The yhcB mutant contains extra membrane and has an altered cell envelope.

(A) Representative transmission electron micrograph (TEM) image of a Δ yhcB mutant cell processed without primary fixation by fast freezing and freeze substitution. Internal membrane structures indicated by red arrows. Scale bar = 200 nm.(B) Phospholipid species extracted from BW25113 (BW) and the Δ yhcB mutant (ΔY) grown in LB broth until stationary phase and separated by thin layer chromatography (TLC) using a chloroform:methanol:water (65:25:4) solvent system. Standards of lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), phosphatidylserine (PS) and phosphatidic acid (PA) were loaded alongside for comparison. The additional spot in the ΔY sample is indicated with an arrow. (C) Analysis of [ 32 P]-labeled lipid A extracted from mid-logarithmic growth and overnight growth cultures of BW25113 and Δ yhcB strains grown in LB, presented as %hepta-acylated lipid A. As a positive control for lipid A palmitoylation, BW25113 cells were treated with 25 mM EDTA for 10 min prior to extraction. The mean percentage of hepta-acylated lipid A and standard deviation were calculated from samples prepared in triplicate. Student’s t -tests: * P < 0.05 compared with BW25113. (D) Optical Density (OD) measurements of BW25113 and a Δ yhcB mutant +/- pLpxC taken after 16 h growth in LB with and without IPTG induction of lpxC . (E) Relative LpxC abundance in BW25113 and a Δ yhcB mutant +/- pLpxC after 16 h growth in LB supplemented with 31.25 μM IPTG, measured by western blot analysis and normalised to RNA polymerase. Statistical significance ( P < 0.05) determined using a one way ANOVA with Tukey’s correction for multiple comparisons, with * P ≤ 0.05 and ** P ≤ 0.01. (F) Comparison of the percentage of peptides in peptidoglycan crosslinks between strains at two stages of growth. Unpaired t-test with Welch’s correction: * P < 0.05. Exponential phase cells were collected at an OD of 0.4, Stationary phase cells were harvested after 16 h at an OD of ~4.0. Abbreviations: phosphatidylethanolamine (PE); ...