1. Cell Biology
  2. Physics of Living Systems

Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions, and cell growth

  1. Nicola Bellotto
  2. Jaime Agudo-Canalejo
  3. Remy Colin
  4. Ramin Golestanian  Is a corresponding author
  5. Gabriele Malengo  Is a corresponding author
  6. Victor Sourjik  Is a corresponding author
  1. Max Planck Institute for Terrestrial Microbiology and Center for Synthetic Microbiology (SYNMIKRO), Germany
  2. Max Planck Institute for Dynamics and Self-Organization, Germany
  3. Rudolf Peierls Centre for Theoretical Physics, University of Oxford, United Kingdom
https://doi.org/10.7554/eLife.82654
Share this article
Cite this article
  1. Nicola Bellotto
  2. Jaime Agudo-Canalejo
  3. Remy Colin
  4. Ramin Golestanian
  5. Gabriele Malengo
  6. Victor Sourjik
(2022)
Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions, and cell growth
eLife 11:e82654.
https://doi.org/10.7554/eLife.82654
  2. Download BibTeX
  3. Download .RIS
10 figures, 46 tables and 1 additional file

Figures

Figure 1 with 10 supplements
Download asset Open asset
Dependence of protein mobility in bacterial cytoplasm on molecular mass and cellular interactions.

(A) Examples of fluorescence microscopy images of Escherichia coli cells expressing either sfGFP or the indicated sfGFP-tagged cytoplasmic proteins. Scale bars are 2 μm. (B) Representative autocorrelation functions (ACFs) measured by FCS for the indicated protein constructs. Data were fitted using the anomalous diffusion model (solid lines). All ACF curves were normalized to their respective maximal values to facilitate comparison. (C) Diffusion times (τD) measured for the indicated protein constructs. Each dot in the box plot represents the value for one individual cell, averaged over six consecutive acquisitions (Figure 1—figure supplement 3). The numbers of cells measured for each construct are shown in Appendix 6. ***p<0.0001 in a two-tailed heteroscedastistic t-test. Exact p-valuescan be found in Appendix 5. (D, E). Dependence of protein mobility (1/τD; D) and apparent anomaly of diffusion (α; E) on molecular mass. Each symbol represents the average value for all individual cells that have been measured for that particular construct and the error bars represent the standard error of the mean. Individual values are shown in Figure 1—figure supplement 5 and the numbers of measured cells for each construct are shown in Appendix 6. Protein constructs with low mobility for which effects of specific interactions were further investigated are highlighted in color and labeled. The values of 1/τD and α for both the original constructs (diamonds) and the constructs where mutations were introduced to disrupt interactions (circles) are shown. For Map, two alternative amino acid substitutions that disrupt its interaction with the ribosome are shown (see Figure 1—figure supplement 10). (F–H) Cartoons illustrating the cellular interactions that could affect mobility of ClpS (F), Map (G), and DnaK (H). ClpS engages with the ClpAP protease and with substrates, cartoon adapted from Figure 1A from Román-Hernández et al., 2011. Map interacts with the actively translating ribosomes, cartoon adapted from Figure 3A from Sandikci et al., 2013. DnaK interacts with unfolded client protein. Amino acidic residues that were mutated to disrupt interactions are highlighted (see text for details). FCS, fluorescence correlation spectroscopy.

Figure 1—source data 1

Individual τD measurements from Figure 1C.

Individual mean and standard errors of the mean of 1/τD values from Figure 1D. Individual mean and standard errors of the mean of α values from Figure 1E.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Download asset Open asset
Expression analysis for all Escherichia coli protein constructs made in this study.

Expression of proteins was analyzed by SDS-PAGE and immunoblotting using a primary antibody specific for GFP. All fusion proteins displayed a dominant band corresponding to the expected molecular mass of the full-length fusion (Table 1).

Figure 1—figure supplement 1—source data 1

Uncropped western blot images for Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-figsupp1-data1-v3.zip
Figure 1—figure supplement 2
Download asset Open asset
Growth curves of Escherichia coli strains expressing tested sfGFP-tagged proteins.

Growth curves of strains expressing sfGFP, sfGFP-tagged proteins, or carrying the control empty vector pTrc99A were measured at 37°C. The optical density of cultures was monitored at 600 nm (OD600) and the measured values were normalized for an optical path of 1 cm.

Figure 1—figure supplement 3
Download asset Open asset
Workflow of a typical FCS experiment.

The focus of the confocal microscope is positioned near one pole in the cell of interest, and six subsequent acquisitions of the fluorescence intensity of 20 s each are performed. The example for sfGFP shows only traces for R1, R3, and R5 measurements (different colors). The autocorrelation functions (ACFs) are then calculated independently for each acquisition. The values for τD and α are extracted independently from the fit to each ACF using the anomalous diffusion model (solid lines) and then averaged to obtain the τD and α for each individual cell.

Figure 1—figure supplement 4
Download asset Open asset
Comparison between fits of the experimental data with Brownian and anomalous diffusion models.

The experimental data (here the example for the R3 measurement from Figure 1—figure supplement 3) were fitted by the model of free Brownian diffusion and by the anomalous diffusion model as indicated by different colors (upper panel), with the corresponding values of residuals (lower panel).

Figure 1—figure supplement 5
Download asset Open asset
Individual measurements of τD and α for all Escherichia coli protein constructs included in the analysis of mass dependence.

The numbers of measured cells for each construct are shown in Appendix 6. Each dot in the box plot represents the values of τD (A) and α (B) for one individual cell. The averages values calculated from these datasets are shown in Figure 1.

Figure 1—figure supplement 5—source data 1

Individual values of τD from Figure 1—figure supplement 5A.

Individual values of α from Figure 1—figure supplement 5B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-figsupp5-data1-v3.xlsx
Figure 1—figure supplement 6
Download asset Open asset
Comparison between one-component and two-components anomalous diffusion fit for DsdA-sfGFP.

(A) Fitting the experimental data for DsdA-sfGFP with a two-component model for anomalous diffusion, where the fast component corresponding to free sfGFP was fixed to 15% (corresponding to the measured degree of fusion protein degradation) with τD = 561 μs and α=0.86, that are, the average values obtained for sfGFP. For comparison, standard fitting with a one-component anomalous diffusion model is also shown. (B) The fitted values of τD with the two-component and one-component model.

Figure 1—figure supplement 6—source data 1

Individual values used to calculate mean and standard error of the mean values of τD and α values from Figure 1—figure supplement 6B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-figsupp6-data1-v3.xlsx
Figure 1—figure supplement 7
Download asset Open asset
Mobility (1/τD) and anomaly of diffusion (α) of sfGFP in individual cells with different width and length.

Values of 1/τD (A, C) and α (B, D) for single cells expressing sfGFP plotted against the length (A, B) or width (C, D) of respective cell. The numbers of measured cells are shown in Appendix 6.

Figure 1—figure supplement 7—source data 1

Individual values of 1/τD and measurements of cell length from Figure 1—figure supplement 7A.

Individual values of α and measurements of cell length from Figure 1—figure supplement 7B. Individual values of 1/τD and measurements of cell width from Figure 1—figure supplement 7C. Individual values of α and measurements of cell width from Figure 1—figure supplement 7D.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-figsupp7-data1-v3.xlsx
Figure 1—figure supplement 8
Download asset Open asset
Comparison of protein mobility in cephalexin-treated and untreated cells.

Mobility of sfGFP measured in single cells treated with cephalexin (red dots) or in control untreated cells (black dots). Only cells of similar length were chosen for this comparison. No significant difference in protein mobility (1/τD; p=0.08) or anomaly of diffusion (α; p=0.67) is observed. The numbers of cells measured for each conditions are shown in Appendix 6.

Figure 1—figure supplement 8—source data 1

Individual values of 1/τD and measurements of cell length from Figure 1—figure supplement 8A.

Individual values of α and measurements of cell length from Figure 1—figure supplement 8B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-figsupp8-data1-v3.xlsx
Figure 1—figure supplement 9
Download asset Open asset
Expression analysis for the mutants with impaired interactions.

The expression of indicated point mutants of ClpS, DnaK, and Map was analyzed by SDS-PAGE and immunoblotting using a primary antibody specific for GFP. All mutants displayed a dominant band corresponding to the expected molecular mass of the full-length fusion, and comparable to that of the wild-type counterpart. ClpSD35A_D36A_H66A-sfGFP was measured in the same ΔclpA background as subsequently used for the FCS experiments.

Figure 1—figure supplement 9—source data 1

Uncropped western blot image for Figure 1—figure supplement 9.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-figsupp9-data1-v3.zip
Figure 1—figure supplement 10
Download asset Open asset
Mobility (1/τD) and anomaly of diffusion (α) of ClpS, Map and DnaK and of indicated mutants with disrupted protein interactions.

Each dot in the box plot represents the values of 1/τD (A) and α (B) for one individual cell. The numbers of cells measured for each construct are shown in Appendix 6. ClpS mutant was measured in ΔclpA background. *** p<0.0001; * p<0.05; NS: no statistically significant difference in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 1—figure supplement 10—source data 1

Individual values of 1/τD from Figure 1—figure supplement 10A.

Individual values of α from Figure 1—figure supplement 10B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig1-figsupp10-data1-v3.xlsx
Figure 2 with 8 supplements
Download asset Open asset
Protein diffusion in bacterial cytoplasm corrected for confinement.

