Abstract
BackgroundEscherichia 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.ResultsTo 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0741-4) contains supplementary material, which is available to authorized users.
Highlights
Escherichia coli K-12 strains contain DNA cytosine methyltransferase (Dcm), which generates 5-methylcytosine at 5′CCWGG3′ sites
Effects of 5-azaC on global DNA methylation levels First, we determined the concentration dependence of DNA methylation inhibition by 5-azaC using digestion of DNA with the restriction enzyme isoschizomers BstNI and PspGI (Fig. 1). Both enzymes cut DNA at Dcm recognition sites (5′CCWGG3′), but PspGI is blocked by Dcm-mediated methylation of the second cytosine
In the absence of 5-azaC, DNA from early stationary phase cells was largely resistant to PspGI indicating that the DNA is heavily methylated at this stage
Summary
Escherichia coli K-12 strains contain DNA cytosine methyltransferase (Dcm), which generates 5-methylcytosine at 5′CCWGG3′ sites. The modified DNA base 5-methylcytosine (5-MeC) plays an important role in transcriptional regulation in higher eukaryotes. The presence of 5-MeC in eukaryotic promoters and CpG islands is generally repressive for transcription, whereas 5-MeC in gene bodies is positively correlated with transcription [1]. Bacteria, such as E. coli, contain 5-MeC [2]. In E. coli K-12 strains, the only known cytosine-5 DNA methyltransferase is DNA cytosine methyltransferase (Dcm) [3, 4]. Dcm methylates the second cytosine in 5′CCWGG3′ sequences [3].
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