FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4229452162> ?p ?o ?g. }

• W4229452162 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Genetic modifications, such as gene deletion and mutations, could lead to significant changes in physiological states or even cell death. Bacterial cells can adapt to diverse external stresses, such as antibiotic exposure, but can they also adapt to detrimental genetic modification? To address this issue, we visualized the response of individual Escherichia coli cells to deletion of the antibiotic resistance gene under chloramphenicol (Cp) exposure, combining the light-inducible genetic recombination and microfluidic long-term single-cell tracking. We found that a significant fraction (∼40%) of resistance-gene-deleted cells demonstrated a gradual restoration of growth and stably proliferated under continuous Cp exposure without the resistance gene. Such physiological adaptation to genetic modification was not observed when the deletion was introduced in 10 hr or more advance before Cp exposure. Resistance gene deletion under Cp exposure disrupted the stoichiometric balance of ribosomal large and small subunit proteins (RplS and RpsB). However, the balance was gradually recovered in the cell lineages with restored growth. These results demonstrate that bacterial cells can adapt even to lethal genetic modifications by plastically gaining physiological resistance. However, the access to the resistance states is limited by the environmental histories and the timings of genetic modification. Editor's evaluation This paper presents the temporal relationships between deletion of a resistance gene, introduction of antibiotic, and cell growth that are intriguing and novel. It will be of interest to researchers studying heterogeneity in antibiotic tolerance and the origins of drug resistance. https://doi.org/10.7554/eLife.74486.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Bacteria in nature are constantly challenged by environmental and genetic perturbations and must be robust against them for survival. Bacterial cells possess programs to adapt or resist such perturbations. For example, under the conditions of nutrient deprivation, E. coli and related bacteria provoke RpoS-mediated general stress response and globally change the metabolic and gene expression profiles to protect themselves from the stress (Battesti et al., 2011). DNA damage also induces SOS response, which promotes both DNA repair and mutagenesis (Walker, 1996; Taddei et al., 1995). If the mutations in essential genetic elements remain unrepaired, their influence will be propagated to cellular phenotypes or even cause cell death. How rapidly new genetic changes alter cellular phenotypes and whether they always give rise to the same phenotypes in given environments are fundamental questions in genetics (Ryan, 1955; Sun et al., 2018). Nevertheless, such time-dependent and redundant genotype-phenotype correspondences are usually deemed negligible or insignificant in most genetics analyses. However, the evidence suggests that biological systems can buffer or compensate for the impact of genetic changes at the cytoplasmic and physiological levels (Waddington, 1942). For example, specific molecular chaperons could hinder genetic variations from manifesting as morphological and growth phenotypes in fruit flies (Rutherford and Lindquist, 1998), plants (Queitsch et al., 2002), and bacteria (Fares et al., 2002). Furthermore, the loss of biological functions caused by genetic changes can be compensated for by modulating the RNA and protein levels of the mutated or other related genes in many organisms (El-Brolosy and Stainier, 2017). In Bacillus subtilis, the expression noise of mutant sporulation regulator results in the partial penetrance of its influence to spore-forming phenotypes (Eldar et al., 2009). These observations across diverse organisms suggest that phenotypic consequences of genetic modifications can be modulated based on environmental and physiological contexts, which may promote the survival and evolution of the organisms. Despite these experimental implications, it remains elusive whether bacterial cells can circumvent even lethal genetic modifications such as antibiotic resistance gene deletion under antibiotic exposure. Furthermore, how cells initially respond to the genetic changes and how their physiological and phenotypic states are modulated