(A) Representative ACFs of simulated fluorescence intensity fluctuations. Simulations were performed in a confined geometry of a cell with indicated length L and diameter d, and dimensions of the measurement volume ω0 and z0, representing an experimental FCS measurement (Inset; see Materials and methods) for two different values of the ansatz diffusion coefficient. Solid lines are fits by the models of unconfined Brownian diffusion, anomalous diffusion and by the Ornstein-Uhlenbeck (OU) model of Brownian diffusion under confinement, as indicated. (B) The exponent α extracted from the fit of the anomalous diffusion model to the ACFs data that were simulated at different values of the cell diameter. Corresponding values of the diffusion coefficient are shown in Figure 2—figure supplement 7. (C, D) Escherichia coli cells treated with cephalexin alone or with cephalexin and 1 µg/ml of A22 (see Materials and methods), show A22-dependent increase in the measured cell diameter (C) and higher values of the exponent α extracted from the fit to the ACF measurements (D). The numbers of cells measured for each condition are shown in Appendix 6. ***p<0.0001 in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5. (E) Dependence of the diffusion coefficient calculated from fitting the experimental ACFs with the OU model of confined diffusion. Only the subset of apparently freely diffusing constructs from Figure 1D has been analyzed with the OU model (see also Table 1). Each circle represents the average value for all individual cells that have been measured for that particular construct (Appendix 6), and the error bars represent the standard error of the mean. Error bars that are not visible are smaller than the symbol size. (F) Fit of the mass dependence with an inverse power law (solid blue line, exponent β=0.56±0.05), and predictions of the Stokes-Einstein relation (black dashed line) and of the model describing diffusion of two linked globular proteins (solid yellow line), both with exponent β=0.4. ACF, autocorrelation function; FCS, fluorescence correlation spectroscopy.

Figure 2—source data 1

Average and error from each simulation in Figure 2B.

Individual measurements of cell diameters from Figure 2C. Individual measurements of α from Figure 2D. Individual mean and standard error of the mean of diffusion coefficient values from Figure 2E and F.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Download asset Open asset
Simulations of particles undergoing fractional Brownian motion.

(A) Examples of simulated autocorrelation functions (ACFs) obtained for particles diffusing under fractional Brownian motion in the confined geometry of a cell of 0.85 μm diameter, with different degrees of anomaly of diffusion (values of α indicated by different colors) and with a fixed diffusion time (τD=[w02/2Γα] 1/α) of 1.2 ms. Data were fitted with the anomalous diffusion model. Similar results were obtained for other values of τD (0.6 and 2 ms). (B) Corresponding anomalous diffusion exponents from the fitting of the simulated ACFs in (A) (αfit, mean values of 5 replicates) compared with the ansatz values of anomalous diffusion exponent for unconfined diffusion (αunconfined) used in simulations for the indicated values of τD. The gray shaded area represents the typical values of α obtained from the fit of experimental ACFs (0.82–0.9). The fitted value of τD was 16%, 10%, and 5% lower than expected from the ansatz τD 0.6, 1.2, and 2 ms, respectively, irrespective of the value of α.

Figure 2—figure supplement 1—source data 1

Mean values and standard errors of the mean from Figure 2—figure supplement 1B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig2-figsupp1-data1-v3.xlsx
Figure 2—figure supplement 2
Download asset Open asset
Mobility of sfGFP in cells treated with cephalexin (CFX) or the combination of cephalexin and A22.

Each dot in the box plot represents the values of 1/τD for one individual cell. The numbers of cells measured for each condition are shown in Appendix 6. Cultures are grown for ~3.5 hr in absence or presence of A22 before being both treated for 45 min with cephalexin. *p<0.05 in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 2—figure supplement 2—source data 1

Individual values of 1/τD from Figure 2—figure supplement 2.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig2-figsupp2-data1-v3.xlsx
Figure 2—figure supplement 3
Download asset Open asset
Sedimentation assay of cellular density for indicated treatments.

Sedimentation assays were performed using non-motile and non-aggregating (ΔfliC Δflu) variant of the Escherichia coli strain used in the other experiments. Cells were grown and treated as described in the correspondent sections and assayed for their density in motility buffer (MB) containing 20% iodixanol to match the density of control untreated cells (A). Treatments with cephalexin (CFX; B), cephalexin and A22 (C), 100 mM NaCl (D), DMSO (E), rifampicin (Rif; F), and chloramphenicol (Cam; G) are shown. Dots represent the cell fraction at each given Z position normalized on the total height of the microfluidic channel (50 μm). The gray shadings indicate the standard deviation of the three technical replicates and the red lines represent the exponential fit to the data used to determine the decay length Z0. (H) Average values of 1/Z0 calculated from three technical replicates for each condition. Error bars represent the standard error of the mean. (I) Estimated cell volume for cells in each of the tested conditions. Error bars represent the standard error of the mean. (J) Calculated values of cellular density mismatch for MB with 20% iodixanol, calculated from the values values of 1/Z0 and cell volume. Error bars represent the standard error of the mean. See Materials and methods for details of calculations and cell volume estimation.

Figure 2—figure supplement 3—source data 1

Mean values and standard errors of the mean from Figure 2—figure supplement 3H.

Mean values and standard errors of the mean from Figure 2—figure supplement 3I. Mean values and standard errors of the mean from Figure 2—figure supplement 3J.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig2-figsupp3-data1-v3.xlsx
Figure 2—figure supplement 4
Download asset Open asset
Apparent anomaly of diffusion and residence time for different pinhole sizes.

FCS measurements for indicated protein constructs were performed at the suboptimal pinhole size of 0.66 Airy units (A.U.) to extract values of the anomaly of diffusion (α; A) and residence time (τD; B). The numbers of cells measured for each condition are shown in Appendix 6. ***p<0.0001; **p<0.001; NS: no statistically significant difference in a two-tailed heteroscedastistic t-test.Exact p-values can be found in Appendix 5.

Figure 2—figure supplement 4—source data 1

Individual values of α from Figure 2—figure supplement 4A.

Individual values of τD from Figure 2—figure supplement 4B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig2-figsupp4-data1-v3.xlsx
Figure 2—figure supplement 5
Download asset Open asset
Apparent anomaly of diffusion (α) extracted from analysis of ACFs at shorter time scales.

ACFs measured for indicated proteins were truncated to shorter time scales and fitted with the anomalous diffusion model. The extracted anomaly of diffusion α is plotted as a function of time to which the analysis was limited. The shortest time lag (10–3 s) was analyzed only for sfGFP because for the slower-diffusing constructs this value was comparable to the inflection point of the ACF curve where α is extrapolated. The numbers of cells measured for each construct are shown in Appendix 6.

Figure 2—figure supplement 5—source data 1

Average values and standard errors of the mean of α from Figure 2—figure supplement 5.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig2-figsupp5-data1-v3.xlsx
Figure 2—figure supplement 6
Download asset Open asset
Residuals of fitting the simulated ACFs with different models.

Simulated fluorescence intensity ACF data were fitted by the models of anomalous and Brownian diffusion, as well as by the OU model of Brownian diffusion under confinement, as indicated.

Figure 2—figure supplement 7
Download asset Open asset
Diffusion coefficients fitted from simulation data.

Diffusion coefficients computed (D= ω02/4τD) from the diffusion times extracted from the fit of the Brownian simulation data with (A) the anomalous diffusion model or (B) the Ornstein-Uhlenbeck (OU) model of Brownian diffusion under confinement at various value of the ansatz D and of the cell diameter d, normalized by the ansatz D. The gray areas represent ±5% accuracy.

Figure 2—figure supplement 7—source data 1

Average and error from simulation in Figure 2—figure supplement 7A.

Average and error from simulation in Figure 2—figure supplement 7B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig2-figsupp7-data1-v3.xlsx
Figure 2—figure supplement 8
Download asset Open asset
Comparison between fits of the experimental data with confined diffusion and anomalous diffusion models.

The experimental data (here the example for the R3 measurement from Figure 1—figure supplement 2) were fitted by the model of confined diffusion and by the anomalous diffusion model as indicated by different colors (upper panel), yielding comparable values of residuals (lower panel).

Figure 3
Download asset Open asset
Comparison between protein diffusion coefficients measured by FCS and FRAP.

(A) Examples of FRAP measurements for two different constructs, sfGFP and AcnA-sfGFP. A 3×3 pixels area close to one cell pole (red circle) was photobleached with a high-intensity laser illumination for 48 ms and the recovery of fluorescence in the bleached area was monitored for 11 s with the time resolution of 18 ms. The scales bars are 2 μm. (B) Representative curves of fluorescence recovery in FRAP experiments and their fitting using simFRAP. The experimental data (colored dots) are used by the simFRAP algorithm to simulate the underlying diffusional process (colored lines). The simulation is then used to compute the diffusion coefficient. The simulation proceeds until the recovery curve reaches a plateau, therefore it is interrupted at a different time for each curve. (C) Correlation between the diffusion coefficients measured in FCS experiments (DFCS, fitting with the OU model; data from Figure 2E) and in FRAP experiment (DFRAP, fitting with simFRAP). The numbers of cells measured for each construct with each technique are shown in Appendix 6. Error bars represent the standard error of the mean. Error bars that are not visible are smaller than the symbol size. FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; OU, Ornstein-Uhlenbeck.

Figure 3—source data 1

Individual mean and standard error of the mean of diffusion coefficient values from Figure 3C.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig3-data1-v3.xlsx
Figure 4 with 1 supplement
Download asset Open asset
Mobility of homologous proteins from other bacterial species in Escherichia coli.

Mass dependence of protein mobility (1/τD; A) and anomaly of diffusion (α; B) of sfGFP fusions to homologues of Adk, Pgk, and AcnA from indicated bacterial species (E.c. = Escherichia coli; Y.e. = Yersinia enterocolitica; V.c. = Vibrio cholerae; C.c. = Caulobacter crescentus; M.x. = Myxococcus xanthus; B.s. = Bacillus subtilis) compared with that of their counterpart from E. coli. Each symbol represents the average value for all individual cells that have been measured for that construct and the error bars represent the standard error of the mean. Error bars that are not visible are smaller than the symbol size. The numbers of cells measured for each construct are shown in Appendix 6.

Figure 4—source data 1

Individual mean and standard error of the mean of 1/τD values from Figure 4A.

