Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

Abstract

In Escherichia coli, cytosine DNA methylation is catalyzed by the Dcm (DNA cytosine methyltransferase) 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. In order to gain insight into the role of cytosine DNA methylation in E. coli, we: (a) 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; (b) examined the same strains for the presence of 5-methylcytosine at 5′CCWGG3′ sites using a restriction enzyme isoschizomer digestion assay; and (c) 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 expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.

Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription

“…Thus, our study includes Dcm as a possible regulator of rpoS expression. This is in addition to the recently discovered role of Dcm in controlling ribosomal gene expression during stationary phase 27 ; we suggest that the expression of these genes begins to increase in late-exponential phase.…”

“…Among the genes differentially expressed only in ∆dcm are 45 genes involved in ribosomal biogenesis and translation (P < 10 − 6 ; Fisher's exact test), most of which are upregulated in late-exponential phase. This, we suggest, precedes the enhanced gene expression of ribosomal genes observable in stationary phase 27 . ; all values were uniformly multiplied by 10 7 and log transformed) of genes that are upregulated in ∆dcm during stationary phase, compared with a control set of all other genes (referred to as 'other') (d) As in c, except that this panel shows the fold change in expression between stationary and exponential phases of growth in the wildtype.…”

“…In bacteria, however, DNA adenine methylation has been recognized as a regulator of gene expression, primarily by influencing the activities of transcription factors whose effects are correlated with the target's methylation status 14,15 . However, until very recently 27 when cytosine methylation was shown to regulate ribosomal gene expression in stationary phase, no role for cytosine methylation in gene expression control had been documented for bacteria, at least for enterobacteria.…”

“…Despite the widespread and apparently 'random' occurrence of the CCWGG motif, a recent study has shown that where present in intergenic regions, several CCWGG sites are clustered together 27 , thus leading to immediate speculation on the role of methylation in gene regulation. However, our analysis of the occurrence of the CCWGG motif across 2,558 experimentally verified transcription factor-binding sites in the RegulonDB database 37 showed that this motif occurs only 69 times in these sequences, which is not significantly different from the trend in the rest of the genome.…”

“…This is because, in the presence of a functional Dcm, EcoRII-target sites will be kept methylated even when the R-M system is disturbed, thus preventing post-segregational killing 24 . To our knowledge, this, an association between Dcm and Tn3 transposition 25, and lambda DNA recombination 26 , and a very recently discovered role in regulating ribosomal gene expression in stationary phase 27 , remain the only phenotypes associated with Dcm.…”