In prokaryotes, DNA methylation is associated with restriction-modification systems and implicated in the regulation of important cellular events such as gene transcription, DNA mismatch repair, initiation of chromosome replication and nucleoid structure. In E. coli, Dam methylation of GATC sequences is the signal to direct the Mismatch repair system (MMRS) to the DNA strand recently synthesized. In order to know the role of DNA methylation in the MMRS of P. aeruginosa (PAO1), we first analyzed the content of N6-methyl-deoxyadenosine (6mdA) and 5-methyl-deoxycytidine (5mdC) in chromosomal DNA of this specie (and in E. coli DNA as a control), by reverse phase HPLC chromatography. The following values were obtained for PAO1: dC: 34.2%; dG: 32.4%; dA: 18.4%; dT: 14.9%; 5mdC: 0.16% and 6mdA:0.03%. Since no homologous of E. coli Dam or Dcm was found in the genome sequence of PAO1 we carried out experiments in order to obtain a knock-out strain for the gene encoding the synthetase of the methyl donor, S-adenosyl-methionine (metK gene). An in vitro mutagenized copy of the metK allele (metK::km, was delivered to the host chromosome via homologous recombination. Exhaustive molecular screening of clones with the appropriate phenotype showed the presence of partial recombination products with and intact copy of metK gene which probably indicates that this gene is essential for PAO1 viability.
