Higher levels of gene regulation in Escherichia coli

Cooke, Karen (2019). Higher levels of gene regulation in Escherichia coli. University of Birmingham. Ph.D.

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Abstract

“Epigenetic” mechanisms result in information being carried by DNA, independent of its base sequence. These are widely studied in eukaryotic cells, but less so in bacterial contexts. DNA methylases, such as DNA adenine methyltransferase (DAM), methylate DNA at ‘5-GATC-3’ sequences. This is known to impact upon DNA replication, DNA repair, and transcriptional regulation at some virulence promoters.
Investigations in to cell methylation were achieved by digestion of plasmid pBR322 with DpnI restriction enzyme; this showed nutrient-dependent differences in methylation levels. DNA topology in wild-type and ∆dam cells was investigated by chloroquine gel electrophoresis, and this revealed a higher degree of supercoiling in ∆dam cells in the exponential phase of growth. To investigate global effects of DAM on transcription in Escherichia coli K-12, a lac28::egfp fusion was inserted at different chromosomal loci, and expression was measured using flow cytometry. Differences in gene expression between wild-type and ∆dam cells were found at these loci, with some differences depending on growth medium. At many loci, ∆dam cell populations exhibited increased fluorescence and, occasionally, less variation than wild-type cells. In further experiments, interactions between DAM and CRP (the catabolite-repression global transcription regulator protein) were explored, and found that DAM methylation could affect the action of CRP at a model promoter. My work implicates DAM methylase as a potent regulator of gene expression in Escherichia coli and an agent of cell-to-cell heterogeneity.
The mechanisms by which DAM methylase affected gene regulation were probed, bioinformatically. These studies revealed that while the absolute number of GATC motifs in the locality of the lac28 promoter did not seem to correlate to overall increases in gene expression, the number of GATC sites upstream of the same promoter may correlate with changes in the variability of GFP expression. This study also highlighted that DAM regulation of some loci may be protein-dependent.
Lastly, the effect of genetic position on the expression of antibiotic resistance genes was investigated in both the chromosomal and plasmid contexts. This work highlighted that chromosomal resistance cassettes may express differently, depending on their location. This in turn, yields different resistance phenotypes within the “same” bacterial strain. This concept was addditionally explored within a plasmid, whereby a transposon encoding a resistance gene was inserted randomly in to different locations in the plasmid genome. Thence, different levels of antibiotic resistance where seen that were location dependent.
In sum, this work aims to further traverse the existence of so-called “higher levels” of genetic regulation within Escherichia coli.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):