Doctoral Dissertations

Date of Award

8-2003

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Kwang W. Jeon

Abstract

The xD strain of Amoeba proteus that arose from the D strain by spontaneous infection of X-bacteria is now dependent on X-bacteria for survival. The Sadenosylmethionine synthetase (SAMS) is the enzyme that catalyzes the synthesis of Sadenosylmethionine (SAM), a major methyl donor in cells. As a result of harboring obligatory bacterial endosymbionts, xD amoebae do not transcribe the sams gene and no longer produce their own 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 4 7% of the SAMS activity, which apparently comes from another source, is still detected. Therefore, it was postulated that X-bacteria suppressed the expression of amoeba's sams and in turn provided the enzyme of SAM for their hosts, forcing host amoebae to become dependent on symbionts themselves. As a part of my work to elucidate the mechanism for the development of mutual dependence between symbionts and hosts, first the sams genes of amoebae and Xbacteria were cloned and characterized (PART II}. The previously reported sams gene of amoebae turned out to be an isoform, so now we call the gene sams 1. The open reading frame of the amoeba's samsl gene has 1,281 nucleotides, encoding SAMS of 426 amino acids with a mass of 48 kDa and pl of 6.5. The 5'-flanking region of amoeba samsl contains consensus-binding sites for several transcription factors that are related to the regulation of sams genes in E. coli and yeast. 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 pl of 6.0. The X-bacteria SAMS has 45% sequence identity with that of A. proteus. Next I tested if symbionts supplied SAMS or SAM to host xD amoebae by localizing X-bacteria SAMS in xD amoebae and measuring SAMS activities in D and xD amoebae (PART Ill). The results show that amoebae have a second sams (sams2) gene, encoding 390 amino acids and that the SAMS activity found in xD amoebae comes from SAMS2 not from the endosymbionts. Interestingly, the expression of two amoeba sams genes was switched from samsl to sams2 by infection with X-bacteria, raising a 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 the switch in the expression of host sams genes by infecting bacteria. In PART IV, the analysis for DNA adenine methylation of samsl shows that the samsl gene is methylated at an internal adenine residue ofGATC site in xD amoebae whereas no methylation occurs in D amoebae. This result implies that the modification might be the reason for the suppression of sams 1 in xD amoebae. The methylation of cytosine residue in CpG sites is the most common modification in eukaryotes. However, it appears that the methylation of cytosine residues is not responsible for the inactivation of samsl in xD amoebae. DNA analysis shows that adenine residues in X-bacteria sams are also methylated, indicating Legionella-like X-bacteria belong to Dam methylasepositive strains. In addition, it appears that SAM and methionine act as a negative regulator for the expression of samsl whereas the expression of sams2 is not affected in amoebae. After the removal ofX-bacteria from xD amoebae, the samsl gene was reactivated and sams2 was down-regulated. The results are reversed in the expression of sams genes when amoebae are infected with X-bacteria. This is the first report to show that a specific eukaryotic gene is modified by DNA adenine methylation.

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