Regeneration of Escherichia coli from Minicells through Lateral Gene Transfer

Abstract

Recently, artificial life has been created with artificial materials and methods. Life can be created when genomic DNA molecules are integrated in liposomes containing biochemical reactions for biogenic needs. However, it is not yet known whether the integration of these parts will be able to occur in nature and constitute a living system. I planned to regenerate bacteria from biologically active liposomes by inserting genomic DNA using only natural materials and methods. Minicells of Escherichia coli, containing plasmids and activated SOS proteins, act as protocells. Four new E. coli strains were regenerated from minicells by inserting the genomes by using the system for conjugation between F^- and Hfr strains. Cells of the four regenerated strains showed the same genetic markers as the two genome donors. Pulse-field gel electrophoresis of their genomes showed admixing of those of both donors. In addition, the genomes of the four regenerated strains had chimeric genome of the two donors. These results show that synthesis of life can occur in nature without artificial arrangement.IMPORTANCE What is the difference between inanimate objects and organisms? Organisms always have genomic DNA. When organisms lose their genomes, they can neither grow nor reproduce. As the result, organisms turn into inanimate objects without their genomes. In this study, I regenerated microbes from cells that had lost their genomes (cell corpses) by inserting another genome. All steps of regeneration used the natural behavior of microbes. The same regeneration of microbes could happen in nature. These primitive lives have plasticity, which accelerates evolution and provides various kinds of life in the world.

Keywords: Hfr; bacterial conjugation; minicells; regeneration of bacteria.

FIG 1

Strategy for regeneration of E. coli cells from minicells. (A) Plan for regeneration from purified minicells of E. coli ME8077 by insertion of the genomes of ME8162 and RC30 through conjugation with F⁻ and Hfr strains. (B) Point of origin and direction of insertion of the genomes of ME8162 and RC30 during conjugation. (C) PCR amplification of the GFP gene and the 16S rRNA gene of ME8162 carrying pTSMb1, purified minicells, and minicells after conjugation with Hfr strains. Lanes: G, GFP gene; 16, 16S rRNA gene. Arrows indicate amplified DNA fragments.

FIG 2

Cell shapes of ME8077, minicells, and regenerated cells. The minicells (arrows) were purified from ME8077 as described in Materials and Methods. All strains were incubated in LB medium at 37°C. Bars indicate 5 μm.

FIG 3

Properties of the regenerated cells. (A) The indicator plasmid, pTSMb1. The cI gene on the genomes of ME8162 and RC30 represses the expression of lacI of pTSMb1 and allows GFP gene expression. The lacI gene on the genome of ME8077 represses cI and GFP gene expression. (B) Expression of the GFP gene in regenerated cells and their parent strains. (C) Genetic markers of the regenerated cells and their parent strains. All strains were streaked on LB medium containing antibiotics or M9 medium containing each of various saccharides as the sole carbon source. (D) Growth of the regenerated cells and their parents. All strains were incubated aerobically (160 rpm) at 37°C, and growth was determined by measuring the optical density at 660 nm. Three experiments were carried out independently, and error bars indicate standard deviations (SD) (n = 3).

FIG 4

Pulsed-field gel electrophoresis of PmeI and AscI digests of the E. coli genome. Electrophoresis was carried out as described in Materials and Methods. M1 and M2 are the low-range PFG marker and lambda ladder marker, respectively. Asterisks show bands of interest, as described in the text.

FIG 5

Comparative analyses of the E. coli genomes. Homologous regions are shown in boxes with the same colors. I used Mauve software for comparative analysis (locally colinear block weight = 22,195). Blue or red dashed boxes indicate regions homologous to RC30 or ME8162, respectively.