in longer timescales are poorly characterized. However, addressing these issues requires precisely defining the timings of genetic changes and tracking individual cells for a long period to unravel their phenotypic transitions and consequences (Sun et al., 2018). In this study, we resolve these technical issues by combining the photoactivatable Cre (PA-Cre) light-inducible genetic recombination technique (Kawano et al., 2016) and microfluidic long-term single-cell tracking (Wang et al., 2010). We induced a pre-designed deletion of chromosomally encoded and fluorescently tagged drug resistance gene in E. coli directly in the microfluidic device. The results show that all of the resistance-gene-deleted cells under continuous drug exposure showed a decline in growth in 5–7 generations, but a fraction of the resistance-gene-deleted cells gradually restored their growth without additional mutations. In contrast, no cells restored growth when the same deletion was introduced 10 hr or more in advance before drug exposure. Therefore, bacterial cells can physiologically adapt to lethal genetic modifications. However, its feasibility depends on environmental histories, the timings of genetic modifications, and the severity of the antibiotic stress. Results Resistance gene deletion in E. coli by blue-light illumination To investigate the response of individual cells to antibiotic resistance gene deletion, we constructed an E. coli strain expressing chloramphenicol acetyltransferase (CAT) tagged with mCherry red fluorescent protein (Figure 1A). CAT confers resistance to chloramphenicol (Cp) by acetylating Cp (Shaw, 1967). The mcherry-cat resistance gene was integrated on the chromosome along with the upstream and downstream loxP sequences so that the resistance gene could be excised by Cre recombinase (Figure 1A). We also introduced the PA-Cre recombination system, which consists of split Cre-recombinase fragments, called CreC and CreN, attached to p-Magnet (p-Mag) and n-Magnet (n-Mag) monomers, respectively (Figure 1B; Kawano et al., 2015). p-Mag and n-Mag are heterodimers derived from the fungal photo-receptor, Vivid (Kawano et al., 2015), which heterodimerize upon blue-light illumination. In the PA-Cre system, blue-light illumination leads to heterodimerizations of p-Mag-CreC and n-Mag-CreN fragments and recovers Cre recombination activity (Figure 1B; Kawano et al., 2016). Therefore, this system allows us to induce the resistance gene deletion at arbitrary timings by blue-light illumination. The original PA-Cre system was designed for use in mammalian cells (Kawano et al., 2016); we thus replaced the plasmid backbone and the promoter for use in E. coli. Figure 1 with 2 supplements see all Download asset Open asset Live-cell monitoring of phenotypic transitions in response to gene deletion. (A) Schematic drawing of an E. coli strain, YK0083. This strain harbors the photo-removable mcherry-cat gene on the chromosome and a low-copy plasmid carrying the pa-cre genes (creN-nmag and pmag-creC). (B) The PA-Cre system. Blue-light illumination provokes the dimerization of two PA-Cre fragments and induces the deletion of the mcherry-cat gene. The micrographs represent the combined images of phase contrast and mCherry-CAT fluorescence channels. Left and right images show the cells before and after blue-light illumination, respectively. (C) Representative time-lapse images of gene-deletion experiments. The upper and lower images show the cells in phase contrast and mCherry-CAT fluorescence channels, respectively. The cell lineages at the closed end of the growth channel (outlined in white) were monitored. A 30-min blue-light illumination starting at t = –0.5 hr led to the loss of mCherry-CAT fluorescence signals in this cell lineage. (D) The transitions of mCherry-CAT fluorescence intensities in resistance-gene-deleted (blue) and non-deleted (red) cell lineages. Lines and shaded areas represent the medians and the 25–75% ranges, respectively. The cyan broken line represents the expected fluorescence decay curve when the fluorescence intensity decreases to half in each generation. (E) Generation time of resistance-gene-deleted and non-deleted cells 10 generations before and after blue-light illumination. The middle line and both edges of the boxes represent the medians and the 25–75% ranges of generation time. Whiskers indicate the minimum and maximum of the data except for the