In the mid-exponential phase (t = 3.5 hr), E. coli MG1655 wild-type and knockout strains underwent (A) streptomycin, (B) gentamicin, and (C) amikacin treatments at a concentration of 50 μg/ml for a …
In the mid-exponential phase (t = 3.5 hr), E. coli MG1655 wild-type and knockout strains underwent gentamicin treatments at a concentration of 5 μg/ml for a duration of 5 hr in test tubes. Following …
(A) Growth of E. coli MG1655 wild type, ΔsucA, ΔgltA, ΔnuoI, and Δicd strains was assessed by measuring the number of cells per ml with flow cytometry. (B) The cells of both the E. coli MG1655 WT …
(A) Redox sensor green (RSG) staining was conducted by suspending wild-type and mutant strains in 0.85% sodium chloride solution during the mid-exponential and early stationary phases, as outlined …
E. coli MG1655 wild-type cells in the exponential phase (t = 3.5 hr) were subjected to staining with 1 μM redox sensor green (RSG) for 10 min at 37°C, followed by analysis using flow cytometry. …
ATP was subjected to 10-fold serial dilutions (10 µM, 1 µM, 100 nM, and 10 nM), starting from an initial concentration of 10 μM in a culture medium. The resulting dilutions were then transferred to …
The pH vs. fluorescence ratio (410/470 nm) was established for E. coli MG1655. The standard curve data were fitted using a Boltzmann sigmoid curve.
(A) Exemplary quantification of Gentamicin-Texas Red (GTTR) uptake in cells during the exponential growth phase (t = 3.5 hr). (B) GTTR assays were performed on cells in the mid-exponential phase for …
Gentamicin-Texas Red (GTTR) assays were performed on cells in the mid-exponential phase (t = 3.5 hr) for both the wild-type and mutant strains, followed by fluorescence measurement using flow …
Deletions of the tricarboxylic acid (TCA) cycle and electron transport chain genes resulted in no significant change in proton motive force (PMF). Mid-exponential and early stationary phase cells of …
This disruption results in the release of DiSC3(5) from the membrane, accompanied by a concurrent increase in fluorescence. Polymyxin B achieves this effect by attaching its polycationic peptide …
The mutant and wild-type strains showed no significant change in PMF. Mid-exponential phase cells of wild-type and mutant strains (t = 3.5 hr) were stained with DiSC3(5), and at specified time …
(A) During the mid-exponential phase (t = 3.5 hr), ATP synthase knockout strains of E. coli BW25113 were subjected to gentamicin treatment at a concentration of 50 μg/ml in test tubes for 5 hr. …
The cells of E. coli MG1655 wild-type and ATP synthase knockout strains were exposed to 5 μg/ml gentamicin at the mid-exponential phase (t = 3.5 hr) for 5 hr in test tubes. After the treatments, …
Cells from both wild-type and mutant strains at the mid-exponential phase (t = 3.5 hr) were collected after which protein extraction and digestion were carried out for mass spectrometry analysis. …
The STRING visual network displays downregulated protein interactions for ΔsucA (A), ΔgltA (B), and ΔnuoI (C) mutants. N = 3.
The STRING visual networks depict both up- and downregulated protein interactions specific to the Δicd mutant (A, B), respectively.
ATP levels of both the mutant and wild-type strains were measured at t=3 hours of cell growth and normalized to cell counts. The figure presents the raw data (a), linear plot (b), and logarithmic …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔsucA mutant | other | Ngo HG, Ngo et al., 2024. Unraveling Crp/cAMP-mediated metabolic regulation in Escherichia coli persister cells. bioRxiv. | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔgltA mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔnuoI mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 Δicd mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔsdhC mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 Δmdh mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔacnB mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔfumA mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔnuoM mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔatpA mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichi. coli MG1655 ΔatpB mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔatpC mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli MG1655 ΔatpD mutant | other | Ngo et al., 2024 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpA mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpB mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpC mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpD mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpE mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpF mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpG mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpH mutant | Horizon Discovery | Catalog # OEC4988 | |
Strain, strain background (Escherichia coli) | Escherichia coli K-12 BW25113 ΔatpI mutant | Horizon Discovery | Catalog # OEC4988 | |
Recombinant DNA Reagent | pGFPR01 | Martinez et al., 2012 | ||
Commercial assay or kit | BacTiter-Glo Microbial Cell Viability assay | Promega Corporation | Catalog # G8230 | |
Commercial assay or kit | BacLight Redox Sensor Green Vitality kit | Thermo Fisher | Catalog # B34954 | |
Chemical compound or drug | Gentamicin-Texas Red Conjugate | AAT Bioquest, Inc | Catalog # 24300 | |
Chemical compound or drug | Polymyxin B Sulfate | Millipore Sigma | Catalog # 52-91-1GM | |
Chemical compound or drug | DiSC3(5) (3,3′-Dipropylthiadicarbocyanine Iodide) | TCI America | Catalog # D4456 | |
Commercial assay or kit | MIC Test Strips | Fisher Scientific | ||
Software, algorithm | Prism (version 10.1.2) | GraphPad | RRID: SCR_002798 | http://www.graphpad.com/ |
Software, algorithm | FlowJo (version 10.10.0) | Becton, Dickinson & Company | RRID: SCR_008520 | https://www.flowjo.com/solutions/flowjo |
The minimum inhibitory concentrations of aminoglycosides were assessed in both wild type and mutant E. coli MG1655 strains.
