Doctoral Dissertations


Jeong Won Pak

Date of Award


Degree Type


Degree Name

Doctor of Philosophy



Major Professor

Kwang W. Jeon

Committee Members

Evans Roth, Bruce McKee, Ranjan Ganguly, Gary Stacey


A recent spontaneously developed amoeba-bacteria symbiosis is a useful system to study the transport and role of symbiont-synthesized proteins in host-symbiont relationships. In the amoeba-bacteria symbiosis, the amoebae showed a different pattern of protein composition from that of non-symbiotic amoebae. By using monoclonal antibodies and some selective inhibitors of prokaryotic and eukaryotic protein synthesis, endosymbiotic X-bacteria in the xD strain of Amoeba proteus were found to produce a large amount of a unique 29-kDa protein (S29x), much of which was transported into the amoeba cytoplasm. The complete nucleotide sequence of the gene (s29x) encoding S29x and its deduced amino acid sequence were determined. The s29x gene had an open reading frame (ORF) of 774 nucleotides, coding for 258 amino acids and this was equivalent to a size of 29,968, which was in good agreement with the 29-kDa size estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A cloned s29x gene was expressed in transformed E.coli with or without isopropyl-β-D-thiogalactoside (IPTG), indicating that the gene was controlled by its own promoter. The transcription initiation point (tsp) was identified by primer extension analyses. The promoter region was found to be located 150-210 bp upstream from the initiation codon, as confirmed by a series of deletion experiments and site-directed mutagenesis. However, the core promoter region did not contain any sequences similar to the consensus recognition hexamers. The S29x protein produced by transformed E. coli was also transported across bacterial membranes into the culture medium. The deduced amino acid sequence of S29x did not contain a typical signal sequence at the N-terminus. The N-terminal amino acid sequences of S29x isolated from transformed E. coli and the culture medium were identical, showing that there was no cleavage of N-terminal peptide during transport. Even when C-terminal hydrophobic region of S29x was deleted, the truncated protein was still efficiently exported. Therefore, S29x was exported without involving a N-terminal signal sequence or an C-terminal hydrophobic region. Pulse-chase experiments using [35S]-methionine showed that S29x was exported as soon as it was synthesized and that export of S29x was insensitive to uncouplers such as CCCP and arsenate. This suggests that export of S29x is energy-independent. A computer-aided search, conducted using the GenBank, EMBL, and PIR databases for both DNA and protein comparison, revealed that S29x was a novel protein with no significant homology to any other proteins in the databases. The S29x was dispersed throughout the host cytoplasm with no noticeable association with cell organelles. An anti-S29x monoclonal antibody (mAb) did not stain X-bacteria without permeabilization by indirect immunofluorescence, indicating that S29x was not present on the cell surface. Although these results give no hint as to the possible cellular function of S29x, the protein may indirectly regulate a gene(s) involved in bacterial infection or the symbiotic relationship since the protein is present inside the amoeba's nucleus as shown by immunoelectron microscopic observations.

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