outliers. The points represent the outliers. No significant differences in generation time were detected between the groups at the significance level of 0.01 (p = 0.47 for before BL, p = 0.027 for after BL, p = 0.055 for before BL vs after BL, Mann-Whitney U test). We first analyzed the response of the constructed strain YK0083 to blue-light illumination under drug-free conditions. Single-cell observations with the mother machine microfluidic device (Wang et al., 2010) and custom stage-top LED illuminator (Figure 1—figure supplement 1) revealed that 30-min blue-light exposure (λ= 464∼474 nm, 6.8 mW at the specimen position) led to the loss of mCherry-CAT fluorescence in 25% (50/200) cells (Figure 1C and D, and Video 1), suggesting the deletion of the mcherry-cat gene in these cell lineages. The fluorescence signals decayed to the background level in 4–5 generations, and the decay kinetics was consistent with the dilution by growth (Figure 1D). Furthermore, the 30 min blue-light illumination and genetic recombination did not affect the growth of individual cells (Figure 1E). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Deletion of mcherry-cat gene by blue-light illumination under drug-free conditions. YK0083 cells were cultured in the mother machine flowing the M9 minimal medium and exposed to blue light from t = –30 min to 0 min (marked with ‘ + BL’). The merged images of phase contrast (grayscale) and mCherry-CAT fluorescence (red) channels are shown. The fluorescence of mCherry-CAT was gradually lost after blue-light illumination in this cell lineage. Scale bar, 5 µm. Extending illumination duration increased the frequency of cells showing loss of fluorescence, reaching 100% (n=296) in 4 hr (Figure 1—figure supplement 2A and Figure 1—figure supplement 2B). We confirmed the correspondence between the loss of mCherry fluorescence and cat gene deletion by colony PCR (Figure 1—figure supplement 2C). Fractional continuation of growth against resistance gene deletion under drug exposure We next induced the deletion of the resistance gene in YK0083 cells in the mother machine, continuously flowing a medium containing 15 µg/mL of Cp. This drug concentration was 1.5-fold higher than the minimum inhibitory concentration (MIC) of the non-resistant strain YK0085 (10 µg/mL), which was constructed by illuminating the YK0083 cells by blue light in batch culture (Figure 2). Therefore, it was expected that this drug concentration would inhibit the growth of resistance-gene-deleted cells. This Cp concentration was significantly lower than the MIC of resistant YK0083 cells (100 µg/mL, Figure 2) and did not influence their elongation rates (Figure 2—figure supplement 1). Figure 2 with 1 supplement see all Download asset Open asset MIC tests. Gray points represent the OD600 of the indicated strains after a 23 hr incubation period in the M9 media containing the corresponding concentrations of Cp. The minimum concentration where OD600 became lower than 0.01 was adopted as the MIC for each strain. A total of 15 µg/mL of Cp was used in the time-lapse experiments (dashed line). The measurements were repeated at least thrice. A 30-min blue-light illumination induced the mcherry-cat gene deletion in 24.5% (343/1399) in YK0083 cells (Figure 3A and Video 2). While non-deleted cells continued to grow with their generation time (interdivision time) and mCherry-CAT fluorescence intensity nearly unaffected (Figure 3B and C), resistance-gene-deleted cells gradually showed a decline in growth (Figure 3D–G). The fluorescence intensity of the resistance-gene-deleted cells decayed to the background levels in 4–5 generations (Figure 3D, F and H), and their generation time also increased correspondingly (Figure 3E, G and I). However, while the growth of 62.7% (163/260) of resistance-gene-deleted cells was eventually stopped, the other 37.3% cells restored and continued their growth over 30 generations without the cat resistance gene (Figure 3H and I). The generation time of these cell lineages recovered from 6.3 hr (the median of 6th-9th generations) to 3.0 hr (the median of 21st-30th generations) under the continuous drug exposure (Figure 3I). The elongation rate transitions across multiple generations also manifested the growth recovery (Figure 3—figure supplement 1). Figure 3 with 5 supplements see all Download asset Open asset Growth continuation under Cp exposure against cat gene