Fig. 5: 5mC methylation modification negatively correlates with tet(X) expression.

a, b Growth curves of a reference strain E. coli MG1655 pBAD or pBAD-tet(X4), cultured with varied concentrations of L-arabinose ranging from 0.1 to 100 mM. Data are presented as mean values ± SEM. c MIC values of engineered E. coli MG1655 (pBAD-tet(X4)) in the presence of varied concentrations of L-arabinose (0.1 to 100 mM). Created with Biorender.com. d Percent survival of E. coli MG1655 (pBAD-tet(X4)) in the presence of Tig or Tig + Met. Tig, 1-fold MIC; Met, 20 mM. e Metabolic networks surveillance on Met degradation pathway in E. coli MG1655 (pBAD-tet(X4)) after exposure to exogenous L-arabinose (1, 10, 50 and 100 mM), corresponding to different expression levels of tet(X4), respectively. The group without Tig was served as a blank control, with the supplement of Tig or Tig + Met was the experimental setting. In 5mC methylation-related pathway, two enzymes (MetK, S-adenosylmethionine synthetase; Dcm, DNA-cytosine methyltransferase) and three metabolites (SAM, S-Adenosyl-L-methionine; SAH, S-Adenosyl-L-homocysteine; L-Hcy, L-homocysteine) were investigated. SAH was recognized as a methyltransferase inhibitor. f–h Effect of different levels of tet(X4) expression on the Met degradation pathway. Box indicates changes in metabolites or proteins when compared E. coli MG1655 (pBAD-tet(X4)) plus Tig with E. coli MG1655 (pBAD-tet(X4)) alone (left), and the right section indicates the comparison between Tig alone and combination treatment. Color coding is based on the level of log₂(fold change) as indicated. i Heatmap of SAM (5mC methylated donors), Dcm (5mC methyltransferases), and SAH (downstream metabolite) level in E. coli MG1655 (pBAD-tet(X4)), in response to the Tig treatment with the increasing concentration of L-arabinose. j mRNA expression of tet(X4) gene of E. coli MG1655 (pBAD-tet(X4)) in the presence of varied concentration of L-arabinose. k Pearson correlation analyses between the survival of E. coli MG1655 (pBAD-tet(X4)) and the mRNA expression of tet(X4) (yellow, Pearson r = 0.9348), the mRNA expression of tet(X4) and the protein expression of Dcm (blue, Pearson r = 0.49). The correlation was fitted alongside linear curves, shown by the grey dashed lines. Colored dots represent the actual values. l A three-dimension bubble chart for summarizing the fitting relationship among the bacterial survival, tet(X4) expression, and Dcm expression. The value of these three were shown by the three axes, and the size of a bubble in was proportional to the survival data that has been normalized according to Z-score. The color intensity from blue to yellow represented the span between death to survival. Data in d, e, and j were displayed as mean ± SEM. Three biological repeats were performed. In d, the P values were determined using an unpaired two-tailed Student’s t-test. In e and j, adjusted P values were determined using two-way ANOVA with Sidak’s multiple comparison test.