Team:GO Paris-Saclay/RNACell
Objectives
According to a common theory about the evolutionary history of life, RNA was the first molecule to carry genetic information and to act as a biochemical catalyst (Alberts, 2002). In other words, in the first cell that evolved, RNA did the job that is nowadays carried out by DNA and proteins. Proteins would have subsequently emerged with better catalytic functions than RNA, and finally DNA would have arisen last. The theory for the “RNA world” is supported by some arguments. Indeed, in current cells:
- RNA is a support for genetic information (through messenger RNA)
- RNA can exhibit enzymatic activity (for example, peptide bond formation is catalyzed by ribosomal RNA)
- Many co-enzymes are nucleotides bound to other molecules
- RNA can bind different ligands and adopt different structures that will regulate its function
- Deoxynucleotides are synthesized from nucleotide precursors, suggesting the biosynthesis of DNA has evolved from preexisting RNA biosynthetic pathways
Moreover, in the laboratory, researchers have been able to synthetize random RNAs and select ribozymes that catalyze many of the known reactions that happen in a cell. Consequently, the theory of an “RNA World” is widely accepted by scientists; however, no experimental evidence supports it (Alberts, 2002). One important aspect of this theory is that the RNA information of “RNA cells” is inherited by daughter cells, suggesting that these cells would have used mechanisms to replicate their RNA using RNA as a template.
As a first step in proving the RNA World concept, we wanted to generate an “RNA cell”, in which replicating RNA, rather than DNA, would be the support of genetic information.
In order to carry out this substitution of DNA with RNA, we first had to eliminate the cellular DNA. Towards this end, we cloned and expressed 3 nucleases and showed that they efficiently degrade bacterial DNA. Cells which have lost their DNA did not lyse for at least 150 minutes.
As the next step in achieving an RNA cell we needed to inject RNA, replicate it and produce proteins from it. We chose to use a bacteriophage (or phage) from the Leviviridae family, a virus that infects bacteria and whose genetic information is carried by RNA. In the infectious virions, RNA is protected by a capsid, a shell made of proteins. Upon infection of “normal” cells, RNA is internalized allowing production of viral proteins. Among these, a special RNA replicase (RNA-dependent RNA polymerase or RdRp) allows replication of the viral genome. New virions are assembled before the host cell is lysed.
In this work, we tested whether cells that have lost their DNA could be infected by the MS2 phage and produce new virions, which would result from the replication of RNA and its translation into proteins. If successful, these cells would transiently be akin to “RNA cells”.
Choice of material
MS2 phage is a positive-sense single-stranded RNA virus that infects Escherichia coli. The MS2 phage binds to a pilus called the fertility (F) factor of the bacterium and the viral RNA penetrates inside the cell by an unknown mechanism. Once internalized, the positive-sense RNA genome is translated by host ribosomes and generates coat and maturation proteins, lysis proteins and RdRp proteins. RdRp synthetizes minus-sense strand RNA which serves as template to produce new positive strand genome. Next, the capsid is assembled with the coat proteins and the viral RNA; once there are enough lysis proteins translated, virions are released (Koning et al. 2016).
Since the receptor for MS2 phages is the F factor, we used E. coli KeioZ1 F’ strain which produces this F pilus. As a negative control, we used the E. coli KeioZ1 strain which does not express the F pilus.
Protocols
To test the infection of a DNA-free cell by the MS2 phage, we performed the following experiment (Figure 1):
Figure 1:
Phage multiplication assay. A) KeioZ1 F’ E. coli
strain was transformed with an empty plasmid (pBAD) or a pBAD plasmid which
carries either nuclease_A1 (BBa_K3027000), nuclease-gp3
(BBa_K3027001) or nuclease_yqcG (BBa_K3027002) genes. Expression
of these genes is under the control of an arabinose-inducible promoter that
can be repressed by glucose (Glc). Following selection on LB agar
supplemented with ampicillin (Amp) and Glc, transformants carrying the
plasmids were shaken at 37°C overnight under shaking in LB medium
supplemented with Glc (0.2%) and Amp (100 µg/mL). Thereafter, strains were
inoculated at an optical density of 0.1 (OD600) in a LB medium
containing Amp, CaCl2 (1 mM) and MgCl2 (1 mM), but no
Glc. Once the OD reached 0.4, arabinose (0.2 %) was added in culture media
and cells were further shaken at 37°C. Five or 15 minutes later (as
mentioned in the results), MS2 phages were added to the cells at a
multiplicity of infection (MOI) of 5. At each timepoint the OD 600 was measured and 500µL of culture were centrifuged.
