Dataset Information

Genomewide Dam Methylation in Escherichia coli during Long-Term Stationary Phase.

ABSTRACT: DNA methylation in prokaryotes is widespread. The most common modification of the genome is the methylation of adenine at the N-6 position. In Escherichia coli K-12 and many gammaproteobacteria, this modification is catalyzed by DNA adenine methyltransferase (Dam) at the GATC consensus sequence and is known to modulate cellular processes including transcriptional regulation of gene expression, initiation of chromosomal replication, and DNA mismatch repair. While studies thus far have focused on the motifs associated with methylated adenine (meA), the frequency of meA across the genome, and temporal dynamics during early periods of incubation, here we conduct the first study on the temporal dynamics of adenine methylation in E. coli by Dam throughout all five phases of the bacterial life cycle in the laboratory. Using single-molecule real-time sequencing, we show that virtually all GATC sites are significantly methylated over time; nearly complete methylation of the chromosome was confirmed by mass spectroscopy analysis. However, we also detect 66 sites whose methylation patterns change significantly over time within a population, including three sites associated with sialic acid transport and catabolism, suggesting a potential role for Dam regulation of these genes; differential expression of this subset of genes was confirmed by quantitative real-time PCR. Further, we show significant growth defects of the dam mutant during long-term stationary phase (LTSP). Together these data suggest that the cell places a high premium on fully methylating the chromosome and that alterations in methylation patterns may have significant impact on patterns of transcription, maintenance of genetic fidelity, and cell survival. IMPORTANCE While it has been shown that methylation remains relatively constant into early stationary phase of E. coli, this study goes further through death phase and long-term stationary phase, a unique time in the bacterial life cycle due to nutrient limitation and strong selection for mutants with increased fitness. The absence of methylation at GATC sites can influence the mutation frequency within a population due to aberrant mismatch repair. Therefore, it is important to investigate the methylation status of GATC sites in an environment where cells may not prioritize methylation of the chromosome. This study demonstrates that chromosome methylation remains a priority even under conditions of nutrient limitation, indicating that continuous methylation at GATC sites could be under positive selection.

SUBMITTER: Westphal LL

PROVIDER: S-EPMC5155068 | biostudies-literature | 2016 Nov-Dec

REPOSITORIES: biostudies-literature

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Genomewide Dam Methylation in <i>Escherichia coli</i> during Long-Term Stationary Phase.

Westphal Lacey L LL Sauvey Peter P Champion Matthew M MM Ehrenreich Ian M IM Finkel Steven E SE

mSystems 20161101 6

DNA methylation in prokaryotes is widespread. The most common modification of the genome is the methylation of adenine at the N-6 position. In <i>Escherichia coli</i> K-12 and many gammaproteobacteria, this modification is catalyzed by DNA adenine methyltransferase (Dam) at the GATC consensus sequence and is known to modulate cellular processes including transcriptional regulation of gene expression, initiation of chromosomal replication, and DNA mismatch repair. While studies thus far have focu ...[more]

PMID: 27981240

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Project description:In natural environments, bacteria survive conditions of starvation and stress. Long-term batch cultures are an excellent laboratory system to study adaptation during nutrient stress because cells can incubate for months to years without the addition of nutrients. During long-term batch culture, cells adapt to acquire energy from cellular detritus, creating a complex and dynamic environment for mutants of increased relative fitness to exploit. Here, we analyzed the genomes of 1,117 clones isolated from a single long-term batch culture incubated for 1,200?days. A total of 679 mutations included single nucleotide polymorphisms, indels, mobile genetic element movement, large deletions up to 64 kbp, and amplifications up to ?500 kbp. During the 3.3-year incubation, two main lineages diverged, evolving continuously. At least twice, a previously fixed mutation reverted back to the wild-type allele, suggesting beneficial mutations may later become maladaptive due to the dynamic environment and changing selective pressures. Most of the mutated genes encode proteins involved in metabolism, transport, or transcriptional regulation. Clones from the two lineages are physiologically distinct, based on outgrowth in fresh medium and competition against the parental strain. Similar population dynamics and mutations in hfq, rpoS, paaX, lrp, sdhB, and dtpA were detected in three additional parallel populations sequenced through day 60, providing evidence for positive selection. These data provide new insight into the population structure and mutations that may be beneficial during periods of starvation in evolving bacterial communities.IMPORTANCE Bacteria have remarkable metabolic capabilities and adaptive plasticity, enabling them to survive in changing environments. In nature, bacteria spend a majority of their time in a state of slow growth or maintenance, scavenging nutrients for survival. Here, a population of Escherichia coli cells was incubated for 1,200?days in long-term batch culture, without the addition of new medium, requiring cells to continuously recycle nutrients. Whole-genome resequencing of cells from the evolving population identified two dominant subpopulations that coexisted while continuously acquiring and fixing new mutations. The population dynamics and alleles identified provide insight into adaptation to nutrient stress. Elucidating mechanisms that allow bacteria to adapt through cycles of feast and famine deepens our understanding of their survival mechanisms in nature.

Project description:Microbes live in complex and constantly changing environments, but it is difficult to replicate this in the laboratory. Escherichia coli has been used as a model organism in experimental evolution studies for years; specifically, we and others have used it to study evolution in complex environments by incubating the cells into long-term stationary phase (LTSP) in rich media. In LTSP, cells experience a variety of stresses and changing conditions. While we have hypothesized that this experimental system is more similar to natural environments than some other lab conditions, we do not yet know how cells respond to this environment biochemically or physiologically. In this study, we began to unravel the cells' responses to this environment by characterizing the transcriptome of cells during LTSP. We found that cells in LTSP have a unique transcriptional program and that several genes are uniquely upregulated or downregulated in this phase. Further, we identified two genes, cspB and cspI, which are most highly expressed in LTSP, even though these genes are primarily known to respond to cold shock. By competing cells lacking these genes with wild-type cells, we show that these genes are also important for survival during LTSP. These data can help identify gene products that may play a role in survival in this complex environment and lead to identification of novel functions of proteins.IMPORTANCE Experimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses that cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study, we began to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more-natural conditions.

Dataset Information

Genomewide Dam Methylation in Escherichia coli during Long-Term Stationary Phase.

Publications

Genomewide Dam Methylation in <i>Escherichia coli</i> during Long-Term Stationary Phase.

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