Abstract

Graphical Abstract

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1. Introduction

The ribonucleic acid (RNA) world hypothesis is widely recognized as one of the ‘origin of life’ (^1–5). In the RNA world, RNA had two functions: information storage and catalytic activities. Woese, Crick, and Orgel showed theoretically that RNA might have had catalytic activity (^6–8). Subsequently, it was experimentally demonstrated that RNA has catalytic activity (^9–12), and Gilbert proposed the concept of the ‘RNA world’ (¹).

It has remained controversial how the RNA world transited to the deoxyribonucleic acid (DNA) world (^{4, 13–15}). It is clear that the storage of information and catalytic functions were passed from RNA to DNA and proteins, respectively, although some catalytic activities remain in RNA as ribozymes (^{11, 12, 16}). The RNA–protein (RNP) world has been proposed to claim that proteins appeared in the RNA world (³). Recently, it has been suggested that various worlds, such as a lipid world, metabolism world and protein world, coexisted and interacted with each other (¹⁷). It was shown that ribonucleotides and deoxyribonucleotides could appear at the same time (¹⁸). Furthermore, an RNA peptide was experimentally suggested to appear after the RNA world (¹⁹). Because DNA replication requires some complex multimer proteins such as polymerase and helicase, it can be assumed that protein synthesis machineries might already have existed before the appearance of the DNA world (¹⁴). When we consider the transition from the RNA world to the DNA world with catalytic functions of proteins, ‘RNA replicons’, in which RNA produces proteins to replicate their coding RNA, and ‘DNA replicons’, in which DNA produces RNA to synthesize proteins which replicate their coding DNA, can be assumed to have coexisted at a certain evolutionary time point (Figure 1). Probably, DNA replicons mostly overtook RNA replicons thereafter.

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Figure 1.

The transition of the replicable information carrier. From the RNA world to the DNA world, an RNA replicon and a DNA replicon coexisted at some intermediate time point.

It is crucial to synthesize a coexistent state of an RNA replicon and DNA replicon to obtain insights into the transition from the RNA world to the DNA world and then to observe the process in which the DNA replicon overtakes the RNA replicon. The DNA genome in present-day organisms is a DNA replicon because it encodes DNA polymerase to replicate its own DNA. The RNA genome of RNA viruses is a replicon, as it encodes its own RNA replicase. Most RNA viruses proliferate explosively to kill the host cells. In other words, the RNA replicons overcome DNA replicons. However, some persistent viruses proliferate in cells but do not kill their hosts (^20–25). The vesicular stomatitis virus (VSV) and the Sendai virus for animal cells have been studied well (^{22, 26, 27}). VSV infected BHK21 cells and was maintained in the cell for over 5 years (²⁸). In plants, the Endornaviridae viruses, which are stably founded as double-stranded RNAs, are vertically transmitted through seeds (^{29, 30}). In fungi, the double-stranded RNA of mycovirus does not exhibit its extracellular transmission route; instead, it is distributed to daughter cells through host cell division (^{29, 31–33}). In bacteria, Escherichia coli infected with the single–stranded RNA (ssRNA) phage f2 and Pseudomonas phaseolicola infected with the double-stranded RNA (dsRNA) phage 6 turned out to be a carrier state (^34–36). These persistent viruses are RNA replicons that parasitize their hosts to coexist with the host DNA genomes.

The experimental setup suitable for getting insights into the transition requires an RNA and DNA replicon coexistence-dependent growth of host cells. The persistent RNA viruses mentioned previously may load a burden that reduces the growth rate of their host cells, and they will be removed if uninfected host cells take over the population of their host cells. Therefore, we need an experimental setup in which an RNA replicon gives the growth advantage to its host cells over uninfected cells. So far, an RNA replicon with genes of interest has been synthesized as RNA vectors by adding selection maker genes to persistent RNA virus genomes (^37–43). The single-stranded (+) RNA genome of the Sindbis virus, which includes a lacZ , and the persistent infectious variants of the (–) stranded RNA Sendai virus genome, with an egfp, were passaged for several generations (^{44, 45}). When the (+) stranded RNA genome of the brome mosaic virus or tobacco mosaic virus with an acetyltransferase gene (cat) was introduced into the protoplast, protein production and RNA genome replication were observed (^{42, 43}). For Pseudomonas syringae pv. phaseolicola, the double-stranded segmented RNA genome of 6 was engineered with kanamycin resistance, β-galactosidase and GFP genes (^{39, 40, 46, 47}). The host cells with these RNA vectors selectively grow to allow the stable coexistence of RNA and DNA replicons.

