Deletions of the TCA cycle and ETC genes enhanced tolerance to aminoglycosides.

In the mid-exponential phase (t=3.5 h), 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 duration of 5 hours in test tubes. Following the treatments, cells were washed to eliminate the antibiotics and then plated on LB agar plates to quantify the surviving cell fractions. CFU: Colony forming units. WT: Wild type. For pairwise comparisons, one-way ANOVA with Dunnett’s post hoc test was used where *, P < 0.05, **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001. N=3. Data points represent mean and standard error.

The observed tolerance in the mutant strains is not linked to cell growth.

(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 and mutant strains were collected from culture flasks (see Materials and Methods for details) at indicated time intervals and then subjected to gentamicin treatment. The figure shows the colony-forming unit (CFU) levels of both the treated and untreated cultures, indicating the surviving and total cell populations, respectively. N=3. Data points represent mean and standard error.

Comparable ATP levels, redox activities, and cytoplasmic pH levels were observed between the wild-type and mutant strains.

(A) 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 in the Materials and Methods section. (B) The ATP levels were measured in wild-type and mutant cells during the mid-exponential and early stationary growth phases. (C) A comparison of the cytoplasmic pH of the WT and mutant cells during the mid-exponential and early stationary phases was performed using the ratiometric pHluorin. For pairwise comparisons, one-way ANOVA with Dunnett’s post hoc test was used where *, P < 0.05, **, P < 0.01, ***, and P < 0.001. N ≥ 3. Data points represent mean and standard error.

Deletion of genes associated with the TCA cycle and ETC did not alter the drug uptake.

(A) Exemplary quantification of GTTR uptake in cells during the exponential growth phase (t=3.5 h). (B) GTTR assays were performed on cells in the mid-exponential phase for both the wild-type and mutant strains, followed by fluorescence measurement using flow cytometry after one hour. The concentration of GTTR is 25 µg/ml. For pairwise comparisons, one-way ANOVA with Dunnett’s post hoc test was used (no statistical significance was detected). N=3. Data points represent mean and standard error.

The dysregulation of membrane potential is not correlated with the observed aminoglycoside tolerance.

Deletions of the TCA cycle and electron transport chain genes resulted in no significant change in PMF. Mid-exponential and early stationary phase cells of wild-type and mutant strains were stained with DiSC3(5), and at specified time intervals their fluorescence was measured using a plate reader. Cells were treated with gentamicin (50 µg/ml) after 20 minutes and fluorescence was measured again. N=4. Data points represent mean and standard error.

Proteomics data on the mutant strains, exhibiting increased gentamicin tolerance, indicates upregulation in proteins linked to energy metabolism.

Cells from both wild-type and mutant strains at the mid-exponential phase (t=3.5 h) were collected after which protein extraction and digestion were carried out for mass spectrometry analysis. The STRING visual network depicts upregulated protein interactions of ΔsucA (A), ΔgltA (B) and ΔnuoI (C) mutants, compared to those of the wild-type strain. The protein clusters and their corresponding gene names are visually distinguished through color-coding on the networks. N= 3.

Proteomics data on the mutant strains, exhibiting increased gentamicin tolerance, reveals downregulation in proteins associated with ribosomes.

The STRING visual network displays down-regulated protein interactions for ΔsucA (A), ΔgltA (B), and ΔnuoI (C) mutants. N= 3.