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.

Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some similarity to RNA interference, and the ability to utilize reconstituted CRISPR systems for genome editing in numerous organisms. In order to introduce CRISPR biology into an undergraduate upper-level laboratory, a five-week set of exercises was designed to allow students to examine the CRISPR status of uncharacterized Escherichia coli strains and to allow the discovery of new repeats and spacers. Students started the project by isolating genomic DNA from E. coli and amplifying the iap CRISPR locus using the polymerase chain reaction (PCR). The PCR products were analyzed by Sanger DNA sequencing, and the sequences were examined for the presence of CRISPR repeat sequences. The regions between the repeats, the spacers, were extracted and analyzed with BLASTN searches. Overall, CRISPR loci were sequenced from several previously uncharacterized E. coli strains and one E. coli K-12 strain. Sanger DNA sequencing resulted in the discovery of 36 spacer sequences and their corresponding surrounding repeat sequences. Five of the spacers were homologous to foreign (non-E. coli) DNA. Assessment of the laboratory indicates that improvements were made in the ability of students to answer questions relating to the structure and function of CRISPRs. Future directions of the laboratory are presented and discussed. V C 2016 by The

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Trypanosoma brucei is the causative agent of African Sleeping Sickness. The regulation of gene expression in T. brucei primarily occurs at the posttranscriptional level, indicating that RNA processing and modification events are extremely important for the parasite's life cycle. Yet, there is little information regarding the collection of covalent RNA modifications that comprise the epitranscriptome in this organism or any protozoan parasite. We focused on cytosine base modifications of tRNAs, as tRNAs are well known to contain a range of modified RNA bases, including 5‐methylcytosine. Small RNAs were isolated from insect‐stage T. brucei parasites and unmodified cytosines were converted to uracils using sodium bisulfite treatment. Sodium bisulfite‐treated small RNAs were analyzed by RNAseq. To confirm the RNAseq data, sodium bisulfite‐treated RNA samples were amplified by RT‐PCR from either total RNA or small RNA fractions, and analyzed by digestion with HpaII or via Sanger sequencing. Overall, our data indicate that tRNA molecules contain between zero to four 5‐methylcytosine residues. The most common location of 5‐methylcytosine in T. brucei tRNA is at the junction between the variable region and TψC arm at positions C48, C49, and C50, although different tRNAs have different numbers of 5‐methylcytosines in this region. There was little evidence for methylation of other sites in the tRNA, including C38 methylation in the anticodon loop. Overall, our data indicated that T. brucei tRNAs contain 5‐methylcytosine at some, but not all standard eukaryotic positions, and the levels of 5‐methylcytosine vary in different tRNA molecules.

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