Bacterial Genome Containing Chimeric DNA–RNA Sequences

Abstract

Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (∼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.

Engineering polymerases for applications in synthetic biology

“…Engineering bacteria . One recent exciting example relevant to the field of synthetic biology is the generation of mutant E. coli strains derived from engineered bacteria that contain significant (~40–50%) ribonucleotide content in their genome (Mehta et al ., 2018). These systems have the potential to provide new insight into the origin of life by offering a better understanding of the transition from the RNA to the DNA world.…”

Synthetic Life with Alternative Nucleic Acids as Genetic Materials

“…Surprisingly, mutated E. coli strains containing chimeric DNA−RNA sequences (40−50% ribonucleotide content) in their genomic DNA were observed during the exploration to improve 5hmC content [95]. It has been described that DNA polymerases can incorporate ribonucleotides and Complete replacement of natural nucleobases with non-canonical ones in the genomes of living organisms is challenging.…”

“…Recent studies postulate that homogeneous DNA genomes may have evolved from heterogeneous chimeric RNA-DNA templates [96]. The detection of this DNA-RNA chimera may provide further insights into the origin of life [95].…”

Toward a Synthetic Yeast Endosymbiont with a Minimal Genome

“…[10][11][12][13] Recently, we developed a synthetic strategy to establish E. coli endosymbionts within host yeast cells to mimic mitochondrial evolution starting from bioenergetically competent and genetically tractable endosymbionts. 14 Briefly, our strategy (Figure 1) involved engineering E. coli strains such that they (i) are auxotrophic for a key central metabolite (present in the host cytosol) for which there are endogenous transporters, (ii) express a functional ADP/ATP translocase to provide ATP to the respiration deficient S. cerevisiae mutant and (iii) express SNARE-like proteins to avoid lysosomal degradation as previously described. [14][15][16][17][18] A respiration deficient mutant of S. cerevisiae, S. cerevisiae cox2-60 (NB97), was used which is unable to synthesize ATP under non-fermentable growth conditions.…”

“…14 Briefly, our strategy (Figure 1) involved engineering E. coli strains such that they (i) are auxotrophic for a key central metabolite (present in the host cytosol) for which there are endogenous transporters, (ii) express a functional ADP/ATP translocase to provide ATP to the respiration deficient S. cerevisiae mutant and (iii) express SNARE-like proteins to avoid lysosomal degradation as previously described. [14][15][16][17][18] A respiration deficient mutant of S. cerevisiae, S. cerevisiae cox2-60 (NB97), was used which is unable to synthesize ATP under non-fermentable growth conditions. [19][20][21] Under selection conditions containing non-fermentable carbon sources, the S. cerevisiae cox2-60 obtains ATP from the engineered E. coli endosymbiont and the S. cerevisiae cox2-60 cytosol in return provides thiamin to the E. coli endosymbiont that is a thiamin auxotroph.…”