The Cyanobacterium Synechocystis sp. Strain PCC 6803 Expresses a DNA Methyltransferase Specific for the Recognition Sequence of the Restriction Endonuclease Pvu I

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 N 6-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 C 5-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 SynMI (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.

Highly plastic genome of Microcystis aeruginosa PCC 7806, a ubiquitous toxic freshwater cyanobacterium

“…To test this hypothesis, the number of occurrences of each 6-mer was counted, and a frequency distribution calculated for Mic-PCC7806, Mic-NIES843, Cwa-WH8501 and Syn-PCC6803 (Table 5 ). The under-represented sites in the three first genomes were not found in Syn-PCC6803, a genome devoid of restriction enzymes [ 29 ], supporting the idea that these rare 6-mers could indeed correspond to restriction enzyme sites. In total, there are 4096 possible 6-mers, 1.5% of which are palindromes.…”

Insensitivity of chloroplast gene expression to DNA methylation

“…The Synechocystis genome also contains at least one functional cytosine methyltransferase ( slr 0214). The gene product has been functionally characterized and shown to methylate cytosines in the recognition sequence of the restriction endonuclease Pvu I (Scharnagl et al 1998 ). This cytosine methylase has been termed M. Ssp 6803I (Scharnagl et al 1998 ) in accordance with the standard nomenclature for DNA methyltransferases, but for simplicity, it will be referred to here as Dcm for deoxycytosine deaminase, although it recognizes a different sequence than the classical Dcm methylase from E. coli .…”

“…Minor differences between transplastomic lines obtained from the same construct are due to unequal loading. b Test for cytosine methylation by DNA digestion with the methylation-sensitive restriction endonuclease Pvu I (Scharnagl et al 1998 ). Pvu I does not cut if the cytosines in its recognition sequence are methylated.…”

“…To test whether cytosine methylation occurs in the Nt-dcm transplastomic lines, we used the methylation-sensitive restriction enzyme Pvu I (Scharnagl et al 1998 ). As Pvu I is a six-base pair cutter and therefore does not cut as frequently in the chloroplast genome as Mbo I and Dpn I, we used a large restriction fragment of the plastid genome as hybridization probe, which detects altogether four Pvu I fragments covering more than 40 kb of the plastid genome (Fig.…”

“…The adenine ( dam ; slr 1803) and cytosine DNA methyltransferase (M. Ssp 6803I; slr 0214; Scharnagl et al 1998 ) genes from Synechocystis sp. strain PCC 6803 (Kaneko et al 1996 ) were cloned as Nco I/ Xba I fragments into plastid transformation vector pRB96 (Wurbs et al 2007 ), a derivative of vector pRB94 (Ruf et al 2001 ).…”