Kwang W. Jeon

The Large, Free‐living Amoebae: Wonderful Cells for Biological Studies

The large, free‐living amoebae have been widely used as model cells for studying a variety of biological phenomena, including cell motility, nucleocytoplasmic interactions, membrane function, and symbiosis. Results of studies by our group on amoebae as moving cells, as material for micrurgical manipulations, and as hosts for intracellular symbionts are summarized here. In particular, our recent studies of the amoeba as a microcosm, in which spontaneously infecting foreign microbes have become integrated as necessary cell components, are described in some detail. These processes have involved an initial microbial infection, mutual adaptation by the host and symbionts, and development of obligatory symbiosis. Evidence is presented to show that symbiont‐derived macromolecules are involved in the protection of symbionts from digestion, the symbionts have acquired regulatory elements on their chromosomal genes to enhance production of beneficial gene products, and symbionts apparently utilize host‐derived macromolecules to their benefit. These studies involved morphological observations both at light and electron microscopic levels, physiological and genetic studies, production and use of poly‐ and monoclonal antibodies, and molecular‐biological approaches including gene cloning and sequencing. It is shown that amoebae are uniquely suited as model cells with which to study these phenomena.

Evidence for Symbiont-induced Alteration of a Host's Gene Expression: Irreversible Loss of SAM Synthetase from Amoeba proteus

ABSTRACT. Symbiont‐bearing xD amoebae no longer produce a 45‐kDa cytoplasmic protein that functions as S‐adenosylmethionine synthetase in symbiont‐free D amoebae. The absence of the protein in xD amoebae is attributable to xD amoebas failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. However, xD amoebae have about half the level of enzyme activity found in D amoebae, indicating that they use an alternative source for the enzyme. xD amoebae originated from D amoebae by bacterial infection and now depend on their symbionts for survival. xD amoebae exhibit irreversible nucleolar abnormalities when their symbionts are removed, suggesting that X‐bacteria supply the needed enzyme. A monoclonal antibody against the 45‐kDa protein was produced and used as a probe in cloning its corresponding cDNA. The product of the cDNA was found to have S‐adenosylmethionine synthetase activity. These results show how symbiotic X‐bacteria may become essential cellular components of amoebae by supplementing a genetic defect for an amoebas house‐keeping gene that is brought about by an action of X‐bacteria themselves. This is the first reported example in which symbionts alter the hosts gene expression to block the production of an essential protein.

A novel strong promoter of the groEx operon of symbiotic bacteria in Amoeba proteus

Gram- symbiotic bacteria (called X-bacteria), present in the xD strain of Amoeba proteus as required cell components, contain a large amount of a 67-kDa protein, a GroEL analog. The complete nucleotide (nt) sequence of the groEx operon of X-bacteria has been determined and it has a high degree of nt identity with those of other bacterial groE operons. The groELx gene is expressed in transformed Escherichia coli and has a novel and potent promoter (P2) in addition to the heat-shock consensus promoter (P1). This is shown by the production of GroELx in Escherichia coli transformed with modified DNA clones lacking P1 and by an enhanced production of a GroELx::beta-galactosidase fusion protein when a portion of groEx containing P2 is linked to the lacZ gene. Primer-extension analyses revealed the presence of possible P2 sequences within the open reading frame of the groESx gene. It is suggested that the presence of a potent P2 in the X-bacterial gene is an adaptation for the endosymbiotic bacteria to survive within a potentially hostile intracellular environment.

Bacterial endosymbiosis in amoebae

The large, free-living amoebae are inherently phagocytic. They capture, ingest and digest microbes within their phagolysosomes, including those that survive in other cells. One exception is an unidentified strain of Gram-negative, rod-shaped bacteria that spontaneously infected the D strain of Amoeba proteus and came to survive inside them. These bacteria established a stable symbiotic relationship with amoebae that has resulted in phenotypic modulation of the host and mutual dependence for survival.

A spectrin-like protein present on membranes of Amoeba proteus as studied with monoclonal antibodies

Monoclonal antibodies against a spectrin-like membrane-associated protein of xD amoebae. (Amoeba proteus) were used to determine the distribution of the protein and some of its characteristics. A total of 34 monoclonal antibodies recognizing different epitopes of the protein were obtained, of which seven stained cell membranes by indirect immunofluorescence. The spectrin-like protein had two subtypes of 225 and 220 kDa and several monoclonal antibodies cross-reacted with human erythrocyte spectrin when checked by indirect immunofluorescence staining and immunoblotting. Some of the antibodies also cross-reacted with antigens in HeLa cells and chick embryo fibroblasts. Polyclonal and monoclonal antibodies against Drosophila and human erythrocyte spectrins cross-reacted with the spectrin-like protein from amoebae. On the basis of these results, it was concluded that the protein is a spectrin. The protein was found on most cellular membranes of amoebae, including the plasma, nuclear, and phagosomal membranes, as well as symbiosome membranes.