Individual mean and standard error of the mean of α values from Figure 4B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Download asset Open asset
Mobility of homologous proteins from other bacterial species in Escherichia coli.

Each dot represents the protein mobility (1/τD; A) or the anomalous diffusion exponent (α; B) from one individual cell expressing the indicated sfGFP fusions to homologues of Adk, Pgk, and AcnA from indicated bacterial species (E.c. = Escherichia coli; Y.e. = Yersinia enterocolitica; V.c. = Vibrio cholerae; C.c. = Caulobacter crescentus; M.x. = Myxococcus xanthus; B.s. = Bacillus subtilis) compared with that of their counterpart from E. coli. The numbers of cells measured for each construct are shown in Appendix 6. ***p<0.0001; **p<0.001; *p<0.05 in a two-tailed heteroscedastistic t-test. When not indicated, no statistically significant difference is observed. Exact p-values can be found in Appendix 5.

Figure 4—figure supplement 1—source data 1

Individual values of 1/τD from Figure 4—figure supplement 1A.

Individual values of α from Figure 4—figure supplement 1B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig4-figsupp1-data1-v3.xlsx
Figure 5 with 7 supplements
Download asset Open asset
Effects of physicochemical perturbations and cell growth on mobility of differently sized proteins.

Each dot represents the average value of protein mobility (1/τD) of all the cells measured for the construct of the indicated molecular mass . The numbers of cells measured for each construct in each condition are shown in Appendix 6. Error bars represent the standard error. Error bars that are not visible are smaller than the symbol size. The solid black lines are the fit with an inverse power law to extract the size dependence of protein mobility (β) in that condition. (A) Protein mobility measured in cells that were resuspended in either tethering buffer (ionic strength of 105 mM; β=0.60±0.01) or in the same buffer but supplemented with additional 100 mM NaCl (total ionic strength of 305 mM; β=0.57±0.05). The measurements were performed in agarose pads prepared at the same ionic strength. (B) Protein mobility at different environmental temperatures. As for the other experiments, Escherichia coli cultures were grown at 37°C and bacterial cells during the measurements were incubated at 25°C (β=0.60±0.01) or at 35°C (β=0.60±0.05), as indicated. (C) Protein mobility in control cells (β=0.58±0.02) and after treatment with chloramphenicol (Cam; 200 µg/ml; β=0.88±0.11), rifampicin (Rif; 200 µg/ml, in 0.1% v/v DMSO; β=0.54±0.04), or DMSO control (0.1% v/v; β=0.62±0.07) as indicated. Antibiotics were added to growing E. coli culture 60 min prior to harvesting. (D) Protein mobility in non-growing cells incubated at 35°C on agarose pads containing only M9 salts (β=0.60±0.05) in comparison with growing cell incubated on pads with M9 salts supplemented with 20 mM glucose and 0.2% casamino acids (Glu+CA; β=0.68± 0.10).

Figure 5—source data 1

Individual mean and standard error of the mean of 1/τD values from Figure 5A.

Individual mean and standard error of the mean of 1/τD values from Figure 5B. Individual mean and standard error of the mean of 1/τD values from Figure 5C. Individual mean and standard error of the mean of 1/τD values from Figure 5D.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Download asset Open asset
Mobility of sfGFP as a function of length and width of individual cells upon indicated perturbations.

The mobility (1/τD) of sfGFP in single cells plotted as a function of the respective cell length (A, C, E, G) or cell width (B, D, F, H) at different ionic strengths (A, B), environmental temperatures (C, D), after treatments with different antibiotics (E, F) and cell growth (G, H). The numbers of measured cells are shown in Appendix 6. Significance analysis was performed for the respective cell dimension. When not indicated, no significant difference is observed. ** p<0.001; * p<0.05 in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 5—figure supplement 1—source data 1

Individual measurements of cell length from Figure 5—figure supplement 1A.

Individual measurements of cell width from Figure 5—figure supplement 1B. Individual measurements of cell length from Figure 5—figure supplement 1C. Individual measurements of cell width from Figure 5—figure supplement 1D. Individual measurements of cell length from Figure 5—figure supplement 1E. Individual measurements of cell width from Figure 5—figure supplement 1F. Individual measurements of cell length from Figure 5—figure supplement 1G. Individual measurements of cell width from Figure 5—figure supplement 1H.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-figsupp1-data1-v3.xlsx
Figure 5—figure supplement 2
Download asset Open asset
Effect of different perturbations on protein mobility (1/τD) in individual cells.

Experiments for ionic strength (A), environmental temperature (B), antibiotics treatment (C), and growth rate (D) are shown for indicated proteins. Individual measurements and significance analysis of data from Figure 5. Each dot in the box plot represents the value of 1/τD for one individual cell measured in the indicated condition. The numbers of cells measured for each construct in each condition are shown in Appendix 6. *** p<0.0001; ** p<0.001; * p<0.05; NS: no statistically significant difference in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 5—figure supplement 2—source data 1

Individual values of 1/τD from Figure 5—figure supplement 2A.

Individual values of 1/τD from Figure 5—figure supplement 2B. Individual values of 1/τD from Figure 5—figure supplement 2C. Individual values of 1/τD from Figure 5—figure supplement 2D.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-figsupp2-data1-v3.xlsx
Figure 5—figure supplement 3
Download asset Open asset
Effect of different perturbations on the anomaly of protein diffusion (α) in individual cells.

Experiments for ionic strength (A), environmental temperature (B), antibiotics treatment (C), and growth rate (D) are shown for indicated proteins. Each dot in the box plot represents the value of α for one individual cell measured in the indicated condition. The numbers of cells measured for each construct in each condition are shown in Appendix 6. ** p<0.001; * p<0.05; NS: no statistically significant difference in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 5—figure supplement 3—source data 1

Individual values of α from Figure 5—figure supplement 3A.

Individual values of α from Figure 5—figure supplement 3B. Individual values of α from Figure 5—figure supplement 3C. Individual values of α from Figure 5—figure supplement 3D.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-figsupp3-data1-v3.xlsx
Figure 5—figure supplement 4
Download asset Open asset
Effect of growth and measurement temperature on protein diffusion.

Mobility (1/τD; A) and anomaly of diffusion (α; B) for sfGFP in cells grown either at 25 °C or at 37 °C and measured either at 25 °C or at 35 °C, as in Figure 4A. The numbers of cells measured for each construct in each condition are shown in Appendix 6. *** p<0.0001; ** p<0.001; NS: no statistically significant difference in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 5—figure supplement 4—source data 1

Individual values of 1/τD from Figure 5—figure supplement 4A.

Individual values of α from Figure 5—figure supplement 4B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-figsupp4-data1-v3.xlsx
Figure 5—figure supplement 5
Download asset Open asset
Influence of nucleoid on protein mobility.

(A) Bacterial cells were treated with chloramphenicol to achieve nucleoid compaction and stained with the DNA-binding dye SYTOX Orange. Scale bars are 2 μm. The mobility (1/τD; B) and anomaly of diffusion (α; C) of sfGFP and of one larger construct (AcnA-sfGFP) was measured in both the cytoplasm and in the nucleoid of chloramphenicol treated cells. The numbers of cells measured for each construct in each condition are shown in Appendix 6. NS: no statistically significant difference in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 5—figure supplement 5—source data 1

Individual values of 1/τD from Figure 5—figure supplement 5B.

Individual values of α from Figure 5—figure supplement 5C.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-figsupp5-data1-v3.xlsx
Figure 5—figure supplement 6
Download asset Open asset
Effect of nutrient availability and growth on protein mobility.

The mobility (1/τD; A) and anomaly of diffusion (α; B) of indicated protein constructs was measured in cells incubated at 35 °C on agarose pads containing either only M9 salts or M9 salts together with 20 mM glucose and 0.2% casamino acids (Glu +CA). Where indicated, chloramphenicol (Cam) was also added to the agarose pads. The numbers of cells measured for each construct in each condition are shown in Appendix 6. *** p<0.0001; ** p<0.001; * p<0.05; NS: no statistically significant difference in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 5—figure supplement 6—source data 1

Individual values of 1/τD from Figure 5—figure supplement 6A.

Individual values of α from Figure 5—figure supplement 6B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-figsupp6-data1-v3.xlsx
Figure 5—figure supplement 7
Download asset Open asset
Effect of DNP on protein mobility.

The mobility (1/τD; A) and the anomaly of diffusion (α; B) of sfGFP and of two constructs with higher molecular mass was measured in bacterial cells treated in batch for 60 minutes with 2 mM DNP and compared with the respective untreated control. The numbers of cells measured for each construct in each condition are shown in Appendix 6. Measurements were performed on 1% agarose pads prepared in tethering buffer and supplemented with 2 mM DNP at the indicated incubation temperature. ** p<0.001; * p<0.05; NS: no statistically significant difference in a two-tailed heteroscedastistic t-test. Exact p-values can be found in Appendix 5.

Figure 5—figure supplement 7—source data 1

Individual values of 1/τD from Figure 5—figure supplement 7A.

Individual values of α from Figure 5—figure supplement 7B.

https://cdn.elifesciences.org/articles/82654/elife-82654-fig5-figsupp7-data1-v3.xlsx
Appendix 2—figure 1
Download asset Open asset
Typical examples of presence or absence of lateral focal drift during FCS measurements.

Substantial lateral drift could be observed for <10% of experiments (upper images), whereas most measurement showed no perceptible lateral drift (lower images). FCS, fluorescence correlation spectroscopy. Scale bars are 2 μm.

Appendix 2—figure 2
Download asset Open asset
Typical traces of fluorescence intensity during FCS measurements.

Examples of fluorescence intensity traces for indicated protein fusions. The vertical red dashed lines separate sequential fluorescence intensity acquisitions on the same cell. FCS, fluorescence correlation spectroscopy.

Appendix 2—figure 3
Download asset Open asset
Results of detrending with multi-segments and local averaging approaches.

