Stochasticity in Gene Expression in a Cell-Sized Compartment

The gene expression in a clonal cell population fluctuates significantly, and its relevance to various cellular functions is under intensive debate. A fundamental question is whether the fluctuation is a consequence of the complexity and redundancy in living cells or an inevitable attribute of the minute microreactor nature of cells. To answer this question, we constructed an artificial cell, which consists of only necessary components for the gene expression (<i>in vitro</i> transcription and translation system) and its boundary as a microreactor (cell-sized lipid vesicle), and investigated the gene expression noise. The variation in the expression of two fluorescent proteins was decomposed into the components that were correlated and uncorrelated between the two proteins using a method similar to the one used by Elowitz and co-workers to analyze the expression noise in <i>E. coli</i>. The observed fluctuation was compared with a theoretical model that expresses the amplitude of noise as a function of the average number of intermediate molecules and products. With the assumption that the transcripts are partly active, the theoretical model was able to well describe the noise in the artificial system. Furthermore, the same measurement for <i>E. coli</i> cells harboring an identical plasmid revealed that the <i>E. coli</i> exhibited a similar level of expression noise. Our results demonstrated that the level of fluctuation found in bacterial cells is mostly an intrinsic property that arises even in a primitive form of the cell

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

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process

<div><p>The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of <i>Escherichia coli</i> were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.</p></div

The linear accumulation of mutations over generations.

<p>The number of non-synonymous (top), synonymous (middle) and non-coding (bottom) mutations were plotted at the time point when the frequency of the mutated genome first became greater than 10%. The mutated genomes that appeared at the latter stage in Line1 and declined at the end, in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005392#pgen.1005392.g003" target="_blank">Fig 3</a>, which correspond to clusters R4 and R5 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005392#pgen.1005392.s003" target="_blank">S2 Table</a>, were omitted to focus on only the lineages dominating at the ends of the two lines. The dashed and solid lines represent the regression lines for non-synonymous and synonymous mutations, respectively (n = 21, including no mutation at generation 5212, P < 10⁻¹⁶ for non-synonymous and P < 10⁻¹⁰ for synonymous).</p

Growth rate changes in the evolution for thermal adaptation.

<p>The duplicated Line1 and Line2 from the final population at generation 7580 were passaged daily. The daily exponential growth rates at various temperatures were calculated based on the absorbance at 600 nm as described in the Materials and Methods section. a) Each color indicates the growth rates of the bacterial cells at 44.8°C (black), 45.0°C (pink), 45.2°C (blue), 45.4°C (orange), 45.6°C (green), 45.8°C (purple), and 46.0°C (red). The cell populations that were finally acquired at 46.0°C were subjected to genome sequencing analysis. b) The populations at generation 7580 (white) and the final populations of Line1 (gray) and Line2 (dark gray) were cultured to observe the growth rates at 20.0°C, 37.0°C, 45.0°C, 46.0°C, 46.5°C and 47.0°C. The average and standard errors were obtained by repeated cultures (n = 5–6).</p