deletion. (A) Time-lapse images of a cell lineage that continued growth and division against mcherry-cat gene deletion. The upper and lower sequences show phase contrast and mCherry fluorescence images, respectively. Blue light was illuminated from t = -0.5 h to 0 hr. The cells at the closed end of the growth channel were monitored during the experiment, which are outlined in white on the images. Scale bar, 5 µm. (B–G) The transitions of mCherry-CAT fluorescence intensities and cell size in single-cell lineages. (B, C) Non-deleted cell lineage. (D, E) Growth-halted resistance-gene-deleted cell lineage. The decrease in cell size after 60 hr was due to the shrinkage of the cell body. (F, G) Growth-restored resistance-gene-deleted cell lineage. (H, I) The transitions of mCherry-CAT fluorescence intensities (H) and generation time (I). The lines and shaded areas represent the medians and the 25–75% ranges, respectively. Red represents non-deleted cell lineages. Blue represents growth-restored resistance-gene-deleted cell lineages. The transitions are shown in generations. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Growth restoration under Cp exposure after resistance gene deletion. YK0083 cells were cultured in the mother machine flowing the M9 minimal medium containing 15 µg/mL of Cp. Blue light was illuminated from t = –30 min to 0 min (marked with ‘ + BL’). The merged images of phase contrast (grayscale) and mCherry-CAT fluorescence (red) channels are shown. The illumination caused the loss of fluorescence signals, that is, the deletion of the mcherry-cat gene, in this cell lineage. Nevertheless, the cell gradually restored growth and division under continuous Cp exposure. Scale bar, 5 µm. We first suspected that the growth recovery under Cp exposure was attributed to the presence of the cat gene untagged from the mcherry gene or by additional unintended resistance-enhancing mutations. To test this hypothesis, we illuminated batch cultures of YK0083 cells by blue light for 30 min under exposure to 15 µg/mL of Cp and plated them on agar plates. Colony PCR confirmed that all the cells that lost mCherry fluorescence were also cat-negative even when the deletion was introduced under Cp exposure (Figure 3—figure supplement 2A). This result strongly suggests that the growth-restored cells do not retain the cat-resistance gene as designed. The fraction of resistance-gene-deleted cells after 30-min blue-light illumination monotonically decreased in batch culture containing 15 µg/mL of Cp (Figure 3—figure supplement 2B), which is consistent with the observation that growth-restored resistance-gene-deleted cells grew more slowly than non-deleted cells. In addition, we sampled the culture media flowing out from the microfluidic device and obtained cell populations derived from a single or few ancestral cells by limiting dilution (Figure 3—figure supplement 3A). PCR analysis confirmed the lack of cat gene in the non-fluorescent cell populations (Figure 3—figure supplement 3B). Furthermore, whole-genome sequencing of the cell populations obtained by limiting dilution showed no additional mutations in four out of five non-fluorescent cell populations tested (Table 1). One point mutation was present in one cellular population, but this mutation did not affect the MIC (Figure 3—figure supplement 3C). To calculate the probability that all of these five non-fluorescent cell populations had originated from resistance-gene-deleted growth-halted cells, we analyzed the regrowing dynamics of both growth-restored and growth-halted cell lineages after removing Cp in the microfluidic device (Figure 3—figure supplement 4A-C and Video 3). We found that 91.3% (73/80) of growth-restored cell lineages recovered fast growth after the exposure to 15 µg/mL of Cp for 72 hr. On the other hand, the proportion of cells that recovered growth was lower for the growth-halted cell lineages; only 69.3% (140/202) of growth-halted cell lineages could resume growth. The growth after first cell divisions were as fast as that for the non-deleted cells and was indistinguishable between the growth-restored and growth-halted cell lineages (Figure 3—figure supplement 4D). Taking the fraction of growth-restored cells among the resistance-gene-deleted cells and the proportions of growth-recovered cells after Cp removal into account, we found that the probability that all the five cell populations were derived