Fig. 4: Exogenous Met promotes intracellular Tig accumulation and 5mC methylation in the promoter region of tet(X4) gene.

a Flow cytometry analysis of the PMF of E. coli B3-1 (tet(X4)) in the presence of Tig (16 μg/mL) with or without Met (20 mM) for 6 h. Cells were incubated with DiOC₂(3) (30 μM) for 30 min and then analyzed using 488 nm excitation and 530 nm emission filter. CCCP (10 μM) was recognized as the negative control to blunt the original PMF in cells. b The changes of bacterial PMF under different treatments, are calculated as follows: PMF= Log(₁₀^3/2*(red fluorescence/green fluorescence)). c Dynamic monitoring of membrane potential (ΔΨ) changes in bacteria was performed using the fluorescent probe DiSC₃(5) (0.5 μM), in the presence of Tig, Met, or both. The fluorescence units were monitored during 40 min. d The activity of efflux pump in bacteria after exposure Tig, Met or both, assessed using a fluorescent probe Ethidium Bromide (EtBr, 5 μM). CCCP was used as a known efflux pump inhibitor. EtBr efflux from cells was detected with the excitation wavelength at 530 nm and emission wavelength at 600 nm within 30 min. e The accumulation of intracellular antibiotics in E. coli B3-1 (tet(X4)) after being treated with Tig (1-fold MIC) with or without metabolites supplement, including Met and SAM. f The accumulation of intracellular antibiotic in E. coli B3-1 (tet(X4)) treated by Tig (1-fold MIC), and Met (20 mM) at different time intervals (the first 2 h before Tig addition or the subsequent 2 h). g mRNA expression of Met degradation-related enzymes in E. coli B3-1 (tet(X4)) under the treatment of Tig (32 μg/mL) with or without Met (20 mM) at 6 h. h Metabolic networks surveillance on Met degradation pathway when exogenous Met was added to Tig-treated E. coli B3-1 (tet(X4)), including 2 enzymes (MetK, S-adenosylmethionine synthetase; Dcm, DNA-cytosine methyltransferase) and 3 metabolites (SAM, S-Adenosyl-L-methionine; SAH, S-Adenosyl-L-homocysteine; L-Hcy, L-homocysteine). SAH was recognized as a methyltransferase inhibitor. i Metabolic networks summarized reprogramming process for exogenous Met in Tig-treated cells. The upper part represents the case of Tig treatment, with the normalized processing shown in gray. The lower part represents the changes under Tig plus Met treatment, with upregulated metabolites or enzymes or genes marked in red, and downregulated metabolites or enzymes or genes marked in green. Metabolites (SAM, SAH, L-Hcy); Enzymes (MetK, Dcm); Genes (mtn, metE, metH). j, k Changes in cytosine methylation rates at the six CG sites in the tet(X) promoter region under different treatments. Bisulfite methylation sequencing was performed on E. coli B3-1 (tet(X4)) after exposure to Tig (32 μg/mL) or Tig-Met combination or Tig-SAH combination for 6 h. l Percent survival of 5mC methylation knockout strain (Δdcm) after 6 h treatment of Tig (8 μg/mL) with or without Met (20 mM). m Relative expression of tet(X4) gene of E. coli B3-1 (tet(X4)) by RT-qPCR analysis. Cells were grown in LB broth and suspended in M9 medium in the treatment of Tig (1-fold MIC) with or without Met (20 mM). n Tet(X4) production in E. coli B3-1 (tet(X4)) cells following coculture with different concentrations of Tig (0, 16, and 32 μg/mL) and Met (0, 20, and 40 mM) by western blotting assays. The expression of internal reference protein GroEL and integrated density were shown in Supplementary Fig. 17. Data were displayed as mean ± SEM. Three biological repeats were carried out. In b, c, e, f, j, l, m, the P values were determined using an unpaired two-tailed Student’s t-test. In g, adjusted P values were determined using two-way ANOVA with Sidak’s multiple comparison test. ns, not significant.