Comparison of experimental ACFs corrected using either multi-segments or local averaging approaches (as indicated) for sfGFP and Adk-sfGFP and different data acquisition segments (R1 vs. R6). ACF, autocorrelation function.

Appendix 2—figure 4
Download asset Open asset
Values of τD or α for the six sequential ACFs.

Values were determined by fitting the anomalous diffusion model to experimental ACFs for the six sequential time segments per individual cell expressing sfGFP (A) or MetH-sfGFP (B). ACF, autocorrelation function.

Author response image 1
Download asset Open asset

Tables

Table 1
Molecular mass, biological function, and measured parameters for all studied sfGFP fusion constructs.

The concentration of expression inducer and the number of cells measured with each technique is also indicated.

Protein nameMolecular mass of sfGFP fusion constructBiological function in E. coliIPTG concentration used for FCS (FRAP)Number of cells analyzed by FCSτD (µs; mean ± SEM)α (mean ± SEM)Diffusion coefficient, FCS (μm2/s, mean ± SEM)Number of cells analyzed by FRAPDiffusion coefficient, FRAP (μm2/s, mean ± SEM)
sfGFP26.9–5 µM (15 µM)52561±140.86±0.0114.7±0.31111.3±1.3
YggX39.2Probable Fe (2+)-trafficking protein5 µM (5 µM)8611±190.85±0.0112.9±0.4109.4±1.6
ClpS39.2ATP-dependent Clp protease adapter protein0 µM111054±330.75±0.01


FolK45.12-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase0 µM8734±240.87±0.0111.6±0.4

Crr45.2Component of glucose-specific phosphotransferase enzyme IIA0 µM141065±360.87±0.01


UbiC45.7Chorismate pyruvate-lyase15 µM141140±580.87±0.01


ThpR46.9RNA 2′,3′-cyclic phosphodiesteraseDiscarded due to instability of sfGFP fusion construct
CoaE49.6Dephospho-CoA kinase0 µM11854±470.87±0.019.8±0.6

Adk50.6Adenylate kinase5 µM (15 µM)23802±260.88±0.0010.6±0.4169.8±1.5
Cmk51.7Cytidylate kinase5 µM161163±580.87±0.01


NagD54.1Ribonucleotide monophosphataseDiscarded due to non-uniform protein localization
KdsB54.63-deoxy-manno-octulosonate cytidylyltransferase0 µM111659±700.84±0.01


Map56.3Methionine aminopeptidase0 µM201830±780.81±0.01


MmuM60.4Homocysteine S-methyltransferase5 µM142241±1380.73±0.01


RihA60.8Pyrimidine-specific ribonucleoside hydrolaseDiscarded due to non-uniform protein localization
PanE60.82-dehydropantoate 2-reductase0 µM (5 µM)181059±260.85±0.017.8±0.2115.2±0.6
SolA67.9N-methyl-L-tryptophan oxidase0 µM7795±310.82±0.019.9±0.5

Pgk68.1Phosphoglycerate kinase0 µM16991±410.90±0.018.6±.0.3

EntC69.9Isochorismate synthase15 µM151777±1190.82±0.01


AroA73.13-phosphoshikimate 1-carboxyvinyltransferase5 µM9995±690.86±0.018.7±0.7

ThrC74.1Threonine synthase0 µM14908±280.87±0.019.1±0.3

MurF74.4UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase0 µM71008±760.85±0.028.3±0.7

DsdA74.9D-serine dehydratase0 µM141017±530.89±0.018.4±0.4107.8±0.7
HemN79.7Oxygen-independent coproporphyrinogen III oxidase0 µM131262±540.86±0.016.7±0.4

PrpD80.92-methylcitrate dehydratase0 µM121866±1400.84±0.01


DnaK96.0Molecular chaperone5 µM102296±780.76±0.01


MalZ96.0Maltodextrin glucosidase0 µM93725±2290.77±0.01


GlcB107.5Malate synthase G5 µM (15 µM)161315±450.86±0.016.4±0.2106.7±1.1
MetE111.75-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase5 µM81137±530.87±0.017.4±0.3