from growth-halted cell lineages was 5.5% (see Materials and methods). Therefore, the probability that unintended genetic changes were responsible for the growth restoration is small. Table 1 Mutations detected by whole-genome sequencing. We obtained isolated cellular populations derived from single or few cells by limiting dilution of the culture media flowing out from the mother machine. We detected only one point mutation in Sample 3. Sample no.MutationPositionBaseAnnotationSample 1No mutationSample 2No mutationSample 3Mutation in leuC site80,471G - > TA132SSample 4No mutationSample 5No mutation Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Growth recovery of resistance-gene-deleted cells after Cp removal. Left, growth-halted resistance-gene-deleted cell; Right, Growth-restored resistance-gene-deleted cells. Cp was removed at t = 0 min (72 hr after blue light illumination). The merged images of phase contrast (grayscale) and mCherry-CAT fluorescence (red) channels are shown. Scale bar, 5 µm. It is also unlikely that the growth restoration was caused by the residual mCherry-CAT proteins because 30 consecutive cell divisions dilute cytoplasmic proteins 230≈109 folds if additional production is prevented; note that even the total number of protein molecules in a bacterial cell are in the order of 106-107 (Milo, 2013) We also detected no significant differences in the mCherry-CAT fluorescence intensity before blue-light illumination between the growth-halted and growth-restored cell lineages (Figure 3—figure supplement 5A). Furthermore, no significant differences were observed even between the resistance-gene-deleted and non-deleted cell lineages (Figure 3—figure supplement 5A). We also examined the influence of elongation rate before blue-light illumination on the gene deletion and the fates after gene deletion, finding no correlations (Figure 3—figure supplement 5B). Therefore, neither the amount of mCherry-CAT proteins at the time of gene deletion nor pre-deletion elongation rate affected the likelihood of gene deletion and the determination of growth-halt and growth-restoration fates under these experimental conditions. We also found that deleting the mcherry-cat gene flowing a medium containing a twofold concentration of Cp (i.e., 30 µg/mL) eliminated growth-restored cell lineages: 33.1% (361/1092) of the cells illuminated by blue light lost the resistance gene, and none of them restored growth. This result suggests that deleting the resistance gene at the concentrations of Cp sufficiently higher than the MIC can prevent physiological adaptation. Resistance gene deletion long before Cp exposure prevents growth restoration The high frequency of growth restoration observed against the resistance gene deletion was unexpected since the MIC of the susceptible strain was below 15 µg/mL (Figure 2 and Figure 3—figure supplement 3C). To understand this discrepancy, we cultured the susceptible YK0085 cells in the mother machine first without Cp and then exposed them to 15 µg/mL of Cp directly in the device. In contrast to the previous observation, all cells stopped growth and division entirely, with no cells restoring growth (“Pre-deleted” in Figure 4A–C and Video 4). This result excludes the hypothesis that the unique cultivation environments in the microfluidics device are the cause of growth restoration. Instead, this observation implies that the timing of gene deletion is crucial for the cells to withstand the Cp exposure without the resistance gene and restore growth. Figure 4 Download asset Open asset History-dependent maintenance of Cp resistance. (A) The schematic diagram of experiments. The duration from the end of blue-light illumination to the onset of Cp exposure (Tc) was varied from 0 to 10 h. Tc≪0 represents the continuous Cp-exposure condition. “Pre-deleted” denotes the results of the experiments performed with the mcherry-cat-deleted YK0085 strain. The non-resistant YK0085 cells were not exposed to blue light. (B) Fractions of mcherry-cat-deleted cell lineages across different Tc conditions. The numbers of resistance-gene-deleted cell lineages among the total numbers of cell lineages observed during the measurements are shown above the bars. Error bars represent standard errors. (C) Fractions of growth-restored cell lineages among resistance-gene-deleted cell lineages. The numbers of growth-restored cell lineages among all the resistance-gene-deleted