Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

Abstract

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.

Figure 1

A) Detection of the dcm gene via PCR. E. coli genomic DNA from different strains was used as a template for PCR using one forward dcm primer and two reverse dcm primers as described in the Materials and Methods. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide straining. The negative sign (−) represents a no DNA control. E. coli GM204 DNA (GM204) contains deletion of the dcm operon. E. coli JM109 DNA (JM109) contains a wild-type dcm allele. EC068 and EC064 are DNAs from E. coli environmental isolates. B) Detection of 5-methylcytosine in 5′CCWGG3′ sequences using restriction enzyme isoschizomer pairs. DNA from E. coli ER2925 (dcm-6), JM109 (dcm⁺), and four environmental strains (EC007-EC010) were left undigested (−), digested with BstNI (B), or digested with PspGI (P). Reactions were analyzed by agarose gel electrophoresis and ethidium bromide staining.

Figure 2. The number and location of 5′CCWGG3′ sites in Escherichia coli K-12 MG1655 promoters

A) 5′CCWGG3′ abundance in E. coli promoters. Promoter sequences available in the Regulon database (http://regulondb.ccg.unam.mx/) were downloaded and queried for the number of 5′CCWGG3′ sites in different E. coli promoters. # represents the number of promoters in each category and total is the total number of promoters with 5′CCWGG3′ sequences. B) Histogram of the frequency of 5′CCWGG3′ sites with respect to the transcription start site. The numbers represent the distance to the transcription start site where the number 1 is the transcription start site. The position refers to the first C in the sequence 5′CCWGG3′.

Figure 3. Expression of ribosomal protein genes in the absence and presence of dcm

E. coli wild-type bacteria with a plasmid containing an inactive dcm truncation (BW25113/pDcm-9), dcm knockout bacteria containing an inactive dcm truncation (JW1944-2/pDcm-9), and dcm knockout bacteria containing a plasmid with a functional dcm gene (JW1944-2/pDcm-21) were grown to early logarithmic phase and early stationary phase. Total RNA was isolated and converted to cDNA. The levels of rplC and rpsJ were measured by qPCR, and normalized to the levels of malate dehydrogenase (mdh) RNA. The BW25113/pDcm-9 samples were set to a value of one. Error bars represent one standard deviation.