Supernatants were serially diluted and used to determine the viral titer
(concentration). For this purpose, dilutions were either (i) spotted on a
plate with a soft-agar overlay containing an E. coli indicator
strain or (ii) mixed with the indicator strain in soft agar and
subsequently overlaid on LB agar. After incubation of the plates at 37°C
overnight, plaques were counted to determine the PFU/mL supernatant. B) Example of overlay agar plates with lysis plaques
formed by phage MS2 on the susceptible indicator strain Hfr.
Results
To test the possibility to infect DNA-less cells with phage MS2, we performed two experiments. In the first one, infection was carried out five minutes after arabinose was added to induce expression of the nuclease. In the second experiment, infection was started fifteen minutes after arabinose addition.
Experiment 1: Nuclease gene expression for five minutes before
infection with RNA phage MS2
Upon infection with phage MS2, E. coli strain KeioZ1, which lacks the receptor for the virus, exhibits a higher growth rate than the susceptible strain KeioZ1 F’ (pBAD) (Figure 2), consistent with a metabolic burden linked to the viral infection. At this stage, the bacteria have not lysed in sufficient numbers to detect a drop in OD600.
Figure 2:
Growth of E. coli strains infected with phage MS2 five minutes after arabinose induction. Time (in min) refers to time since infection.
Strains expressing the 3 different nucleases show either an arrest in growth within 30 min (nuclease_gp3 and nuclease_yqcG) or a severe decrease in their multiplication rate (nuclease_A1) (Figure 2). This result suggests that, as was observed in the absence of infection, most cells in this experiment have lost their capacity to divide, probably due to the production of the nucleases.
Figure 3:
MS2 phage titer after incubation with resistant strain KeioZ1 or strain KeioZ1 F’ pBAD which produces the MS2 receptor (F pilus).
The incubation of the MS2 phage with resistant host strain KeioZ1 shows an initial reduction in titer 30 minutes after the addition of phages possibly through phage binding to the plastic tube surface or phage inactivation in the culture medium, then, the titer remains constant (Figure 3). Thus, there is no increase of PFU/mL over time. In conclusion, MS2 does not multiply in KeioZ1 cells which is consistent with the fact that MS2 needs the F pilus to infect a cell.
When MS2 was incubated with strain KeioZ1 F’ pBAD, the phage titer initially decreased at 30 min post-infection similarly to KeioZ1 strain (Figure 3). However, between 60 and 120 min the titer skyrocketed by 4 logs (Figure 3), consistent with the lytic cycle of 80 min and a burst size around 1000 virions per infected cell previously described for MS2 (Jenkins, 1974).
Figure 4:
Evolution of MS2 phage titer after incubation with KeioZ1 F’ pBAD cells (no nuclease expression, blue bars) or KeioZ1 F’ cells harbouring a pBAD plasmid which encodes the nuclease A1 (A), Gp3 (B) and YqcG (C).
Upon infection of cells expressing nuclease genes A1 or yqcG, we observed ca. 100-fold increase in MS2 phage titer released in the supernatant (Figure 4A and 4C, respectively). In this experiment, the increase in MS2 titer was more modest in cells expressing nuclease_gp3, with a mere 10-fold increase between 60 et 120 min of infection (Figure 4B).
Taken together, our results indicate that cells undergoing a sudden genomic DNA loss could be infected with the RNA phage MS2 and produce new virions.