LeuS124.2Leucine--tRNA ligase0 µM141637±750.86±0.015.1±0.2

AcnA124.7Aconitate hydratase A5 µM (15 µM)191415±560.86±0.016.1±0.2104.3±0.4
MetH163.0Methionine synthase0 µM (5 µM)91402±450.81±0.015.8±0.1154.0±0.5
Appendix 1—table 1
List of primers used in this study.
Primer nameSenseNucleotide sequenceDescription
NBp1RWACCCATGGCACACTCCTTCACTAGAmplify pTrc99A
NBp2RWCTTGGACATGCTACCTCCGCCCCCTTAGTACAACGGTGACGCCGGAmplify ubiC gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp3FWGGGGGCGGAGGTAGCATGTCCAAGGGTGAAGAGCTATTTACAmplify pTrc99A
NBp4FWGTACTAGTGAAGGAGTGTGCCATGGGTATGTCACACCCCGCGTTAACAmplify ubiC gene of E. coli MG1655
and fuse it to trc promoter
NBp5FWTTGACAATTAATCATCCGGCTCGSequence pTrc99A
NBp7RWCTTGGACATGCTACCTCCGCCCCCGTACAACGGTGACGCCGGAmplify ubiC gene from K12 and fuse it to linker-sfGFP
NBp8FWGTACTAGTGAAGGAGTGTGCCATGGGTATGCGTATCATTCTGCTTGGCGAmplify adk gene of E. coli MG1655
and fuse it to trc promoter
NBp9RWCTTGGACATGCTACCTCCGCCCCCGCCGAGGATTTTTTCCAGATCAGAmplify adk gene of E. coli MG1655 and fuse it to linker-sfGFP
NBp10FWGTACTAGTGAAGGAGTGTGCCATGGGTATGTCGCAGAATAATCCGTTAmplify mmuM gene of E. coli MG1655
and fuse it to trc promoter
NBp11RWCTTGGACATGCTACCTCCGCCCCCGCTTCGCGCTTTTAACGAmplify mmuM gene of E. coli MG1655 and fuse it to linker-sfGFP
NBp12FWGTACTAGTGAAGGAGTGTGCCATGGGTATGGAAAACGCTAAAATGAACTCGAmplify dsdA gene of E. coli MG1655
and fuse it to trc promoter
NBp13RWCTTGGACATGCTACCTCCGCCCCCACGGCCTTTTGCCAGATATTGAmplify dsdA gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp14FWGTACTAGTGAAGGAGTGTGCCATGGGTATGTCTGTACAGCAAATCGACTGGGAmplify hemN gene from K12 genome and fuse it to trc promoter
NBp15RWCTTGGACATGCTACCTCCGCCCCCAATCACCCGAGAGAACTGCTGCAmplify hemN gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp16FWGTACTAGTGAAGGAGTGTGCCATGGGTATGAGTCAAACCATAACCCAGAGAmplify glcB gene of E. coli MG1655
and fuse it to trc promoter
NBp17RWCTTGGACATGCTACCTCCGCCCCCATGACTTTCTTTTTCGCGTAAACAmplify glcB gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp18RWGATTTAATCTGTATCAGGSequence pTrc99A
NBp19FWGTACTAGTGAAGGAGTGTGCCATGGGTATGACAGTGGCGTATATTGCAmplify folK gene of E. coli MG1655
and fuse it to trc promoter
NBp20RWCTTGGACATGCTACCTCCGCCCCCCCATTTGTTTAATTTGTCAAAmplify folK gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp21FWGTACTAGTGAAGGAGTGTGCCATGGGTATGGCTATCTCAATCAAGACCCCAmplify map gene of E. coli MG1655
and fuse it to trc promoter
NBp22RWCTTGGACATGCTACCTCCGCCCCCTTCGTCGTGCGAGATTATCGAmplify map gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp23FWGTACTAGTGAAGGAGTGTGCCATGGGTATGAAACTCTACAATCTGAAAGAmplify thrC gene of E. coli MG1655
and fuse it to trc promoter
NBp24RWCTTGGACATGCTACCTCCGCCCCCCTGATGATTCATCATCAATTTACAmplify thrC gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp25FWGTACTAGTGAAGGAGTGTGCCATGGGTATGTCAGCTCAAATCAACAACATCCGAmplify prpD gene of E. coli MG1655
and fuse it to trc promoter
NBp26RWCTTGGACATGCTACCTCCGCCCCCAATGACGTACAGGTCGAGATACTCAmplify prpD gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp27FWGTACTAGTGAAGGAGTGTGCCATGGGTATGTTAAATGCATGGCACCTGCAmplify malZ gene of E. coli MG1655
and fuse it to trc promoter
NBp28RWCTTGGACATGCTACCTCCGCCCCCGTTCATCCATACCGTAGCCGAAATGAmplify malZ gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp29FWGTACTAGTGAAGGAGTGTGCCATGGGTATGTCTGAACCGCAACGTCTGAmplify thrP gene of E. coli MG1655
and fuse it to trc promoter
NBp30RWCTTGGACATGCTACCTCCGCCCCCTTGCGTTAGCGCCCAGCAmplify thrP gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp31FWGTACTAGTGAAGGAGTGTGCCATGGGTATGTCTGTAATTAAGATGACCGATCAmplify pgk gene of E. coli MG1655
and fuse it to trc promoter
NBp32RWCTTGGACATGCTACCTCCGCCCCCCTTCTTAGCGCGCTCTTCGAmplify pgk gene of E. coli MG1655
and fuse it to linker-sfGFP
NBp33FWGTACTAGTGAAGGAGTGTGCCATGGGTATGAGGTATATAGTTGCCTTAACGGAmplify coaE gene of E. coli MG1655
and fuse it to trc promoter
NBp35FWGTACTAGTGAAGGAGTGTGCCATGGGTATGACGGCAATTGCCCCAmplify cmk gene of E. coli MG1655
and fuse it to trc promoter
NBp37FWGTACTAGTGAAGGAGTGTGCCATGGGTATGGATACGTCACTGGCTGAGAmplify entC gene of E. coli MG1655
and fuse it to trc promoter
NBp39FWGTACTAGTGAAGGAGTGTGCCATGGGTATGATTAGCGTAACCCTTAGCCAmplify murF gene of E. coli MG1655
and fuse it to trc promoter
NBp41FWGTACTAGTGAAGGAGTGTGCCATGGGTATGAAAATTACCGTATTGGGATGCGAmplify panE gene of E. coli MG1655
and fuse it to trc promoter
NBp53FWTCCAAGGGTGAAGAGCTATTTACTGGGDeletion of ATG from sfgfp in dsdA-sfgfp, ubiC-sfgfp, thrC-sfgfp, malZ-sfgfp *
NBp54RWGCTACCTCCGCCCCCACGDeletion of ATG from sfgfp in dsdA-sfgfp *
NBp55FWTCCAAGGGTGAAGAGCTATTTACTGGGGTTGDeletion of ATG from sfgfp in adk-sfgfp *
NBp56RWGCTACCTCCGCCCCCGCCDeletion of ATG from sfgfp in adk-sfgfp *
NBp57FWTCCAAGGGTGAAGAGCTATTTACTGGGGDeletion of ATG from sfgfp in mmuM-sfgfp and folK-sfgfp *
NBp58RWGCTACCTCCGCCCCCGCTDeletion of ATG from sfgfp in mmuM-sfgfp *
NBp59RWGCTACCTCCGCCCCCGTADeletion of ATG from sfgfp in ubiC-sfgfp *
NBp60FWTCCAAGGGTGAAGAGCTATTTACTGGDeletion of ATG from sfgfp in glcB-sfgfp *
NBp61RWGCTACCTCCGCCCCCATGDeletion of ATG from sfgfp in glcB-sfgfp *
NBp62FWTCCAAGGGTGAAGAGCTATTTACTGDeletion of ATG from sfgfp in hemN-sfgfp, map-sfgfp, prpD-sfgfp *
NBp63RWGCTACCTCCGCCCCCAATDeletion of ATG from sfgfp in hemN-sfgfp and prpD-sfgfp *
NBp64RWGCTACCTCCGCCCCCTTCDeletion of ATG from sfgfp in map-sfgfp *
NBp65RWGCTACCTCCGCCCCCCTGDeletion of ATG from sfgfp in thrC-sfgfp *
NBp66RWGCTACCTCCGCCCCCCCADeletion of ATG from sfgfp in folK –sfgfp *
NBp67RWGCTACCTCCGCCCCCGTTDeletion of ATG from sfgfp in malZ-sfgfp *
NBp68FWGGGGGCGGAGGTAGCTCCAAGGGTGAAGAGCTATTTACTGAmplification of backbone
flexible linker-sfgfp without ATG
NBp81RWGCTCTTCACCCTTGGAGCTACCTCCGCCCCCTGCGAGAGCCAATTTCTGGAmplify cmk gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp82RWGCTCTTCACCCTTGGAGCTACCTCCGCCCCCCGGTTTTTCCTGTGAGACAAACAmplify coaE gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp83RWGCTCTTCACCCTTGGAGCTACCTCCGCCCCCATGCAATCCAAAAACGTTCAACATAmplify entC gene of E. coli MG1655
and fuse it to linker -sfgfp deleted STOP
NBp84RWGCTCTTCACCCTTGGAGCTACCTCCGCCCCCACATGTCCCATTCTCCTGTAAAGAmplify murF gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp85RWGCTCTTCACCCTTGGAGCTACCTCCGCCCCCTTGCGTTAGCGCCCAGCAmplify thrP gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp86RWGCTCTTCACCCTTGGAGCTACCTCCGCCCCCCCAGGGGCGAGGCAAACAmplify panE gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp87RWGCTCTTCACCCTTGGAGCTACCTCCGCCCCCCTTCTTAGCGCGCTCTTCGAmplify pgk gene gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp88FWGTACTAGTGAAGGAGTGTGCCATGGGTATGGGTTTGTTCGATAAACTGAmplify crr gene of E. coli MG1655
