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First published online 6 January 2004
doi: 10.1242/jcs.00894

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Gene switching in Amoeba proteus caused by endosymbiotic bacteria

Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996, USA

View larger version (30K): [in a new window]   Fig. 1. Growth rates of amoebae at different concentrations of AdoMet. (A) Growth rates of D and xD amoebae at 22°C; (B) growth rates at 27°C. Each point represents the average (± s.d.) from 24 cells grown singly in two different experiments.

View larger version (16K): [in a new window]   Fig. 2. Graphs to show the uptake of radiolabeled AdoMet (S-adenosyl-L-[methyl-3H]methionine) by D amoebae (A) and xD amoebae (B). S-Adenosyl-L-[methyl-3H]methionine was added to 2.0x103 amoebae/ml in Chalkley's solution to a final concentration of 2 µCi/ml. Total uptake () was determined from collected amoebae before sonication. After sonication and centrifugation, incorporated AdoMet into macromolecules () was estimated from the pellets, and net transport () from supernatants. Results are expressed as percentage of the initial radioactivity of AdoMet. Values are averages from two separate experiments.

View larger version (33K): [in a new window]   Fig. 3. Graphs to show SAMS activities in whole-cell proteins of D and xD ameobae as measured using L-[methyl-H3]methionine. To remove X-bacteria from xD amoebae, cells were grown for 8 days at 27°C. The values for xD amoebae represent only the SAMS activities of the host amoebae, not including that of the symbionts, since X-bacteria were excluded during extraction of proteins from xD amoebae. The data represent mean ± s.d. from three separate experiments.

View larger version (28K): [in a new window]   Fig. 4. Fractionation of SAMS from D and xD ameobae. (A) Results of Sephadex G-150 chromatography of D and xD amoebae. (B) Results of DEAE-cellulose chromatography of D and xD amoebae. Cytosolic extracts (2-3 ml) of D and xD ameobae were chromatographed, and 25 µl aliquot from each fraction was used in the assay of SAMS activity. The Sephadex G-150 column was previously calibrated with the following molecular markers (arrows): a, catalase (232 kDa); b, aldolase (158 kDa) and c, albumin (67 kDa).

View larger version (14K): [in a new window]   Fig. 5. Results of western blot analyses of proteins fractionated by Sephadex G-150 chromatography. Fractions containing SAMS enzyme activities (Fig. 4) were subjected to western blotting using pAb against amoeba SAMS1. A 15 µl aliquot from each fraction was separated by SDS-PAGE (10%), and transferred to a membrane for western blotting. Whole-cell proteins of D amoebae were loaded in the first lane as a size marker for amoeba SAMS1.

View larger version (51K): [in a new window]   Fig. 6. Complete nucleotide and deduced amino acid sequences of the sams2 gene (GenBank accession no. AY324626). The ORF of sams2 is 1,173 nt long and encodes 390 amino acids. The start and stop codons are underlined.

View larger version (56K): [in a new window]   Fig. 7. Alignment of the deduced amino acid sequence of amoeba SAMS2 with SAMS sequences of other organisms. Periods represent amino acids identical to those of MetK of E. coli, and dashes show gaps inserted for an optimal alignment of amino acids. *1, ATP-binding motif; *2, glycine-rich nanopeptide; *3, metal-binding sites; *4, a site for Cys-121 of human MAT1A, characteristic of liver enzymes; EC, E. coli; XB, X-bacteria; AC, Acanthamoeba castellanii; S2, SAMS2 of amoebae; PI, Phytophthora infestans; AP, Amoeba proteus. Complete genomic or cDNA sequences of SAMS proteins are available in GenBank, for E. coli (accession no. 1708999), A. castellanii (6016547), and P. infestans (23394401). Amino acid sequences of amoeba SAMS1 (U91602) and X-bacteria SAMS (AY324627) are from Choi et al. (Choi et al., 1997) and Jeon and Jeon (Jeon and Jeon, 2003).

View larger version (38K): [in a new window]   Fig. 8. Northern blot analyses of sams1 and sams2 genes following infection of D amoebae with X-bacteria. Total RNAs (20 µg) isolated from amoebae collected every 4 days after infection were separated in formamide gels and then transferred to nylon membranes. The signal was detected subsequently on the same blot with 32P-labeled sams1, sams2 and myosin probes. The myosin probe was used as a loading control.

View larger version (31K): [in a new window]   Fig. 9. Graphs to show the size of intracellular pools of AdoMet in amoebae and X-bacteria. Graphs for xD amoebae include the AdoMet of the symbiotic X-bacteria, as measured by using a Dowex 50W-X8 resin. The data represent mean ± s.d. from three separate experiments.

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