(a) The minimum inhibitory concentration (MIC) levels of streptomycin, gentamicin, and amikacin were examined in tricarboxylic acid cycle (TCA) and electron transport chain (ETC) mutants as well as the wild type.
Proteomics data analysis identified upregulated and downregulated proteins in mutant E. coli MG1655 strains with subsequent pathway analysis for these proteins compared to the wild type.
(a) Upregulated proteins at mid-exponential phase (t = 3.5 hr) in the ΔsucA mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05, based on F- and t-statistics (see Materials and methods), is applied. (b) Upregulated proteins at mid-exponential phase (t = 3.5 hr) in the ΔgltA mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05 is applied. (c) Upregulated proteins at mid-exponential phase (t = 3.5 hr) in the ΔnuoI mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05 is applied. (d) Upregulated proteins at mid-exponential phase (t = 3.5 hr) in the Δicd mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05 is applied. (e) Downregulated proteins at mid-exponential phase (t = 3.5 hr) in the ΔsucA mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05 is applied. (f) Downregulated proteins at mid-exponential phase (t = 3.5 hr) in the ΔgltA mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05 is applied. (g) Downregulated proteins at mid-exponential phase (t = 3.5 hr) in the ΔnuoI mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05 is applied. (h) Downregulated proteins at mid-exponential phase (t = 3.5 hr) in the Δicd mutant strain relative to the wild type. FC: fold change. A significance threshold of p < 0.05 is applied. (i) The pathway analysis for the upregulated proteins in the ΔsucA strain compared to the wild type. This analysis integrates statistical analysis across the entire genome and includes various functional pathway classification frameworks such as Gene Ontology annotations, KEGG pathways, Uniprot, and STRING. Count in network: The first number indicates how many proteins in our network are annotated with a particular term. The second number indicates how many proteins in total (in our network and the background) have this term assigned. Strength: Log10(observed/expected). This measure describes how large the enrichment effect is. It is the ratio between (1) the number of proteins in our network that are annotated with a term and (2) the number of proteins that we expect to be annotated with this term in a random network of the same size. False discovery rate: This measure describes how significant the enrichment is. Shown are p-values corrected for multiple testing within each category using the Benjamini–Hochberg procedure. Note: When discussing our findings in the manuscript, we primarily reference the local network cluster (STRING), as it offers the advantage of broader coverage, including potential novel modules that might not yet be classified as pathways. (j) The pathway analysis for the upregulated proteins in the ΔgltA strain compared to the wild type. See the legend of Supplementary file 2i for further details. (k) The pathway analysis for the upregulated proteins in the ΔnuoI strain compared to the wild type. See the legend of Supplementary file 2i for further details. (l) The pathway analysis for the upregulated proteins in the Δicd strain compared to the wild type. See the legend of Supplementary file 2i for further details. (m) The pathway analysis for the downregulated proteins in the ΔsucA strain compared to the wild type. See the legend of Supplementary file 2i for further details. (n) The pathway analysis for the downregulated proteins in the ΔgltA strain compared to the wild type. See the legend of Supplementary file 2i for further details. (o) The pathway analysis for the downregulated proteins in the ΔnuoI strain compared to the wild type. See the legend of Supplementary file 2i for further details. (p) The pathway analysis for the downregulated proteins in the Δicd strain compared to the wild type. See the legend of Supplementary file 2i for further details.
The raw proteomics data included quantified protein levels in samples from both wild type and mutant strains.