and fuse it to trc promoter
NBp89RWTCACCCTTGGAGCTACCTCCGCCCCCCTTCTTGATGCGGATAACCAmplify crr gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp90FWGTACTAGTGAAGGAGTGTGCCATGGGTATGCAAGAGCAATACCGCCAmplify leuS gene of E. coli MG1655
and fuse it to trc promoter
NBp91RWTTCACCCTTGGAGCTACCTCCGCCCCCGCCAACGACCAGATTGAGGAmplify leuS gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp92FWGTACTAGTGAAGGAGTGTGCCATGGGTATGGCACTGCCAATTCTGTTAGAmplify rihA gene of E. coli MG1655
and fuse it to trc promoter
NBp93RWTTCACCCTTGGAGCTACCTCCGCCCCCAGCGTAAAATTTCAGACGATCAGAmplify rihA gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp94FWGTACTAGTGAAGGAGTGTGCCATGGGTATGACCATTAAAAATGTAATTTGCGATATCGAmplify nagA gene of E. coli MG1655
and fuse it to trc promoter
NBp95RWTTCACCCTTGGAGCTACCTCCGCCCCCGATAACGTCGATTTCAGCGACTGAmplify nagA gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp96FWGTACTAGTGAAGGAGTGTGCCATGGGTATGGGTAAAACGAACGACTGAmplify clpS gene of E. coli MG1655
and fuse it to trc promoter
NBp97RWTTCACCCTTGGAGCTACCTCCGCCCCCGGCTTTTTCTAGCGTACACAGAmplify clpS gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp98FWGTACTAGTGAAGGAGTGTGCCATGGGTATGGAATCCCTGACGTTACAACCAmplify aroA gene of E. coli MG1655
and fuse it to trc promoter
NBp99RWTTCACCCTTGGAGCTACCTCCGCCCCCGGCTGCCTGGCTAATCCGAmplify aroA gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp100FWGTACTAGTGAAGGAGTGTGCCATGGGTATGAGTTTTGTGGTCATTATTCCCGAmplify kdsB gene of E. coli MG1655
and fuse it to trc promoter
NBp101RWTTCACCCTTGGAGCTACCTCCGCCCCCGCGCATTTCAGCGCGAACAmplify kdsB gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp102FWGTACTAGTGAAGGAGTGTGCCATGGGTATGAAATACGATCTCATCATTATTGGCAGAmplify solA gene of E. coli MG1655
and fuse it to trc promoter
NBp103RWTTCACCCTTGGAGCTACCTCCGCCCCCTTGGAAGCGGGAAAGCCTGAmplify solA gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp107RWCACCCTTGGAGCTACCTCCGCCCCCCCATTTGTTTAATTTGTCAAATGCTCAmplify folK gene of E. coli MG1655
and fuse it to linker-sfgfp
NBp122FWACTAGTGAAGGAGTGTGCCATGGGTGTGAGCAGCAAAGTGGAACAACAmplify metH gene of E. coli MG1655
and insert it into pTrc99A fused to sfgfp
NBp123RWCACCCTTGGAGCTACCTCCGCCCCCGTCCGCGTCATACCCCAGATTC
NBp124FWACTAGTGAAGGAGTGTGCCATGGGTATGTCGTCAACCCTACGAGAmplify acnA gene of E. coli MG1655
and insert into pTrc99A fused to sfgfp
NBp125RWCACCCTTGGAGCTACCTCCGCCCCCCTTCAACATATTACGAATGACATAATGC
NBp126FWACTAGTGAAGGAGTGTGCCATGGGTATGACAATATTGAATCACACCCTCAmplify metE gene of E. coli MG1655
and insert into pTrc99A fused to sfgfp
NBp127RWCACCCTTGGAGCTACCTCCGCCCCCCCCCCGACGCAAGTTC
NBp177FWACTAGTGAAGGAGTGTGCCATGGGTATGAGCAGAACGATTTTTTGTACAmplify yggX of E. coli MG1655
and insert into pTrc99A fused to sfgfp
NBp178RWCACCCTTGGAGCTACCTCCGCCCCCTTTTTTATCTTCCGGCGTATAG
NBp179FWACTAGTGAAGGAGTGTGCCATGGGTATGAATCTGATCCTGTTCGGAmplify adk gene from Caulobacter crescentus and insert into pTrc99A fused to sfgfp
NBp180RWCACCCTTGGAGCTACCTCCGCCCCCTCCTGCAGCGACG
NBp181FWCAGACCATGTACTAGTGAAGGAGTGTGCCATGGGTATGACCTTCCGCACCCTCAmplify pgk gene from Caulobacter crescentus and insert into pTrc99A fused to sfgfp
NBp182RWCACCCTTGGAGCTACCTCCGCCCCCGGATTCGAGCGCCGC
NBp183FWACTAGTGAAGGAGTGTGCCATGGGTATGGCGTCTGTGGACAGCAmplify acnA gene from Caulobacter crescentus and insert into pTrc99A fused to sfgfp
NBp184RWCACCCTTGGAGCTACCTCCGCCCCCGTCGGCCTTGGCCAGG
NBp185FWACTAGTGAAGGAGTGTGCCATGGGTATGAACTTAGTCTTAATGGGGAmplify adk from Bacillus subtilis and insert into pTrc99A fused to sfgfp
NBp186RWCACCCTTGGAGCTACCTCCGCCCCCTTTTTTTAATCCTCCAAGAAGATCC
NBp187FWACTAGTGAAGGAGTGTGCCATGGGTATGAATAAAAAAACTCTCAAAGACATCGAmplify pgk from Bacillus subtilis and insert into pTrc99A fused to sfgfp
NBp188RWCACCCTTGGAGCTACCTCCGCCCCCTTTATCGTTCAGTGCAGCTAC
NBp189FWACTAGTGAAGGAGTGTGCCATGGGTATGGCAAACGAGCAAAAAACAmplify acnA from Bacillus subtilis and insert into pTrc99A fused to sfgfp
NBp190RWCACCCTTGGAGCTACCTCCGCCCCCGGACTGCTTCATTTTTTCACG
NBp191FWACTAGTGAAGGAGTGTGCCATGGGTATGAACCTGATCCTGTTGGGGAmplify adk from Myxococcus xanthus and insert into pTrc99A fused to sfgfp
NBp192RWCACCCTTGGAGCTACCTCCGCCCCCGGCCTTGCCCGCAG
NBp193FWACTAGTGAAGGAGTGTGCCATGGGTATGATCCGTTACATCGATGATCTGCAmplify pgk from Myxococcus xanthus and insert into pTrc99A fused to sfgfp
NBp194RWCACCCTTGGAGCTACCTCCGCCCCCCCGCGTCTCCAGCG
NBp195FWACTAGTGAAGGAGTGTGCCATGGGTATGACCGACAGTTTCGGCAmplify acnA from Myxococcus xanthus and insert into pTrc99A fused to sfgfp
NBp196RWCACCCTTGGAGCTACCTCCGCCCCCGCCCTTGGCCAGTTG
NBp197FWACTAGTGAAGGAGTGTGCCATGGGTATGCGCATCATTCTTCTCGGAmplify adk from Vibrio cholerae and insert into pTrc99A fused to sfgfp
NBp198RWCACCCTTGGAGCTACCTCCGCCCCCAGCCAACGCTTTAGCAATGTC
NBp199FWACTAGTGAAGGAGTGTGCCATGGGTATGTCTGTAATCAAGATGATTGACCTGGAmplify pgk from Vibrio cholerae and insert into pTrc99A fused to sfgfp
NBp200RWCACCCTTGGAGCTACCTCCGCCCCCCGCTTTAGCGCGTGCTTC
NBp201FWACTAGTGAAGGAGTGTGCCATGGGTATGAACAGTCTGTATCGTAAAGCAmplify acnA from Vibrio cholerae and insert into pTrc99A fused to sfgfp
NBp202RWCACCCTTGGAGCTACCTCCGCCCCCCTGCGCCAAAAAGTCTTG
NBp216FWACTAGTGAAGGAGTGTGCCATGGGTATGCGTATCATTCTGCTGGamplify adk from Yersinia enterocolitica and insert into pTrc99A fused to sfgfp
NBp217RWCACCCTTGGAGCTACCTCCGCCCCCACCGAGAATAGTCGCCAG
NBp218FWCTAGTGAAGGAGTGTGCCATGGGTATGTCTGTAATTAAGATGACCGATCTGGAmplify pgk from Yersinia enterocolitica and insert into pTrc99A fused to sfgfp
NBp219RWCACCCTTGGAGCTACCTCCGCCCCCCTGCTTAGCGCGCTCTTC
NBp220FWACTAGTGAAGGAGTGTGCCATGGGTATGTCGTTGGATTTGCGGAAAACAmplify acnA from Yersinia enterocolitica and insert into pTrc99A fused to sfgfp
NBp221RWCACCCTTGGAGCTACCTCCGCCCCCCAACATTTTGCGGATCACATAATGC
NBp227FWGATGGCTGGACGGTAGAAACCGAAGATCGCAGCTTGTCTGCACSite-directed mutagenesis of Lys to Glu in map-sfgfp
NBp228RWTTCCATGGTGCGGATCTCTTTTTCACCCGCGTTGACCATTGG
NBp229FWGATGGCTGGACGGTAGCAACCGCAGATCGCAGCTTGTCTGCACSite-directed mutagenesis of Lys to Ala in map-sfgfp
NBp230RWTGCCATGGTGCGGATCTCTTTTGCACCCGCGTTGACCATTGG
NBp231FWACTAGTGAAGGAGTGTGCCATGGGTATGGGTAAAATAATTGGTATCGAmplify dnaK from E. coli MG1655
and insert into pTrc99A fused to sfgfp
NBp232RWCACCCTTGGAGCTACCTCCGCCCCCTTTTTTGTCTTTGACTTCTTC
NBp234FWCCAGTCTGCGTTTACCATCCATGSite-directed mutagenesis of V436F in dnaK-sfgfp
NBp235RWTTGTCTTCAGCGGTAGAG
NBp240FWTTTTCTTATGATGTAGAACGTGCAACGCAATTGATGCTCGCTGTTGCGTACCAGGGGAAGGCCATTSite-directed mutagenesis of D35A, D36A, H66A in clpS-sfgfp
NBp241RWGAATTTTTGTAACACGTCAATAACAAACTCCATCGGAGTGTACGCCGCATTGACTAATATCACTTTATACATAGATGGC
Eri121FWCAGTCATAGCCG
AATAGCCT		Checking insertion of KanR cassette
Eri122RWCGGTGCCCTGAA
TGAACTGC
  1. *