It is desirable to generate a simple RNA replicon that can be stably maintained with a selection maker in E. coli since the bacterium is the most-studied model organism and is more suitable for studying the ‘origin of life’ because of its simplicity than animal and plant cells are. The dsRNA genome of phage 6, which has been successfully engineered in P. syringae pv. phaseolicola, as mentioned previously, has a complex segmented structure. A simple RNA replicon with a minimal size is needed for an experimental approach to the ‘origin of life’. Although it is not an RNA replicon, Mills developed an RNA molecule that could be maintained in E. coli (⁴⁸). The RNA molecule was replicated by Qβ RNA replicase supplied from a plasmid to produce its complementary strand, which encoded the λN protein, a selection maker for E. coli, thus allowing the cells with the RNA molecule to grow selectively.

In this study, we produced an E. coli strain in which a ‘mini-RNA replicon’ and a DNA replicon coexisted. The mini-RNA replicon in our system was an RNA molecule that encoded exclusively the β subunit of the Qβ replicase derived from the RNA bacteriophage Qβ. The β subunit gene was inserted into the middle of MDV-1, a short ssRNA sequence replicable using Qβ replicase (^{49, 50}). The translated β subunit constitutes Qβ replicase with elongation factor (EF)-Tu, EF-Ts and S1 from E. coli. The matured enzyme replicated the original RNA, which was a mini-RNA replicon. By replacing the λN protein on the RNA molecule prepared by Mills (as described previously) with the α fragment of LacZ, we synthesized another RNA molecule to allow the E. coli cells with the two RNA molecules to grow selectively in a lactose minimal medium. One problem in Mills’ system is that chloramphenicol resistance, the selective advantage brought about by the λN protein, is lost for a few passages with spontaneous mutations.

The synthesized coexistent state of the mini-RNA replicon and DNA replicon (E. coli genome) was confirmed to be persistent in E. coli. As our system involves present well-evolved macromolecules, it may not be very similar to the one that might have existed before the transition from the RNA world to the DNA world. However, our system and the ones present before the transition involve common competition between the two replicons for resources, translation machinery and others. Therefore, our system is an experimental setup toward obtaining experimental insights into how the RNA world transited to the DNA world.

2. Materials and methods

3. Results

3.1. E. coli growth system dependent on Qβ replicase

To maintain the mini-RNA replicon encoding the β subunit of Qβ replicase stably in E. coli cells, we constructed another RNA molecule that allowed the cells with Qβ replicase to grow selectively. As described in Section 1, we replaced the λN protein in the system prepared by Mills with the α fragment of LacZ for α complementation (⁵⁶). The complementary strand of the sequence encoding the α fragment of LacZ was sandwiched with two recognition sites for Qβ replicase and placed under the trp promoter (pLacZα(–)) so that the complementary strand of the coding sequence of the lacZα (LacZα(−)RNA) was transcribed (Figure 2a). The transcribed LacZα(−)RNA was replicated by Qβ replicase into its complementary strand (LacZα(+)RNA) to synthesize α fragment of LacZ. The translated fragment was associated with the ω fragment from the E. coli genome to allow the cells to grow in a lactose minimal medium. Qβ replicase was constituted by the β subunit supplied from the other plasmid, pβ, together with EF-Tu, EF-Ts and S1 from the E. coli genome.

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Figure 2.

The E. coli growth dependent on Qβ replicase. (a) The operation principle of E. coli growth in the lactose minimal medium by Qβ replicase. E. coli was transformed with double plasmids, pLacZα(−) and pβ. The complementary strand of the LacZα-encoding gene with Qβ replicase recognition sites at both ends (LacZα(−)RNA) was transcribed by RNA polymerase of E. coli. The β subunit was produced from pβ and constituted Qβ replicase by assembling with three subunits from E. coli. The LacZα-encoding RNA strand (LacZα(+)RNA) was obtained by replication by Qβ replicase using the LacZα(−)RNA as a template. The LacZα protein was translated from the LacZα(+)RNA and assembled with LacZω from E. coli to produce LacZ (α complementation). Then, E. coli could grow in the lactose minimal medium. (b) Left: The growth of E. coli with pLacZα(−) and pβ or pLacZα(−) and pΔβ that did not have β subunit gene. Right: The growth of E. coli with empty vector (without LacZα(−)) and pβ or empty vector and pΔβ.

E. coli cells with pLacZα(−) and pβ grew in the lactose minimal medium (Figure 2b). As a control experiment, we cultured the cells with pLacZα(−) and the pΔβ plasmid; however, they did not grow in the lactose minimal medium. Furthermore, we introduced a vector that did not have the lacZα (empty vector). The cells with the empty vector and pβ or empty vector and pΔβ did not grow in the lactose minimal medium. This result indicated that pLacZα(–) afforded Qβ-replicase-dependent growth to E. coli.

