Adaptive Evolution of an Artificial RNA Genome to a Reduced Ribosome Environment

The reconstitution of an artificial system that has the same evolutionary ability as a living thing is a major challenge in the <i>in vitro</i> synthetic biology. In this study, we tested the adaptive evolutionary ability of an artificial RNA genome replication system, termed the translation-coupled RNA replication (TcRR) system. In a previous work, we performed a study of the long-term evolution of the genome with an excess amount of ribosome. In this study, we continued the evolution experiment in a reduced-ribosome environment and observed that the mutant genome compensated for the reduced ribosome concentration. This result demonstrated the ability of the TcRR system to adapt and may be a step toward generating living things with evolutionary ability

Effects of Compartment Size on the Kinetics of Intracompartmental Multimeric Protein Synthesis

The cell contents are encapsulated within a compartment, the volume of which is a fundamental physical parameter that may affect intracompartmental reactions. However, there have been few studies to elucidate whether and how volume changes alone can affect the reaction kinetics. It is difficult to address these questions <i>in vivo</i>, because forced cell volume changes, e.g., by osmotic inflation/deflation, globally alters the internal state. Here, we prepared artificial cell-like compartments with different volumes but with identical constituents, which is not possible with living cells, and synthesized two tetrameric enzymes, β-glucuronidase (GUS) and β-galactosidase (GAL), by cell-free protein synthesis. Tetrameric GUS but not GAL was synthesized more quickly in smaller compartments. The difference between the two was dependent on the rate-limiting step and the reaction order. The observed acceleration mechanism would be applicable to living cells as multimeric protein synthesis in a microcompartment is ubiquitous <i>in vivo</i>

Relation between growth and translation.

<p><b>A.</b> Translation activity of ribosomes. The maximal translation rates of the purified ribosomes were estimated according to GFP biosynthesis curves. The ribosomes from R0, R1 and R5 are indicated in blue, purple and green, respectively. Solid lines indicated linear regressions with the slopes of 0.064, 0.016 and 0.007 for R0, R1 and R5, respectively. The standard errors of repeated tests (n = 3–4) are indicated. <b>B.</b> Correlation between growth rate and translation rate. The translation rates at 1 nM ribosomes (data from A) are plotted against the theoretically estimated maximal growth rates <i>r</i>_<i>max</i> (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135639#pone.0135639.g003" target="_blank">Fig 3C</a>). Blue, purple and red represent R0, R1 and R5, respectively. Standard errors are indicated as described in A and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135639#pone.0135639.g003" target="_blank">Fig 3C</a>. <b>C.</b> A scheme of the changes in the growth economics for maintaining cellular activity. The purple shading represents the intracellular resource (histidine) concentration balanced between incorporation (orange arrow) and consumption (green arrows). The green arrows indicate the changes in resource (histidine) redistribution for translation under poor conditions. The green shading highlights the reorganized protein translation enabling survival. The orange shading suggests the changes caused by the mutations related to membrane and transport, which required further demonstration and were excluded from the present study.</p

Reduced growth rate and improved sustainability.

<p><b>A.</b> Competence under starved conditions. The control strain (open circles) was co-cultured with R0, R1, R5 and R7 under starved conditions. The temporal changes of cell concentrations were monitored by a CFU assay. The standard errors of four to eight assay plates are indicated. <b>B.</b> Growth in the presence of histidine. The temporal changes in cell concentrations of R0, R1, R5 and R7 in the presence of 1, 10, and 100 μM histidine were evaluated by flow cytometry. Blue circles, purple squares, green rhombuses and red triangles represent R0, R1, R5 and R7, respectively. The standard errors of three independent test tubes (cultures) are indicated.</p

Damage in ribosomes.

<p><b>A.</b> The relative abundance of the ribosomal protein S1 in cells. The ribosomal protein S1 was detected by western blotting, and its relative abundance was calculated according to the band intensity by image analysis using the purified S1 protein as the positive control. Crosses indicate the disappearance of the band. The standard errors of three independent Western blot analyses are shown. <b>B.</b> The relative abundance of the ribosomal protein S9. The ribosomal protein S9 was detected by western blotting, and its relative abundance was calculated according to the band intensity by image analysis. Standard errors of triplicates are shown. <b>C.</b> Ribosomal RNAs. An example RNA electrophoresis pattern of the total RNAs is shown. 16S and 23S rRNAs are indicated. The truncated rRNAs are indicated with asterisks. <b>D.</b> The ratio of 16S and 23S rRNAs. The ratios of 16S and 23S rRNAs were calculated according to the band intensity and area as shown in C. Standard errors of the RNA samples purified from three independent cell cultures are indicated.</p

Genome mutations accumulated from R1 to R7.

<p>The mutation accumulation is represented in color from R0 to R7, which indicate the rounds of repeated starvation and re-growth. 1 and 0 indicate the mutant and the wild type, respectively. Genome position, Change in DNA and Gene refer to AP012306 (DDBJ). Change in protein, Description and Cell location are according to GenoBase and RegulonDB. The mutations that occurred in the non-coding regions are indicated with parentheses in the “Cell location” column. The two mutations fixed in R1 were maintained in R2–R7. The additional mutations were detected in R5, R6, and R7. The cell location of the mutations indicated as outer/integral membrane or periplasmic suggest these mutations play a role in transport, and the mutations corresponding to <i>rnr</i>, <i>rpsI</i> and <i>rpsA</i> participate in translation. Asterisks and plus marks in Description indicate essential genes and gene functions related to translation.</p

Growth rate and population size in response to various amounts of histidine.

<p><b>A.</b> Saturated population densities detected by FCM. The maximal cell concentrations experimentally acquired in the various concentration of histidine are shown. Blue circles, purple squares, green rhombuses and red triangles represent R0, R1, R5 and R7, respectively. Standard errors of triplicates are indicated. <b>B.</b> Experimentally evaluated exponential growth rates. The growth rates of the growing cells were calculated according to the average cell concentrations of any three out of 12–15 test tubes at the defined time scales. The data fit to the Monod equation are shown as solid lines. Open marks represent the experimental measurements. The concentrations of histidine varied from 0.1 nM to 100 μM. <b>C.</b> Theoretically estimated <i>Ks</i> and <i>r</i>_<i>max</i>. The parameters <i>Ks</i> (right) and <i>r</i>_<i>max</i> (left) were calculated according to the Monod equation using the experimentally acquired growth rates shown in B. The standard errors derived from the theoretical fitting are indicated.</p

Repeated starvation and re-growth.

<p><b>A.</b> Scheme of the evolution experiment. <b>B.</b> Temporal changes in the population density. The cell concentrations were counted by flow cytometry (FCM count, upper panel), and the active cells were detected with a colony formation unit (CFU) assay (bottom panel). The blue- and orange-filled circles represent the sampling points, and the white circles indicate the cell concentrations after 100-fold dilutions.</p