    Indicated constructs were erroneously generated omitting deletion of ATG start codon of sfgfp gene and thus corrected with site-directed mutagenesis.

Appendix 1—table 2
List of plasmids generated for this study.
PlasmidRelevant genotypeReference or source
pTrc99AAmpr; expression vector; pBR ori; trc promoter, IPTG inducibleAmann et al., 1988
pCP20Ampr, Camr; flpCherepanov and Wackernagel, 1995
pNB1Ampr; sfGFP in pTrc99AThis work
pNB3Ampr; Adk-sfGFP in pTrc99AThis work
pNB4Ampr; CoaE-sfGFP in pTrc99AThis work
pNB5Ampr; Cmk-sfGFP in pTrc99AThis work
pNB6Ampr; Pgk-sfGFP in pTrc99AThis work
pNB7Ampr; MmuM-sfGFP in pTrc99AThis work
pNB8Ampr; PrpD-sfGFP in pTrc99AThis work
pNB9Ampr; DsdA-sfGFP in pTrc99AThis work
pNB11Ampr; GlcB-sfGFP in pTrc99AThis work
pNB13Ampr; HemN-sfGFP in pTrc99AThis work
pNB14Ampr; MapWT-sfGFP in pTrc99AThis work
pNB15Ampr; ThrC-sfGFP in pTrc99AThis work
pNB16Ampr; MalZ-sfGFP in pTrc99AThis work
pNB17Ampr; EntC-sfGFP in pTrc99AThis work
pNB18Ampr; ThpR-sfGFP in pTrc99AThis work
pNB19Ampr; AroA-sfGFP in pTrc99AThis work
pNB20Ampr; ClpSWT-sfGFP in pTrc99AThis work
pNB21Ampr; Crr-sfGFP in pTrc99AThis work
pNB22Ampr; KdsB-sfGFP in pTrc99AThis work
pNB23Ampr; LeuS-sfGFP in pTrc99AThis work
pNB24Ampr; MurF-sfGFP in pTrc99AThis work
pNB25Ampr; NagD-sfGFP in pTrc99AThis work
pNB26Ampr; RihA-sfGFP in pTrc99AThis work
pNB27Ampr; SolA-sfGFP in pTrc99AThis work
pNB28Ampr; UbiC-sfGFP in pTrc99AThis work
pNB29Ampr; PanE-sfGFP in pTrc99AThis work
pNB30Ampr; FolK-sfGFP in pTrc99AThis work
pNB39Ampr; AcnA-sfGFP in pTrc99AThis work
pNB40Ampr; MetE-sfGFP in pTrc99AThis work
pNB42Ampr; MetH-sfGFP in pTrc99AThis work
pNB44Ampr; YggX-sfGFP in pTrc99AThis work
pNB45Ampr; AdkC.c.-sfGFP in pTrc99AThis work
pNB46Ampr; AdkV.c.-sfGFP in pTrc99AThis work
pNB47Ampr; AdkM.x.-sfGFP in pTrc99AThis work
pNB48Ampr; AcnAM.x.-sfGFP in pTrc99AThis work
pNB49Ampr; AcnAV.c.-sfGFP in pTrc99AThis work
pNB51Ampr; AdkB.s.-sfGFP in pTrc99AThis work
pNB52Ampr; AcnAB.s.-sfGFP in pTrc99AThis work
pNB54Ampr; AdkY.e.-sfGFP in pTrc99AThis work
pNB56Ampr; PgkC.c.-sfGFP in pTrc99AThis work
pNB58Ampr; PgkV.c.-sfGFP in pTrc99AThis work
pNB59Ampr; PgkM.x.sfGFP in pTrc99AThis work
pNB60Ampr; AcnAY.e.-sfGFP in pTrc99AThis work
pNB61Ampr; DnaKWT-sfGFP in pTrc99AThis work
pNB62Ampr; MapK211E_K218E_K224E_K226E-sfGFP in pTrc99AThis work
pNB63Ampr; MapK211A_K218A_K224A_K226A-sfGFP in pTrc99AThis work
pNB64Ampr; DnaKV436F -sfGFP in pTrc99AThis work
pNB66Ampr; ClpSD35A_D36A_H66A-sfGFP in pTrc99AThis work
Appendix 5—table 1
Figure 1C.
Testing pairP-value
sfGFP vs Adk-sfGFP0.000000010
Adk-sfGFP vs AcnA-sfGFP0.00000000044
Appendix 5—table 2
Figure 1—figure supplement 10A.
Testing pairP-value
ClpSWT-sfGFP versus ClpSD35A_D36A_H66A-sfGFP0.000094
MapWT-sfGFP versus MapLys→Ala-sfGFP0.0000012
MapWT-sfGFP versus MapLys→Glu-sfGFP0.000000016
MapLys→Ala-sfGFP versus MapLys→Glu-sfGFP0.10
DnaKWT-sfGFP versus DnaKV436F-sfGFP0.00023
Appendix 5—table 3
Figure 1—figure supplement 10B.
Testing pairP-value
ClpSWT-sfGFP versus ClpSD35A_D36A_H66A-sfGFP0.000014
MapWT-sfGFP versus MapLys→Ala-sfGFP0.0092
MapWT-sfGFP versus MapLys→Glu-sfGFP0.065
MapLys→Ala-sfGFP versus MapLys→Glu-sfGFP0.37
DnaKWT-sfGFP versus DnaKV436F-sfGFP0.00000019
Appendix 5—table 4
Figure 2C.
Testing pairP-value
Untreated versus A22 treatment0.0000000000007
Appendix 5—table 5
Figure 2D.
Testing pairP-value
Untreated versus A22 treatment0.000001
Appendix 5—table 6
Figure 2—figure supplement 2.
Testing pairP-value
Untreated versus A22 treatment0.002
Appendix 5—table 7
Figure 2—figure supplement 4A.
Testing pairP-value
sfGFP, 1 A.U. versus 0.66 A.U.0.00001
DnaK-sfGFP 1 A.U. versus 0.66 A.U.0.60
AcnA-sfGFP, 1 A.U. versus 0.66 A.U.0.000002
Appendix 5—table 8
Figure 2—figure supplement 4B.
Testing pairP-value
sfGFP, 1 A.U. versus 0.66 A.U.0.24
DnaK-sfGFP 1 A.U. versus 0.66 A.U.0.50
AcnA-sfGFP, 1 A.U. versus 0.66 A.U.0.002
Appendix 5—table 9
Figure 4—figure supplement 1A.
Testing pairP-value
AdkE.c.-sfGFP versus AdkY.e.-sfGFP0.17
AdkE.c.-sfGFP versus AdkV.c. -sfGFP0.056
AdkE.c.-sfGFP versus AdkC.c.-sfGFP0.93
AdkE.c.-sfGFP versus AdkM.x.-sfGFP0.21
AdkE.c.-sfGFP versus AdkB.s.-sfGFP0.23
PgkE.c.-sfGFP versus PgkV.c.-sfGFP0.75
PgkE.c.-sfGFP versus PgkC.c.-sfGFP0.26
PgkE.c.-sfGFP versus PgkM.x.-sfGFP0.33
AcnAE.c.-sfGFP versus AcnAY.e.-sfGFP0.093
AcnAE.c.-sfGFP versus AcnAV.c.-sfGFP0.084
AcnAE.c.-sfGFP versus AcnAM.x.-sfGFP0.0000000023
AcnAE.c.-sfGFP versus AcnAB.s.-sfGFP0.069
Appendix 5—table 10
Figure 4—figure supplement 1B.
Testing pairP-value
AdkE.c.-sfGFP versus AdkY.e.-sfGFP0.000060
AdkE.c.-sfGFP versus AdkV.c. -sfGFP0.18
AdkE.c.-sfGFP versus AdkC.c.-sfGFP0.042
AdkE.c.-sfGFP versus AdkM.x.-sfGFP0.070
AdkE.c.-sfGFP versus AdkB.s.-sfGFP0.0029
PgkE.c.-sfGFP versus PgkV.c.-sfGFP0.11
PgkE.c.-sfGFP versus PgkC.c.-sfGFP0.0082
PgkE.c.-sfGFP versus PgkM.x.-sfGFP0.0087
AcnAE.c.-sfGFP versus AcnAY.e.-sfGFP0.035
AcnAE.c.-sfGFP versus AcnAV.c.-sfGFP0.16
AcnAE.c.-sfGFP versus AcnAM.x.-sfGFP0.083
AcnAE.c.-sfGFP versus AcnAB.s.-sfGFP0.00024
Appendix 5—table 11
Figure 5—figure supplement 2A.
Testing pairP-value
sfGFP ionic strength 105 mM versus 305 mM0.0000036
Adk-sfGFP ionic strength 105 mM versus 305 mM0.000044
AroA-sfGFP ionic strength 105 mM versus 305 mM0.035
AcnA-sfGFP ionic strength 105 mM versus 305 mM0.0018
Appendix 5—table 12
Figure 5—figure supplement 2B.
Testing pairP-value
sfGFP 25°C versus 35°C0.00015
Adk-sfGFP 25°C versus 35°C0.0000025
AcnA-sfGFP 25°C versus 35°C0.00077
Appendix 5—table 13
Figure 5—figure supplement 2C.
Testing pairP-value
sfGFP Untreated versus Chloramphenicol0.40
sfGFP DMSO versus Rifampicin0.012
Adk-sfGFP Untreated versus Chloramphenicol0.12
Adk-sfGFP DMSO versus Rifampicin0.00048
Pgk-sfGFP Untreated versus Chloramphenicol0.17
Pgk-sfGFP DMSO versus Rifampicin0.0000012
AcnA-sfGFP Untreated versus Chloramphenicol0.000011
AcnA-sfGFP DMSO versus Rifampicin0.0085
Appendix 5—table 14
Figure 5—figure supplement 2D.
Testing pairP-value
sfGFP M9 salts versus M9 salts, Glu+CA0.000023
Adk-sfGFP M9 salts versus M9 salts, Glu+CA0.000070
AroA-sfGFP M9 salts versus M9 salts, Glu+CA0.00000015
AcnA-sfGFP M9 salts versus M9 salts, Glu+CA0.0022
Appendix 5—table 15
Figure 5—figure supplement 3A.
Testing pairP-value
sfGFP ionic strength 105 mM versus 305 mM0.097
Adk-sfGFP ionic strength 105 mM versus 305 mM0.54
AroA-sfGFP ionic strength 105 mM versus 305 mM0.077
AcnA-sfGFP ionic strength 105 mM versus 305 mM0.31
Appendix 5—table 16
Figure 5—figure supplement 3B.
Testing pairP-value
sfGFP 25°C versus 35°C0.12
Adk-sfGFP 25°C versus 35°C0.005
AcnA-sfGFP 25°C versus 35°C0.26
Appendix 5—table 17
Figure 5—figure supplement 3C.
Testing pairP-value
sfGFP Untreated versus Chloramphenicol0.32
sfGFP DMSO versus Rifampicin0.50
Adk-sfGFP Untreated versus Chloramphenicol0.53
Adk-sfGFP DMSO versus Rifampicin0.32
Pgk-sfGFP Untreated versus Chloramphenicol0.17
Pgk-sfGFP DMSO versus Rifampicin0.59
AcnA-sfGFP Untreated versus Chloramphenicol0.008
AcnA-sfGFP DMSO versus Rifampicin0.42
Appendix 5—table 18
Figure 5—figure supplement 3D.
Testing pairP-value
sfGFP M9 salts versus M9 salts, Glu+CA0.44
Adk-sfGFP M9 salts versus M9 salts, Glu+CA0.035
AroA-sfGFP M9 salts versus M9 salts, Glu+CA0.69
AcnA-sfGFP M9 salts versus M9 salts, Glu+CA0.31
Appendix 5—table 19
Figure 5—figure supplement 4A.
Testing pairP-value
sfGFP Grown 25°C, measured 25°C versus 35°C0.0020
sfGFP Measured 25°C, grown 25°C versus 37°C0.060
sfGFP Measured 35°C, grown 25°C versus 37°C0.98
sfGFP Grown 37°C, measured 25°C versus 35°C0.000040
Appendix 5—table 20
Figure 5—figure supplement 4B.
Testing pairP-value
sfGFP Grown 25°C, measured 25°C versus 35°C0.26
sfGFP Measured 25°C, grown 25°C versus 37°C0.45
sfGFP Measured 35°C, grown 25°C versus 37°C0.44
sfGFP Grown 37°C, measured 25°C versus 35°C0.12
Appendix 5—table 21
Figure 5—figure supplement 5A.
Testing pairP-value
