RESUMEN
BACKGROUND: Escherichia coli K-12 strains contain DNA cytosine methyltransferase (Dcm), which generates 5-methylcytosine at 5'CCWGG3' sites. Although the role of 5-methylcytosine in eukaryotic gene expression is relatively well described, the role of 5-methylcytosine in bacterial gene expression is largely unknown. RESULTS: To identify genes that are controlled by 5-methylcytosine in E. coli, we compared the transcriptomes of cells grown in the absence and presence of the DNA methylation inhibitor 5-azacytidine. We observed expression changes for 63 genes. The majority of the gene expression changes occurred at early stationary phase and were up-regulations. To identify gene expression changes due to a loss of DNA methylation, we compared the expression of selected genes in a wild-type and dcm knockout strain via reverse transcription quantitative PCR. CONCLUSIONS: Our data indicate that 5-azacytidine can influence gene expression by at least two distinct mechanisms: DNA methylation loss and a mechanism that is independent of DNA methylation loss. In addition, we have identified new targets of 5-methylcytosine-mediated regulation of gene expression. In summary, our data indicate that 5-azacytidine impacts the composition of the bacterial transcriptome, and the primary effect is increased gene expression at early stationary phase.
Asunto(s)
Azacitidina/antagonistas & inhibidores , Metilación de ADN/efectos de los fármacos , Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Transcriptoma/efectos de los fármacos , 5-Metilcitosina/metabolismo , 5-Metilcitosina/fisiología , Secuencia de Bases , Técnicas de Cultivo de Célula , Citosina , ADN Bacteriano , Escherichia coli K12/crecimiento & desarrollo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Técnicas de Inactivación de Genes , Genes Bacterianos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Bacteriano/aislamiento & purificación , Análisis de Secuencia , Regulación hacia ArribaRESUMEN
In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35 years ago, the biological role of 5-methylcytosine in E. coli remains unclear. To gain insight into the role of cytosine DNA methylation in E. coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence of the full-length dcm gene using the polymerase chain reaction; (2) examined the same strains for the presence of 5-methylcytosine at 5'CCWGG3' sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2'-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.