Molecular studies on a symbiont gene coding for the protein required for the survival of host amoebae

The purpose of this study was to clone and characterize a symbiotic bacterial gene encoding a protein which appears to be essential for the survival of host Amoeba proteus. A genomic expression library was constructed from symbiotic bacterial DNA using the bacteriophage vector Lambda-Zap. The library was immunoscreened with a monoclonal antibody (mab29kD) specific to the symbiont-originated, symbiont-bearing-strain specific protein (xD protein). The original lambda clone was subcloned into various expression vectors including pBluescript, pTacll, and pKK223-3, and expression of xD protein by transformed Escherichia coli was studied by SDS-polyacrylamide electrophoresis (SDS-PAGE), two-dimensional PAGE (2D-PAGE), Western blot analysis, and indirect immunofluorescence microscopy. More than 20% of the total proteins of transformed E. coli were xD proteins. The expressed protein comigrated with the xD protein from the symbiont-carrying strain of amoebae in SDS-PAGE and was immunologically reactive with mab29kD. The expressed protein was also resolved to the same position as xD protein on a 2D-PAGE. Culture media collected after growing transformed E. coli contained a 29-kD protein immunologically reactive with mab29kD, indicating that the protein was exported across the bacterial membrane after synthesis. The newly synthesized xD protein appeared in the culture medium as early as 20 min after the addition of a labeled precursor.

A restriction map of the original 8.2 kb/EcoRI lambda clone was generated and used to subclone a 1.8 kb/EcoRV fragment which fully expressed the 29-kD protein. A deletion series of the 1.8 kb fragment was generated with exonuclease III and mung bean nuclease for nucleotide-sequence analysis by the dideoxychain-termination method. The gene for xD protein has an open reading frame of 759 nucleotides that encode 253 amino acids with a total molecular weight of 29,431. The xD gene does not share sequence homolgy with any known genes. A string of nucleotides at +18 to +79, however, does show a high degree of sequence similarity with the GCN4 gene in Saccharomyces cerevisiae and the cytochrome b short gene in yeast mitochondria. The predicted amino-acid sequence showed that 41% of the total amino-acid residues were hydrophobic. In particular two regions of the deduced primary sequence of the xD protein were composed mainly of hydrophobic residues. These regions were between amino-acid residues 2-15 and 176-193. Serine was one of the major amino acids and comprised 11% of the total amino acids of the xD protein.