sfGFP cytoplasm versus nucleoid0.20
AcnA-sfGFP cytoplasm versus nucleoid0.062
Appendix 5—table 22
Figure 5—figure supplement 5B.
Testing pairP-value
sfGFP cytoplasm versus nucleoid0.09
AcnA-sfGFP cytoplasm versus nucleoid0.09
Appendix 5—table 23
Figure 5—figure supplement 6A.
Testing pairP-value
sfGFP M9 salts versus M9 salts, Cam0.82
sfGFP M9 salts versus M9 salts, Glu+CA0.000023
sfGFP M9 salts, Cam vs M9 salts, Cam, Glu +CA0.019
sfGFP M9 salts, Glu +CA versus M9 salts, Cam, Glu +CA0.019
AcnA-sfGFP M9 salts versus M9 salts, Cam0.37
AcnA-sfGFP M9 salts versus M9 salts, Glu+CA0.0022
AcnA-sfGFP M9 salts, Cam versus M9 salts, Cam, Glu +CA0.0035
AcnA-sfGFP M9 salts, Glu +CA versus M9 salts, Cam, Glu +CA0.014
Appendix 5—table 24
Figure 5—figure supplement 6B.
Testing pairP-value
sfGFP M9 salts versus M9 salts, Cam0.33
sfGFP M9 salts versus M9 salts, Glu+CA0.44
sfGFP M9 salts, Cam versus M9 salts, Cam, Glu+CA0.91
sfGFP M9 salts, Glu+CA versus M9 salts, Cam, Glu+CA0.80
AcnA-sfGFP M9 salts versus M9 salts, Cam0.099
AcnA-sfGFP M9 salts versus M9 salts, Glu+CA0.31
AcnA-sfGFP M9 salts, Cam versus M9 salts, Cam, Glu+CA0.0085
AcnA-sfGFP M9 salts, Glu+CA versus M9 salts, Cam, Glu+CA0.56
Appendix 5—table 25
Figure 5—figure supplement 7A.
Testing pairP-value
sfGFP untreated versus DNP treatment, 25°C0.52
sfGFP untreated versus DNP treatment, 35°C0.66
Adk-sfGFP untreated versus DNP treatment, 25°C0.46
Adk-sfGFP untreated versus DNP treatment, 35°C0.03
AcnA-sfGFP untreated versus DNP treatment, 25°C0.59
AcnA-sfGFP untreated versus DNP treatment, 35°C0.98
Appendix 5—table 26
Figure 5—figure supplement 7B.
Testing pairP-value
sfGFP untreated versus DNP treatment, 25°C0.013
sfGFP untreated versus DNP treatment, 35°C0.32
Adk-sfGFP untreated versus DNP treatment, 25°C0.006
Adk-sfGFP untreated versus DNP treatment, 35°C0.25
AcnA-sfGFP untreated versus DNP treatment, 25°C0.94
AcnA-sfGFP untreated versus DNP treatment, 35°C0.57
Appendix 6—table 1
Figure 1 and Figure 1—figure supplement 5.
ConstructNumerosity (n)ConstructNumerosity (n)
sfGFP52EntC-sfGFP15
YggX-sfGFP8AroA-sfGFP9
ClpSWT-sfGFP11ThrC-sfGFP14
FolK-sfGFP8MurF-sfGFP7
Crr-sfGFP14DsdA-sfGFP14
UbiC-sfGFP14HemN-sfGFP13
CoaE-sfGFP11PrpD-sfGFP12
Adk-sfGFP23DnaKWT-sfGFP10
Cmk-sfGFP16MalZ-sfGFP9
KdsB-sfGFP22GlcB-sfGFP16
MapWT-sfGFP20MetE-sfGFP8
MmuM-sfGFP14LeuS-sfGFP14
PanE-sfGFP18AcnA-sfGFP19
SolA-sfGFP7MetH-sfGFP9
Pgk-sfGFP16
Appendix 6—table 2
Figure 1—figure supplement 7.
ConstructNumerosity (n)
sfGFPSame as Appendix 6—table 1
Appendix 6—table 3
Figure 1—figure supplement 8.
ConditionNumerosity (n)
Untreated5
Cephalexin6
Appendix 6—table 4
Figure 1—figure supplement 10.
ConstructNumerosity (n)
ClpSWT-sfGFPSame as Appendix 6—table 1
ClpSD35A_D36A_H66A-sfGFP10
MapWT-sfGFPSame as Appendix 6—table 1
MapLys→Glu-sfGFP10
MapLys→Ala-sfGFP12
DnaKWT-sfGFPSame as Appendix 6—table 1
DnaKV436F-sfGFP10
Appendix 6—table 5
Figure 2C and D and Figure 2—figure supplement 2.
ConditionNumerosity (n)
UntreatedSame as Appendix 6—table 1
A22-treated12
Appendix 6—table 6
Figure 2—figure supplement 4.
ConditionNumerosity (n)
sfGFP 1 A.U.Same as Appendix 6—table 1
sfGFP 0.66 A. U.12
DnaK-sfGFP 1 A.U.Same as Appendix 6—table 1
DnaK-sfGFP 0.66 A.U,10
AcnA-sfGFP 1 A.U.Same as Appendix 6—table 1
AcnA-sfGFP 0.66 A. U.10
Appendix 6—table 7
Figure 2—figure supplement 5.
ConstructNumerosity (n)
sfGFPSame as Appendix 6—table 1
Adk-sfGFPSame as Appendix 6—table 1
DnaK-sfGFPSame as Appendix 6—table 1
DnaKV436-sfGFPSame as Appendix 6—table 4
AcnA-sfGFPSame as Appendix 6—table 1
Appendix 6—table 8
Figure 3C.
ConstructNumerosity (n)ConstructNumerosity (n)
sfGFP11DsdA-sfGFP10
YggX-sfGFP10GlcB-sfGFP10
Adk-sfGFP16AcnA-sfGFP10
PanE-sfGFP11MetH-sfGFP15
Appendix 6—table 9
Figure 4 and Figure 4—figure supplement 1.
ConstructNumerosity (n)ConstructNumerosity (n)
AdkE.c.-sfGFPSame as Appendix 6—table 1PgkC.c.-sfGFP5
AdkY.e.-sfGFP5PgkM.x.-sfGFP11
AdkV.c.-sfGFP10AcnAE.c.-sfGFPSame as Appendix 6—table 1
AdkC.c.-sfGFP5AcnAY.e.-sfGFP5
AdkM.x.-sfGFP11AcnAV.c.-sfGFP10
AdkB.s.-sfGFP10AcnAM.x.-sfGFP10
PgkE.c.-sfGFPSame as Appendix 6—table 1AcnAB.s.-sfGFP10
PgkV.c.-sfGFP10
Appendix 6—table 10
Figure 5A, Figure 5—figure supplement 1A, B, Figure 5—figure supplement 2, and Figure 5—figure supplement 3A.
Construct and conditionNumerosity (n)Construct and conditionNumerosity (n)
sfGFP, 105 mMSame as Appendix 6—table 1AroA-sfGFP, 105 mMSame as Appendix 6—table 1
sfGFP, 305 mM11AroA-sfGFP, 305 mM12
Adk-sfGFP, 105 mMSame as Appendix 6—table 1AcnA-sfGFP, 105 mMSame as Appendix 6—table 1
Adk-sfGFP, 305 mM11AcnA-sfGFP, 305 mM6
Appendix 6—table 11
Figure 5B, Figure 5—figure supplement 1C, D, Figure 5—figure supplement 2, and Figure 5—figure supplement 3B.
Construct and conditionNumerosity (n)Construct and conditionNumerosity (n)
sfGFP, 25°CSame as Appendix 6—table 1AcnA-sfGFP, 25°CSame as Appendix 6—table 1
sfGFP, 35°C14AcnA-sfGFP, 35°C18
Adk-sfGFP, 25°CSame as Appendix 6—table 1
Adk-sfGFP, 35°C21
Appendix 6—table 12
Figure 5C, Figure 5—figure supplement 1E, F, Figure 5—figure supplement 2C, and Figure 5—figure supplement 3C.
Construct and conditionNumerosity (n)Construct and conditionNumerosity (n)
sfGFP, untreatedSame as Appendix 6—table 1Pgk-sfGFP, untreatedSame as Appendix 6—table 1
sfGFP, chloramphenicol10Pgk-sfGFP, chloramphenicol10
sfGFP, DMSO15Pgk-sfGFP, DMSO10
sfGFP, rifampicin15Pgk-sfGFP, rifampicin10
Adk-sfGFP, untreatedSame as Appendix 6—table 1AcnA-sfGFP, untreatedSame as Appendix 6—table 1
Adk-sfGFP, chloramphenicol10AcnA-sfGFP, chloramphenicol10
Adk-sfGFP, DMSO10AcnA-sfGFP, DMSO10
Adk-sfGFP, rifampicin10AcnA-sfGFP, rifampicin10
Appendix 6—table 13
Figure 5D, Figure 5—figure supplement 1G, H, Figure 5—figure supplement 2D, and Figure 5—figure supplement 3D.
Construct and conditionNumerosity (n)Construct and conditionNumerosity (n)
sfGFP, M9 salts10AroA-sfGFP, M9 salts11
sfGFP, M9 salts, Glu+CA15AroA-sfGFP, M9 salts, Glu+CA11
Adk-sfGFP, M9 salts11AcnA-sfGFP, M9 salts10
Adk-sfGFP, M9 salts, Glu+CA12AcnA-sfGFP, M9 salts, Glu+CA10
Appendix 6—table 14
Figure 5—figure supplement 4.
Construct and conditionNumerosity (n)
sfGFP, grown 25°C, measured 25°C10
sfGFP, grown 25°C, measured 35°C10
sfGFP, grown 37°C, measured 25°CSame as Appendix 6—table 1
sfGFP, grown 37°C, measured 35°CSame as Appendix 6—table 11
Appendix 6—table 15
Figure 5—figure supplement 5.
Construct and conditionNumerosity (n)
sfGFP, cytoplasm10
sfGFP, nucleoid10
AcnA-sfGFP, cytoplasm10
AcnA-sfGFP, nucleoid10
Appendix 6—table 16
Figure 5—figure supplement 6.
Construct and conditionNumerosity (n)Construct and conditionNumerosity (n)
sfGFP, M9 saltsSame as Appendix 6—table 12AcnA-sfGFP, M9 saltsSame as Appendix 6—table 12
sfGFP, M9 salts, Cam13AcnA-sfGFP, M9 salts, Cam8
sfGFP, M9 salts, Glu+CASame as Appendix 6—table 12AcnA-sfGFP, M9 salts, Glu+CASame as Appendix 6—table 12
sfGFP, M9 salts, Cam, Glu+CA13AcnA-sfGFP, M9 salts, Cam, Glu+CA10
Appendix 6—table 17
Figure 5—figure supplement 7.
Construct and conditionNumerosity (n)
sfGFP, untreated, 25°CSame as Appendix 6—table 1
sfGFP, 2 mM DNP, 25°C5
sfGFP, untreated, 35°CSame as Appendix 6—table 10
sfGFP, 2 mM DNP, 35°C6
Adk-sfGFP, untreated, 25°CSame as Appendix 6—table 1
Adk-sfGFP, 2 mM DNP, 25°C5
Adk-sfGFP, untreated, 35°CSame as Appendix 6—table 10
Adk-sfGFP, 2 mM DNP, 35°C6
AcnA-sfGFP, untreated, 25°CSame as Appendix 6—table 1
AcnA-sfGFP, 2 mM DNP, 25°C5
AcnA-sfGFP, untreated, 35°CSame as Appendix 6—table 10
AcnA-sfGFP, 2 mM DNP, 35°C6

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/82654/elife-82654-mdarchecklist1-v3.docx

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Nicola Bellotto
  2. Jaime Agudo-Canalejo
  3. Remy Colin
  4. Ramin Golestanian
  5. Gabriele Malengo
  6. Victor Sourjik
(2022)
Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions, and cell growth
eLife 11:e82654.
https://doi.org/10.7554/eLife.82654