Genetic and Physiological Interactions in the Amoeba-Bacteria Symbiosis1

Abstract Amoebae of the xD strain of Amoeba proteus that arose from the D strain by spontaneous infection of Legionella-like X-bacteria are now dependent on their symbionts for survival. Each xD amoeba contains about 42,000 symbionts within symbiosomes, and established xD amoebae die if their symbionts are removed. Thus, harmful infective bacteria changed into necessary cell components. As a result of harboring X-bacteria. xD amoebae exhibit various physiological and genetic characteristics that are different from those of symbiont-free D amoebae. One of the recent findings is that bacterial symbionts control the expression of a hosts house-keeping gene. Thus, the expression of the normal amoeba sams gene (sams1) encoding one form of S-adenosylmethionine synthetase is switched to that of sams2 by endosymbiotic X-bacteria. Possible mechanisms for the switching of sams genes brought about by endosymbionts and its significance are discussed.

Gene switching in Amoeba proteus caused by endosymbiotic bacteria

The expression of genes for S-adenosylmethionine synthetase (SAMS), which catalyzes the synthesis of S-adenosylmethionine (AdoMet), a major methyl donor in cells, was studied in symbiont-free (D) and symbiont-bearing (xD) amoeba strains to determine the effect of bacterial endosymbionts. The symbionts suppressed the expression of the gene in host xD amoebae, but amoebae still exhibited about half the enzyme activity found in symbiont-free D amoebae. The study was aimed at elucidating mechanisms of the suppression of the amoebas gene and determining the alternative source for the gene product. Unexpectedly, we found a second sams (sams2) gene in amoebae, which encoded 390 amino acids. Results of experiments measuring SAMS activities and amounts of AdoMet in D and xD amoebae showed that the half SAMS activity found in xD amoebae came from the amoebas SAMS2 and not from their endosymbionts. The expression of amoeba sams genes was switched from sams1 to sams2 as a result of infection with X-bacteria, raising the possibility that the switch in the expression of sams genes by bacteria plays a role in the development of symbiosis and the host-pathogen interactions. This is the first report showing such a switch in the expression of host sams genes by infecting bacteria.

A symbiont-produced protein and bacterial symbiosis in Amoeba proteus.

ABSTRACT. Gram‐ symbiotic X‐bacteria present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29‐kDa protein (S29x) into the hosts cytoplasm across bacterial and symbiosome membranes. The S29x protein produced by E. coli transformed with the s29x gene is also rapidly secreted into the culture medium. Inside amoebae, S29x enters the hosts nucleus as detected by confocal and irnmunoelectron microscopy, although it is not clear if S29x is selectively accumulated inside the nucleus. The deduced amino‐acid sequence of S29x has a stretch of basic amino acids that could act as a nuclear localization signal, but there is no signal peptide at the N‐terminus and the transport of S29x is energy independent. The functions of S29x are not known, but in view of its prominent presence inside the amoebas nucleus, S29x is suspected to be involved in affecting the expression of amoebas nuclear gene(s).

Characterization of sams Genes of Amoeba proteus and the Endosymbiotic X-Bacteria

Abstract As a result of harboring obligatory bacterial endosymbionts, the xD strain of Amoeba proteus no longer produces its own S-adenosylmethionine synthetase (SAMS). When symbiont-free D amoebae are infected with symbionts (X-bacteria), the amount of amoeba SAMS decreases to a negligible level within four weeks, but about 47% of the SAMS activity, which apparently comes from another source, is still detected. Complete nucleotide sequences of sams genes of D and xD amoebae are presented and show that there are no differences between the two. Long-established xD amoebae contain an intact sams gene and thus the loss of xD amoebas SAMS is not due to the loss of the gene itself. The open reading frame of the amoebas sams gene has 1,281 nucleotides, encoding SAMS of 426 amino acids with a mass of 48 kDa and pI of 6.5. The amino acid sequence of amoeba SAMS is longer than the SAMS of other organisms by having an extra internal stretch of 28 amino acids. The 5′-flanking region of amoeba sams contains consensus-binding sites for several transcription factors that are related to the regulation of sams genes in E. coli and yeast. The complete nucleotide sequence of the symbionts sams gene is also presented. The open reading frame of X-bacteria sams is 1,146 nucleotides long, encoding SAMS of 381 amino acids with a mass of 41 kDa and pI of 6.0. The X-bacteria SAMS has 45% sequence identity with that of A. proteus.

Role of spectrin in Amoeba proteus, as studied by microinjection of anti-spectrin monoclonal antibodies

Spectrin is a major protein accounting for about 5% of whole-cell proteins in Amoeba proteus, and the precipitation of spectrin by intracellular injection of purified anti-spectrin monoclonal antibodies has a profound effect on cell morphology, motility, and movement-related cell activities in amoebae. Thus, amoebae injected with anti-spectrin antibodies show drastic changes in their shape and movement, suggesting that amoeba spectrin plays an important structural role, unlike nonerythroid spectrins in other cells. However, precipitation of spectrin does not affect the distribution of F-actin in amoebae.