3.3. Preparation of the host cell strain for the mini-RNA replicon

We obtained an E. coli strain that exhibited high growth with MDVβ(+)RNA and LacZα(−)RNA through two passage experiments to circumvent this problem. For the first-passage experiment, we introduced the I-SceIdownstream of the tetA promoter and an I-SceI recognition site into pLacZα(−), designated as pLacZα(−)_I-SceI_I-SceI site (Figure 3a). We also introduced an I-SceI recognition site into pβ, which was designated as the pβ_I-SceI site. The E. coli cells with the two plasmids were passaged four times to increase their growth rate in the lactose minimal medium. After the growth rate adaptation, followed by induction of I-SceI, we picked colonies that grew on LB+Amp agar medium but did not grow on LB +Km agar medium, to obtain the adapted strain only with pLacZα (−)_I-SceI_I-SceI site. Before the second-passage experiment, the adapted strain with pLacZα(−)_I-SceI_I-SceI site was transformed with pMDVβ_I-SceI site. The pMDVβ_ISceI site was constructed by introducing an I-SceI recognition site into pMDVβ (Figure 3b). Throughout three passages in lactose minimal medium, the cells with the two plasmids showed a high growth rate, indicating the circumvention of the problem of the lack of growth of the original cells with MDVβ(+)RNA and LacZα(−)RNA. After the induction of I-SceI for curing both plasmids, we picked colonies that could not grow on LB+Amp agar medium or LB+Km agar medium but grew on LB agar medium. Thus, we obtained an adapted E. coli strain that could be used as a host cell for the mini-RNA replicon in our system (designated as Adapted MDS42Blue).

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Figure 3.

The adaptation of E. coli with pLacZα(−) and pMDVβ_I-SceI site to the lactose minimal medium. (a) E. coli with pLacZα(−)_I-SceI_I-SceI site and pβ_I-SceI site was passaged in lactose medium four times. The pβ_I-SceI site was cured by endonuclease I-SceI upon Dox induction at the time indicated by the circle. (b) The cells were transformed with pMDVβ_I-SceI site and passaged in the lactose medium three times. pLacZα (−)_I-SceI_I-SceI site and pMDVβ_I-SceI site were cured upon Dox induction at the time indicated by the circle.

A whole-genome sequence was conducted using original and Adapted MDS42Blue to determine the mutation sites of Adapted MDS42Blue. Four point mutations were observed; two were nonsynonymous, and two were on intergenic regions (Table S2). One of the synonymous mutations was a substitution in 34 bases upstream of lacZω, and one of the nonsynonymous mutations was a substitution of chaperon groL that assists protein folding.

3.4. Persistent coexistence of the mini-RNA replicon and the DNA replicon in E. coli

We investigated whether the mini-RNA replicon was replicated in the adapted E. coli strain, Adapted MDS42Blue. We introduced pMDVβ_I-SceI site and pLacZα(−)_I-SceI into Adapted MDS42Blue (Figure 4a). pLacZα(−)_I-SceI was constructed by eliminating the I-SceI site from pLacZα(−) _I-SceI_I-SceI site (Figure S1). First, we passaged the cells with the two plasmids in the lactose minimal medium without the induction of I-SceI (Figure 4b, black dots). We divided the culture into two tubes and added Dox (for the induction of I-SceI) into one of the two tubes. After 24h of cultivation, we confirmed that only 0.07–0.08% of the cells held pMDVβ_I-SceI site by taking the ratio of colony forming unit (CFU) on LB+Amp+Km agar medium over CFU on LB+Amp agar medium (Figure 4c at 119h). Although ~99.9% of the cells lost pMDVβ_I-SceI site, they showed a high growth rate of 0.16 ±0.01h⁻¹ (n=7), comparable to the growth rate of the cells without the induction of I-SceI 0.36±0.02h⁻¹ (n=5), implying that MDVβ(+)RNA was kept without its continuous transcription from the pMDVβ_I-SceI site. We measured the MDVβ(+)RNA molecules per cell by RT-qPCR. The RNA molecules of cells with pMDVβ_I-SceI site were 980±310 molecules/cell (n=3), and the RNA molecules of cells without pMDVβ_I-SceI site were 23±24 molecules/cell (n=3). The coexistent state of the mini-RNA replicon and DNA replicon in E. coli was successfully synthesized.

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Figure 4.

The growth of E. coli with the mini-RNA replicon in the lactose minimal medium. (a) Adapted MDS42Blue was transformed with pLacZα(−)_I-SceI and pMDVβ_I-SceI site. (b) Cells were passaged in with Km in two lines and divided into two, respectively. One was treated with 20nM Dox (white and gray triangles) and the other was not (white and gray circles). The (×) shows the detection limit (OD₆₀₀=0.0025) and the one-fourth dilution by passage. (c) The ratio of pMDVβ_I-SceI site-carrying cells. The ratio of CFU grown on LB with Km to CFU grown on LB with Amp and Km is shown.

