on symbionts of protozoa published since 1941. Both in flagellates and amoebae, the suspected presence of some of the small bacterial symbionts had to be confirmed later by more sophisticated methods such as electron microscopy and specific staining. Amoebae and flagellates represent two very diverse groups of protozoa and it is not possible to cover all known prokaryotic symbionts in depth. In this chapter, we shall simply list known symbionts described in the above two reviews in a tabular form (Tables 1 and 2) and then consider newly found symbionts or results of recent studies on earlier symbionts in some detail. The significance of symbiotic relationships remains obscure in most cases and in only a few instances has the host-symbiont relationship been studied in detail. Some disagreements remain about whether the term “symbiosis” should be limited to associations where definite benefits have been proven to exist or not. In this chapter, we shall use the broader definition of symbiosis to include parasitism, commensalism, and mutualism, as did Kirby (1941a) and Hall (1969). Thus, the list of prokaryotic symbionts will include those whose relationships to their hosts are not known or may not be mutually beneficial. The heightened interest in cellular symbiosis in recent years has been stimulated, in part, by the notion that eukaryotic cell organelles such as mitochondria, chloroplasts, and microtubules may have originated from endosymbionts, i.e., the Serial Endosymbiosis Theory (Sagan, 1967; Margulis, 1970, 1981; Taylor, 1974; papers in Lee and Fredrick, 1987). Some authors have felt that the role of endosymbiosis in the origin of eukaryotic cell organelles has not yet been clearly established (Gray and Doolittle, 1982), but the theory is gaining wider support in view of recent results on the close relationship between the ribosomal RNAs of prokaryotes and those of chloroplasts and mitochondria (e.g., Watson et al., 1987). It should be noted that an opposing view has existed, according to which such organelles evolved as a result of autogenous intracellular differentiation without involving symbionts (Cavalier-Smith, 1975; Raff and Mahler, 1972; Uzzell and Spolsky, 1974, 1981). Meanwhile, it is interesting to note that Pelomyxa palustris, which does not have mitochondria (Daniels et al., 1965; Leiner and Wohlfeil, 1953) contains several types of intracellular symbionts (Daniels, 1973), and the suggestion has been made that such symbionts may carry out metabolic functions in place of mitochondria (Chapman-Andresen, 1971). Bacteria present in P. palustris have been found to be methanogenic (van Bruggen et al., 1983, 1985; see also The Methanogenic Bacteria) and may function as electron sinks related to energy production, comparable to mitochondrial function in aerobic eukaryotic cells. In the case of amoeba-bacteria symbiosis, the D strain of Amoeba proteus became spontaneously infected with a large number (60,000–150,000 bacteria per amoeba) of rod-shaped Gram-negative bacteria (Jeon and Lorch, 1967). Initially, the bacteria were harmful and brought about damaging effects to their hosts, called xD amoebae, such as reduced cell size, slower cell growth, increased membrane fragility, sensitivity to starvation, and a poor clonability. When introduced into symbiont-free D amoebae, the bacteria multiplied and killed their new hosts within a few host cell generations. However, adverse effects of infection gradually diminished over a period of about 1 year, and the bacteria became less virulent, bacteria-bearing xD amoebae growing well with near-normal growth rates. Also, some of the newly infected D amoebae survived, indicating a reduced virulence as compared to earlier infection. Within a few years, host amoebae became dependent on their endosymbionts (Jeon, 1972). Thus, xD amoebae lost viability when they were deprived of endosymbionts either by nuclear transplantation (Jeon and Jeon, 1976), by treatment with antibiotics (Jeon and Hah, 1977), or by raising the culture temperature (Jeon and Ahn, 1978). Aposymbiotic xD amoebae could be resuscitated only by reintroducing live X-bacterial symbionts (Lorch and Jeon, 1980). Newly infected amoebae became dependent on their symbionts after about 200 cell generations or 18 months. The reason for the hosts’ dependence is not known, but preliminary evidence suggests that a symbiont-synthesized protein may be required for the survival of hosts. When xD amoebae are grown in the presence of chloramphenicol (100–700 �g/ml) or rifampicin (125�g/ml), the synthesis of a unique 29-kDa polypeptide by endosymbionts is instantly suppressed and xD amoebae die much sooner than do symbiont-free D amoebae (Kim and Jeon, 1986, 1987a). The symbiont’s gene coding for the xD-specific protein has been cloned (Park and Jeon, 1988) and its nucleotides sequenced (Park and Jeon, 1989). The symbiotic bacteria were found to accumulate host actin selectively (Kim and Jeon, 1987b), as studied using a monoclonal antibody against the amoeba actin. Thus, in this example, the transition of spontaneously infecting parasites to required cell components was observed while it occurred (Jeon, 1980, 1983, 1986, 1987). While the host’s dependence on symbionts developed over 200 host cell generations, some physiological characters changed after a few host cell divisions (Lorch and Jeon, 1981, 1982). In Blastocrithidia culicis and Crithidia oncopelti, symbiotic bacteria were found to supply their hosts with lysine (Gill and Vogel, 1962, 1963), hemin (Guttman and Eisenman, 1965; Chang and Trager, 1974; Newton, 1956, 1957), and other nutritional factors. An aposymbiotic host, produced by growing symbiont-bearing cells in the presence of chloramphenicol, required exogenous hemin for growth. The ectosymbiotic spirochetes on Myxotricha were found to help their host move by their coordinated undulation while the host’s flagella functioned only to steer its movement (Cleveland and Grimstone, 1964). Flagella of prokaryotic symbionts attached to Cryptotermes were also found to help propel the host cell (Tamm, 1978b). These are a few examples in which protozoan hosts and prokaryotic symbionts have developed an intimate relationship, and symbiont integration and host-symbiont interactions have been experimentally studied.