Journal Articles

Permanent URI for this collectionhttps://mro.massey.ac.nz/handle/10179/7915

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Author: search.filters.author.Silander OKSubject: search.filters.subject.Escherichia coli

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    Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background
    (John Wiley and Sons, Ltd, 2021-08-24) Vlková M; Morampalli BR; Silander OK
    The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between cis- or trans-genetic elements affect the behavior of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context-dependent cis-interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self-splicing ribozyme called RiboJ, which can be used to decouple upstream 5' UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans- and cis-genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis-effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis- nor trans-genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis- and trans-genetic changes.
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    Growth condition-dependent differences in methylation imply transiently differentiated DNA methylation states in Escherichia coli
    (Oxford University Press on behalf of the Genetics Society of America, 2023-02) Breckell GL; Silander OK
    DNA methylation in bacteria frequently serves as a simple immune system, allowing recognition of DNA from foreign sources, such as phages or selfish genetic elements. However, DNA methylation also affects other cell phenotypes in a heritable manner (i.e. epigenetically). While there are several examples of methylation affecting transcription in an epigenetic manner in highly localized contexts, it is not well-established how frequently methylation serves a more general epigenetic function over larger genomic scales. To address this question, here we use Oxford Nanopore sequencing to profile DNA modification marks in three natural isolates of Escherichia coli. We first identify the DNA sequence motifs targeted by the methyltransferases in each strain. We then quantify the frequency of methylation at each of these motifs across the entire genome in different growth conditions. We find that motifs in specific regions of the genome consistently exhibit high or low levels of methylation. Furthermore, we show that there are replicable and consistent differences in methylated regions across different growth conditions. This suggests that during growth, E. coli transiently differentiate into distinct methylation states that depend on the growth state, raising the possibility that measuring DNA methylation alone can be used to infer bacterial growth states without additional information such as transcriptome or proteome data. These results show the utility of using Oxford Nanopore sequencing as an economic means to infer DNA methylation status. They also provide new insights into the dynamics of methylation during bacterial growth and provide evidence of differentiated cell states, a transient analog to what is observed in the differentiation of cell types in multicellular organisms.