We confirmed the persistence of the mini-RNA replicon in E. coli. We performed an additional eight to nine passages with and without Dox induction of I-SceI (Figure 4b and c). Regardless of the induction, the fraction of cells without the pMDVβ_I-SceI site reached 60% at the end of the passage experiments. This may have stemmed from imperfect plasmid curing by I-SceI and the burden of the presence of the plasmid. The convergence of the ratio indicated that the cells without the pMDVβ_I-SceI site showed the same specific growth rate as that observed with the pMDVβ_I-SceI site, implying that MDVβ(−)RNA without continued transcription from pMDVβ_I-SceI was kept during at least nine passages at the same concentration as in the cells with transcription from the plasmid. The coexistence of the mini-RNA replicon with the DNA replicon in E. coli cells was persistent.

4. Discussion

In this study, we constructed a persistent coexistence state of a mini-RNA replicon and DNA replicon in E. coli. The mini-RNA replicon encoding the β subunit of Qβ replicase was replicated by Qβ replicase in E. coli. To maintain the mini-RNA replicon persistently in E. coli cells, we constructed a system of α complementation that was dependent on Qβ replicase, thus allowing the E. coli cells with the mini-RNA replicon to grow in the lactose minimal medium selectively.

We modified Mills’ Qβ replicase-dependent E. coli growth system to allow the cells with our mini-RNA replicon selectively grow through α complementation. Mills constructed a system in which E. coli grew depending on Qβ replicase (⁴⁸). He inserted the complementary RNA strand of the N protein gene between 145 bases of the complementary strand of the 3′ end of the Qβ genome sequence and 147 bases of the MDV sequence. The system encompassed another two plasmids; one encoded the β subunit of Qβ replicase, and the other carried a cat located downstream of a transcription terminator. After the replication of the complementary RNA in the presence of Qβ replicase made of a β subunit supplied from the first plasmid together with EF-Tu, EF-Ts and S1 from the E. coli genome, the N protein was translated. With the translated N protein, the mRNA of cat was transcribed from the second plasmid to produce CAT, thus allowing Qβ replicase-dependent cell growth in the presence of chloramphenicol.

It should be noted that Mills’ RNA is not an RNA replicon because it does not encode any protein for its replication. One problem in Mills’ system is that the E. coli cells quickly become chloramphenicol resistant after a few passages. To circumvent this problem, we replaced the N protein with the α fragment of LacZ to allow the cells with our mini-RNA replicon to grow stably. The cells with our mini-RNA replicon were stably passaged for >10days in the lactose minimal medium. We confirmed that no mutations that damage our Qβ replicase-dependent cell growth system appeared on the E. coli genome during the passages (data not shown).

We synthesized a mini-RNA replicon encoding only the β subunit of Qβ replicase. Some RNA replicon-type RNA vectors have been developed by modifying persistently infecting RNA virus and phage genomes (^37–43). The constructed coexistent state of the mini-RNA replicon and the DNA genome in E. coli cells is suitable for research on the ‘Origin of life’ because of its simplicity compared with the previously developed RNA replicon-type RNA vectors. We can observe the evolutionary dynamic of the coexistent state with passages of many generations and whole-genome sequencing of both the RNA replicon and the DNA replicon (E. coli genome).

The next challenge in our coexistence state is the expansion of the size of the RNA replicon. Two routes of expansion are possible. One consists of inserting several genes from the E. coli genome into the mini-RNA replicon obtained in this study. The other way consists of modifying persistent or pseudolysogenized Qβ phage mutants in the same way as for developing RNA replicon-type RNA vectors. It is known that Qβ phage enters a pseudolysogeny state if it loses the function of the A2 protein for cell lysis (⁵⁷). After a substantial expansion of the size of the RNA replicon, we will be able to obtain insights regarding what happened on the long RNA replicon in the ancient RNP world that coexisted with the DNA world.

Using the coexistence state of the mini-RNA replicon and DNA replicon, it is possible to obtain insight into the transition process from the RNA world to the DNA world. Questions related to the transition process, such as how did some of the functional information on the RNA replicon move to the DNA replicon and to what extent were the suitable mutation rates of the RNA replicon and DNA replicon dedicated to enhancing the transition process or keeping the coexistence state, can be addressed using the approaches of experimental evolution and synthetic biology. Addressing these questions experimentally will contribute to progress in the research field of the origin of life.

Supplementary Material

Acknowledgments

Contributor Information

Akiko Kashiwagi, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan.

Tetsuya Yomo, School of Life Sciences, East China Normal University, Shanghai, People’s Republic of China.

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