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. 2016 Jun 21;11(6):e0157842. doi: 10.1371/journal.pone.0157842

Fig 3. Fatty acid analysis of HMW O-Ag capsule.

Fig 3

A) Elution of [14C] labeled species during deoxycholate/Sephacryl S-200 chromatography of radiolabeled EtOHp (larger sample input). B) SDS-PAGE followed by immunoblot (O-Ag; FB11, top pane) and autoradiogram (bottom pane) representing capsule- and LMW-rich species (fractions 25–33 and 46–51, respectively). C) Percent of cpm of the indicated samples recovered in the CHCl3 phase after chemical hydrolysis and Bligh-Dyer extraction are indicated. * denotes that recovery from capsule is significantly lower than that from both EtOHp and LMW samples, per Tukey’s test for multiple comparisons (P < 0.001). D) HPLC profile of recovered [14C] species in CHCl3 phase from EtOHp, nearly O-Ag-free LMW species, and HMW O-Ag capsule. Each curve was normalized so that the peak of the most abundant species (peak 3) was set at 1.0. E) Normal-phase TLC (top panels) and reverse-phase TLC (bottom panels) of the 4 major peaks derived from EtOHp (left) and capsule (right). Francisella-derived and purified commercial FFA were used to identify individual fatty acids, as indicated. NFA, non-hydroxylated fatty acids. 3-OH FA, 3-OH fatty acids. Note that C14:0 and 3-0H-18:0 (peaks 2 and 3) closely migrate on reverse-phase TLC but are readily distinguished by normal phase TLC. F) Relative content of the 4 major FA substituents of the EtOHp, LMW, and capsule samples, expressed as percent of total cpm recovered. These results represent analyses of fractions derived from the same population of metabolically labeled bacteria. G) Similar analyses were performed on three separate EtOHp and capsule preparations from independent batches of labeled bacteria to demonstrate that FA compositional differences between the EtOHp (in which free lipid A is most abundant) and HMW O-Ag capsule are reproducible and, where indicated (*, P < 0.05 and ** P < 0.001 for paired T tests) significant. Left panel: ratio of [14C] 3-OH-16:0 to [14C] 3-OH-18:0; center panel: ratio of [14C] C14:0 to combined [14C] 3-OH-16:0 + 3-OH-18:0; right panel ratio of 14[C] C16:0 to combined [14C] 3-OH-16:0 + 3-OH-18:0.