Fig. 7: The wt ObgE protein is involved in the regulation of E. coli cell envelope synthesis.

A Schematic representation of the set-up of the CRISPRi screen performed in exponential phase. B Schematic representation of the set-up of the CRISPRi screen performed in the stationary phase. C KEGG pathway enrichment results are shown for the comparison of ObgE to vector control in the stationary phase CRISPRi screen. As input, genes were used for which our gene-level analysis showed significant enrichment of sgRNAs targeting this gene. Enrichment was determined by Fisher’s exact test. Two-sided p-values were FDR adjusted. Ara, arabinose. D Both ObgE and ObgE* synergize with LPS inhibitor PF-04753299. E. coli carrying pBAD33Gm, pBAD33Gm-obgE, or pBAD33Gm-obgE* were induced with arabinose, and PF-04753299 was added at a concentration of 1/8x MIC (0.03125 µg/ml). CFUs/ml were monitored for a growth period of 6 h. Data are represented as the mean ± SEM, number of biological replicates n = 3. E E. coli wt and lpxA mutants carrying pBAD33Gm, pBAD33Gm-obgE, or pBAD33Gm-obgE* were induced with arabinose and treated with PF-04753299 at a concentration of 1/8x MIC (0.03125 µg/ml for lpxA_wt, lpxA_V197H, lpxA_I199S, and 0.0078 µg/ml for lpxA_R216C). After 6 h, CFUs/ml were determined, and survival was calculated by dividing CFUs/ml with PF-04753299 treatment by those without PF-04753299. Bar graphs and error bars represent the mean ± SEM, number of biological replicates n = 3. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test was performed against the lpxA_wt control condition. F CRISPRi depletion of ObgE using four different sgRNAs leads to slightly increased LPS levels, similar to depletion of known LPS inhibitors FtsH and YciM with CRISPRi⁷⁴. Quantitative interpretation of this gel-based assay was performed and values were normalized to the vector control sample. Bar graphs and error bars represent the mean ± SEM, number of biological replicates n ≥ 3. A one-sample, two-sided t test was performed to assess if samples display a normalized LPS content that deviates from one. Samples were tested individually, and a sample where all obgE-targeting sgRNAs were taken together was also considered.