5-azacytidine induces transcriptome changes in Escherichia coli via DNA methylation-dependent and DNA methylation-independent mechanisms.

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.

Blue genes: An integrative laboratory to differentiate genetic transformation from gene mutation for underclassmen.

The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by which the genetic material can be altered: genetic transformation and gene mutation. In the first week of the laboratory, students incubate a plasmid DNA with calcium chloride-treated Escherichia coli JM109 cells and observe a phenotype change from ampicillin sensitive to ampicillin resistant and from white color to blue color on plates containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) and isopropyl ß-D-thiogalactopyranoside (IPTG). Over the course of the next three weeks, students use a battery of approaches including plasmid DNA isolation experiments, restriction maps, and PCR to differentiate between mutation and transformation. The students ultimately come to the conclusion that the changes in phenotypes are due to genetic transformation and not mutation based on the evidence generated over the four-week period. Pre-laboratory tests and post-laboratory tests indicate that this set of exercises is successful in helping students differentiate between transformation and mutation. The laboratory is designed for underclassmen and is a good prerequisite for an apprentice-based research opportunity, although it is not designed as a class based research experience. Potential modifications and future directions of the laboratory based upon student experiences and assessment are presented.

Cytosine DNA methylation influences drug resistance in Escherichia coli through increased sugE expression.

Escherichia coli K-12 strains contain the orphan cytosine-5 DNA methyltransferase enzyme Dcm (DNA cytosine methyltransferase). Two recent reports indicate that Dcm has an influence on stationary phase gene expression in E. coli. Herein, we demonstrate that dcm knockout cells overexpress the drug resistance transporter SugE, which has been linked to ethidium bromide (ETBR) resistance. SugE expression also increased in the presence of the DNA methylation inhibitor 5-azacytidine, suggesting that Dcm-mediated DNA methylation normally represses sugE expression. The effect of Dcm on sugE expression is primarily restricted to early stationary phase, and RpoS is required for robust sugE expression. Dcm knockout cells are more resistant to ETBR than wild-type cells, and complementation with a plasmid-borne dcm gene restores ETBR sensitivity. SugE knockout cells are more sensitive to ETBR than wild-type cells. These data indicate that Dcm influences the sensitivity to an antimicrobial compound through changes in gene expression.

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli.

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.