cell lineages are shown above the bars. Error bars represent standard errors. The numbers of resistance-gene-deleted cell lineages are different from those in B because some cell lineages were flushed away from the growth channels at the later time points of the measurements, and their fates could not be determined. (D, E) The distributions of the mCherry-CAT fluorescence intensities at the onset of Cp exposure across the different Tc conditions represented by histograms (D) and box plots (E). (F) Fractions of growth-restored cell lineages and their dependence on the mCherry-CAT fluorescence at the onset of Cp exposure. Blue and orange bars indicate the fractions of growth-restored cells among cell lineages whose mCherry-CAT fluorescence intensities were higher and lower than the median, respectively. Error bars represent standard errors. The growth-restored cell lineages were not detected under the Tc = 10 h and “Pre-deleted” conditions. Fractional differences between the top 50% and the bottom 50% of cell lineages were statistically significant only for the Tc = 6 h condition (p = 0.86 for Tc = 0 hr; p = 0.28 for Tc = 3 hr; and p = 8.6 × 10–3 for Tc = 6 hr, two proportional z-test). Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg No growth restoration of YK0085 strain under Cp exposure. YK0085 cells were cultured in the mother machine flowing the M9 minimal medium and exposed to 15 µg/mL of Cp at t = 0 min (marked with ‘ + Cp’). The Cp exposure caused growth arrest, and, unlike the YK0083 cells, the cell did not restore growth. We found no growth-restored cell lineages in this experimental condition. Scale bar, 5 µm. To further investigate the importance of the timing of gene deletion, we performed microfluidic single-cell measurements with the resistant YK0083 strain, varying the duration from blue-light illumination to the onset of Cp exposure (Tc) between 0 h to 10 h (Figure 4A). The proportions of cells that lost the mcherry-cat gene in response to blue-light illumination almost remained unchanged among all conditions, ranging from 24% to 28% (Figure 4B). However, the proportions of growth-restored cell lineages among resistance-gene-deleted cells strongly depended on Tc (Figure 4C). When Cp exposure was initiated immediately after the blue-light illumination (Tc= 0 hr) or after 3 h (Tc= 3 hr), the proportions of growth-restored cells were nearly equivalent to those observed under continuous Cp exposure conditions (Figure 4C). In contrast, the proportions of growth-restored cells were reduced to 18.5% (34/184) when Tc=6 hr, and we did not detect any growth restoration when Tc = 10 hr (0/195; Figure 4C). The almost equivalent frequencies of growth-restored cell lineages under the Tc = 0 hr and Tc = 3 hr conditions and those observed under continuous exposure exclude the possibility that growth restoration requires prior exposure to Cp before resistance gene deletion. We next conjectured that a low amount of mCherry-CAT proteins is required at the onset of Cp exposure to withstand and restore growth without the resistance gene. In fact, mcherry-cat gene deletion before Cp exposure led to the dilution of mCherry-CAT proteins by growth by the time of Cp exposure (Figure 4D and E). To examine whether the mCherry-CAT concentration in individual cells affects growth restoration, we divided the resistance-gene-deleted cell lineages into two groups under each condition (top 50% and bottom 50%) based on their mCherry-CAT fluorescence intensities at the onset of Cp exposure. While we found no significant differences in the Tc = 0 and 3 hr conditions, the top 50% group produced 2.4-fold more growth-restored cells than the bottom 50% group in the Tc = 6 hr condition (Figure 4F). In the Tc = 10 hr condition, we could barely detect the fluorescence signals from any cells (Figure 4D and E), and no cells showed growth restoration as mentioned above (Figure 4F). These results suggest that a low level of residual mCherry-CAT proteins is required at the onset of Cp exposure, but high amounts do not necessarily ensure the growth restoration in all resistance-gene-deleted cells. Moreover, 40% could be considered as the maximum frequencies at which cells can restore growth without the resistance gene. Growth restoration accompanies the recovery of stoichiometric balance of ribosomal subunits The results above demonstrated that the residual mCherry-CAT proteins