Fig 5. yhdP, tamB mutants shed OMVs that are rich in LPS.

(A) ³H-labeled membrane separation. Listed strains were grown to mid-log phase in the presence of ³H-glycerol to label GPLs. Inner and outer membrane fractions were separated by a 3-step sucrose gradient and OMVs were collected separately (error bars in red). IM peak fractions are indicated by NADH oxidase and OM peak fractions are indicated by LPS levels (S2 Fig). The % of ³H-incorporation to indicate GPL levels were performed in biological triplicate. (B) Kdo levels measured from collected OMVs. Growth supernatants were collected, filtered, and OMVs collected by centrifugation. Purpald reagent was used to determine Kdo levels. Error bars represent SD from technical triplicate and is representative of two biological experiments. (C) Total lipid levels of ³²P_i labeled OM and OMV content. WT and yhdP, tamB cells were grown to mid-log phase and the OM fraction and OMVs were collected and total lipids were extracted for TLC analysis (S3 Fig). Error bars represent SD from biological triplicates. 0.001>P***, 0.0001>P****, NS = not significant. (D) Scanning electron microscopy (SEM) of yhdP, tamB mutants. Top row 20,000x zoom. Bottom row 50,000x zoom. White scale bar set to 5 or 3 μm.