Experiment 2: Nuclease gene expression for fifteen minutes before
infection with RNA phage MS2
Next, we wanted to carry the experiment one step further. We used the same protocol, but nucleases were produced for 15 min before MS2 phages were added and, at some time points, we extracted genomic DNA from infected cells to ensure that the infected cells had lost their DNA.
Figure 5:
Figure 5: Growth of E. coli strains infected with phage MS2 fifteen minutes after arabinose induction. Time (in min) refers to time since infection.
Figure 6:
Figure 6: Comparison of MS2 phage titer after incubation with KeioZ1 F’ pBAD cells or KeioZ1 F’ cells harbouring a pBAD plasmid which encodes a nuclease.
While the resistant strain KeioZ1 grew well in the presence of the phages, strain KeioZ1 F’ pBAD stopped to grow after 30min of infection and the optical density of the culture dropped at 120min (Figure 5). This diminution of OD600 correlated with a strong increase in MS2 titer (more than 4 logs) (Figure 6). Together, these results indicate that MS2 phages multiply well in our positive control strain and lead to cells lysis.
Cells expressing the three nucleases for 15 min before infection showed no growth in the presence of MS2 phage, most likely due to DNA degradation. We did not observe a drop of OD for strains as the control cells (Figure 5). Titers of MS2 increased 10 to 20-fold between 90 and 120 minutes when phages were incubated with KeioZ1 F’ pBAD-nuclease_gp3 or KeioZ1 F’ pBAD-nuclease_yqcG (Figure 6). Most importantly, upon infection of cells expressing the nuclease gene A1, MS2 titers soared by more than 4 logs between the same lapse of time, following the very same trend as KeioZ1 F’ pBAD (Figure 6). While we cannot exclude that some of the RNA phages are replicating within some of the “cheater cells” that still have some DNA, these cells only represent 0.3 % of the total cells and cannot account for all the phages released into the supernatant. To control that the cells used in this experiment lost their genomic DNA, we prepared genomic DNA during infection. While genomic DNA could be recovered in control cells 90 and 120 minutes after addition of phages, no DNA was recovered from the three strains expressing the nucleases (Figure 7).
It is intriguing to see that DNA-free cells can still produced virions but much less than normal cells. For this reason, we mathematically modelled the phage infection in DNA-less and DNA-proficient bacteria. We showed that the lower phage production in DNA-less bacteria is due to a lower quantity of host ribosomes.
Figure 7:
Electrophoresis gel of gDNA prepared from KeioZ1 F’ cells expressing different nucleases and infected by MS2 phage at different times of infection (in min). Lane 1: KeioZ1 F’ pBAD (negative control without nuclease), lane 2: KeioZ1 F’ pBAD-nuclease_A1, lane 3: KeioZ1 F’ pBAD-nuclease_gp3, lane 4: KeioZ1 F’ pBAD-nuclease_yqcG.
Conclusions
Taken together, our results suggest that MS2 is able to multiply in cells without DNA. Therefore, for a short amount of time, we generated cells where the replicating genetic information was carried by RNA. Although ephemeral we were able to produce an RNA cell, resembling a cell that may have existed in the RNA world, before DNA evolved (Figure 8).
Figure 8:
Model of our ephemeral RNA-cell. With a little help from RNA phage MS2, we generated a DNA-free cell where the replicating genetic material is RNA.
References
Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. The RNA World and the Origins of Life. https://www.ncbi.nlm.nih.gov/books/NBK26876/
Jenkins, Beard and Howe. 1974. « Male-Specific Bacteriophage MS2 Propagation in Fluorophenylalanine-Resistant Escherichia coli K12». J Virol. 14(1): 50.
Koning, Roman I, Josue Gomez-Blanco, Inara Akopjana, Javier Vargas, Andris Kazaks, Kaspars Tars, José María Carazo, et Abraham J. Koster. 2016. « Asymmetric Cryo-EM Reconstruction of Phage MS2 Reveals Genome Structure in Situ ». Nature Communications 7 (1): 12524. https://doi.org/10.1038/ncomms12524 .
