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. 1998 Aug;180(16):4116-22.
doi: 10.1128/JB.180.16.4116-4122.1998.

The cyanobacterium Synechocystis sp. strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI

M Scharnagl  1 , S Richter, M Hagemann
Affiliations

The cyanobacterium Synechocystis sp. strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI

M Scharnagl et al. J Bacteriol. 1998 Aug.

Erratum in

  • J Bacteriol 1998 Dec;180(24):6794

Abstract

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N6-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C5-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5'-CGATCG-3', corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation M.Ssp6803I [corrected] (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.

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Figures

FIG. 1
FIG. 1
Amino acid sequence comparison between the Slr0214 protein (SynMI) (11) and the C5-methylcytosine-synthesizing methyltransferases XorII (5), DdeIM (34), NgoII (33), and HphIM (21). Uppercase letters and a shaded background indicate amino acids identical to those in SynMI. The conserved motifs characteristic for C5-cytosine methyltransferases (26) are indicated by Roman numerals.
FIG. 2
FIG. 2
Schematic drawing showing the genetic organization, restriction map, and protein-encoding regions of the chromosomal region encoding Slr0214 (SynMI) in Synechocystis (11). (A and B) The insertion of the aphII gene in selected sites to generate mutants is shown. (A) BalI was used to obtain the slr0214Δ mutant. (B) HincII was used to obtain the slr0214 mutant. (C) Protein-encoding region. (D) The binding sites of the primers are represented by triangles under the arrows indicating the protein-encoding regions in panel C.
FIG. 3
FIG. 3
Southern blot experiments for characterization of complete segregation of the slr0214 mutant and the slr0214Δ mutant. The digoxigenin-labelled PCR fragment containing slr0214 (A) or the digoxigenin-labelled aphII gene (B) was used as a probe for hybridization to HindIII digested (lanes 1 to 3) and NcoI-digested (lanes 4 to 6) chromosomal DNAs from cells of the WT (lanes 1 and 4), the slr0214Δ mutant (lanes 2 and 5), and the slr0214 mutant (lanes 3 and 6) of Synechocystis. M, fragment sizes of digoxigenin-labelled HindIII-digested λ DNA.
FIG. 4
FIG. 4
Separation of fragments generated during a restriction analysis of chromosomal DNAs of the WT (lanes 1, 3, 5, 7, 9, 11, 13, and 15) and the slr0214 mutant (lanes 2, 4, 6, 8, 10, 12, 14, and 16) of Synechocystis obtained by agarose gel electrophoresis. The following restriction endonucleases were applied in this experiment: lanes 1 and 2, uncut control (n. c.); lanes 3 and 4, SgfI; lanes 5 and 6, PvuI; lanes 7 and 8, NcoI; lanes 9 and 10, HaeIII; lanes 11 and 12, MboI; lanes 13 and 14, Sau3AI; and lanes 15 and 16, DpnI. M, fragment sizes of HindIII-digested λ DNA.
FIG. 5
FIG. 5
Comparison of the total methyltransferase activity in and the growth of cells of the WT (columns 1 and 3) and of the slr0214 mutant (columns 2 and 4) of Synechocystis after cultivation in CO2-gassed cultures with the medium of Allen and Arnon (1) at reduced light intensities (about 40 μmol of photons s−1 m−2). The data represent the mean values from two independent experiments (each done in duplicate).
FIG. 6
FIG. 6
Overexpression of the Slr0214 protein in E. coli BL21 by use of the GST gene fusion system (Pharmacia). (A) Coomassie blue staining of proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lanes: 1, total extract from E. coli cells after induction by IPTG; 2, crude extract after sonication and centrifugation at 25,000 × g for 20 min; 3, isolated Slr0214 protein after thrombin cleavage; M, prestained broad-range markers (Bio-Rad). (B) Methyltransferase activity of the purified Slr0214 protein in in vitro assays with buffers containing different NaCl concentrations. Lanes: 1, assays done with 100 mM NaCl; 2, assays done with 50 mM NaCl; 3, assays done under NaCl-free conditions. (C) Restriction analysis of a DNA fragment with (lanes 4 to 6) and without (lanes 1 to 3) methylation by SynMI in vitro. Lanes: 1 and 4, uncut; 2 and 5, PvuI; 3 and 6, ClaI; M, fragment sizes of EcoRI/HindIII-digested λ DNA.
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