Genome-wide GATC methylation in long-term cultures of Escherichia coli K-12

Abstract

DNA Adenine Methyltransferase (Dam) catalyzes the addition of a methyl group to the N6 position of adenine at 5’-GATC-3’ sequences in Escherichia coli. Dam is involved in a variety of cellular processes, including the timing and initiation of chromosomal replication, epigenetic transcriptional regulation of several genes (including virulence factors), and methyl-directed mismatch repair (MMR). Not only does Dam methylation of the chromosome have the potential to cause mutation due to deamination at methylated adenine, the loss of Dam methylation can cause mutation due to MMR errors. Because Dam methylation can alter mutation and transcription, it is potentially a promising mechanism for adaptation in the cell. In this work, I have characterized the extent of Dam methylation through the five phases of the bacterial life cycle using gel-based methods, liquid chromatography combined with mass spectroscopy (LCMS), and Single-Molecule Real-Time (SMRT) sequencing. Additionally, this work investigates the role that mutation may play in adaptation and whether a mutator phenotype is advantageous under any conditions. Our data demonstrate that GATC sites are always methylated over time. However, there are several sites that change significantly over time within the population, including three sites involved in sialic acid transport and catabolism. This work indicates that maintaining methylation is important to the cell and has been under positive selection, and that a mutator phenotype such as seen in mutS, mutL, and dam strains is deleterious under most conditions.