are important for initiating the transition to growth restoration without the resistance gene. However, the residual proteins cannot directly support the growth in later generations under Cp exposure as their levels are eventually diluted with growth. Indeed, the amounts of mCherry-CAT proteins during growth restoration were below the detection limit (Figure 3F and H). Therefore, we speculated that the other physiological changes were responsible for growth restoration. Because Cp targets the 50 S ribosomal subunit (Pongs, 1979), it is plausible that ribosomal states were modulated in the duration beginning from resistance gene deletion to growth restoration. Hence, we constructed an E. coli strain YK0136 that expressed fluorescently-tagged dual ribosomal reporters, RplS-mCherry and RpsB-mVenus (Figure 5A; Nikolay et al., 2014). RplS and RpsB are ribosomal proteins in the 50 S and 30 S subunits, respectively. Their fluorescent reporters were utilized to probe the ribosomal subunit balance in living cells (Nikolay et al., 2014). YK0136 also harbors the cat resistance gene (not tagged with mcherry) between the upstream and downstream loxP sequences on the chromosome, and the PA-Cre recombination system was expressed via the plasmid (Figure 5A). We confirmed that YK0136 showed MIC values almost equivalent to those of the YK0083 strain (Figure 2 and Figure 5—figure supplement 1). Furthermore, 30-min blue-light illuminations provoked cat-gene deletion in 28.6% (452/1587) of YK0136 cells, which was comparable to the frequency of mcherry-cat-gene deletion in YK0083 (Figure 4B). Figure 5 with 5 supplements see all Download asset Open asset Disruption and restoration of ribosomal proteins’ stoichiometry. (A) Schematic diagram of the ribosome reporter strain, YK0136. Fluorescently tagged ribosomal protein genes (rplS and rpsB) are expressed from the native loci on the chromosomes. Additionally, the photo-removable cat gene was integrated into the intC locus on the chromosome. The PA-Cre fragments were expressed via low-copy plasmids. (B) Transitions of elongation rates. Lines represent the medians of elongation rates at each time point, and shaded areas show the 95% error ranges of the medians estimated by resampling the cell lineages 1000 times. Red represents non-delete" @default.
• W4229452162 created "2022-05-11" @default.
• W4229452162 creator A5013172910 @default.
• W4229452162 creator A5032795137 @default.
• W4229452162 date "2022-03-18" @default.
• W4229452162 modified "2023-10-18" @default.
• W4229452162 title "Author response: History-dependent physiological adaptation to lethal genetic modification under antibiotic exposure" @default.
• W4229452162 doi "https://doi.org/10.7554/elife.74486.sa2" @default.
• W4229452162 hasPublicationYear "2022" @default.
• W4229452162 type Work @default.
• W4229452162 citedByCount "0" @default.
• W4229452162 crossrefType "peer-review" @default.
• W4229452162 hasAuthorship W4229452162A5013172910 @default.
• W4229452162 hasAuthorship W4229452162A5032795137 @default.
• W4229452162 hasBestOaLocation W42294521621 @default.
• W4229452162 hasConcept C139807058 @default.
• W4229452162 hasConcept C169760540 @default.
• W4229452162 hasConcept C501593827 @default.
• W4229452162 hasConcept C54355233 @default.
• W4229452162 hasConcept C86803240 @default.
• W4229452162 hasConceptScore W4229452162C139807058 @default.
• W4229452162 hasConceptScore W4229452162C169760540 @default.
• W4229452162 hasConceptScore W4229452162C501593827 @default.
• W4229452162 hasConceptScore W4229452162C54355233 @default.
• W4229452162 hasConceptScore W4229452162C86803240 @default.
• W4229452162 hasLocation W42294521621 @default.
• W4229452162 hasOpenAccess W4229452162 @default.
• W4229452162 hasPrimaryLocation W42294521621 @default.
• W4229452162 hasRelatedWork W1991523530 @default.
• W4229452162 hasRelatedWork W2002128513 @default.
• W4229452162 hasRelatedWork W2020824267 @default.
• W4229452162 hasRelatedWork W2093140091 @default.
• W4229452162 hasRelatedWork W2099966111 @default.
• W4229452162 hasRelatedWork W2616738127 @default.
• W4229452162 hasRelatedWork W2944732678 @default.
• W4229452162 hasRelatedWork W4232340583 @default.
• W4229452162 hasRelatedWork W4245780361 @default.
• W4229452162 hasRelatedWork W2092874662 @default.
• W4229452162 isParatext "false" @default.
• W4229452162 isRetracted "false" @default.
• W4229452162 workType "peer-review" @default.