Lipid a mutants of Vibriocholerae: Isolation and partial characterization

Presence of exposed phospholipids in the outer membrane of Vibrio cholerae

“…V . cholerae strain 569B and its detergent-resistant mutant, lpa-1 (Paul et al, 1990) were grown in complex media with shaking at 37 "C as described previously (Lohia et al, 1984). A low phosphate medium was used for the induction of alkaline phosphatase (Roy et al, 1982).…”

“…Phospholipids were extracted from whole cells and isolated membranes by chloroform/methanol(2 : 1, v/v) and quantitatively analysed after their separation by TLC as described previously (Lohia et al, 1985). The phospholipids were essentially free of LPS, as no 3-hydroxytetradecanoic acid could be detected by GLC (Paul et al, 1990).…”

“…Isolation of LPS from crude cell envelopes, and subsequent separation of the lipid A and polysaccharide moieties were carried out as described previously (Paul et al, 1990). LPS was labelled by growing cells in the presence of [14C]acetate [specific activity: 56.7 mCi (2.1 GBq) mmol-l, 0.2 pCi (7.4 kBq) ml-l] for 3 generations.…”

“…Free amino groups were assayed by 2,4-dinitrofluorobenzene using Dglutamic acid as a standard (Ghuysen & Strominger, 1963). Glucosamine in lipid A was assayed as described earlier (Paul et al, 1990). Protein was estimated by the method of Markwell et al (1978).…”

“…The ability of the probe to react selectively with exposed amino groups of PE is shown by its failure to bind PE of the inner membrane of V. cholerae 569B and membrane lipids (both outer and inner) of E. coli C-600 (Table 4). It is important to note that membrane phospholipids of the mutant strain lpa-1 which is resistant to hydrophobic compounds and detergents (Paul et al, 1990) were not labelled by CNBractivated dextran under identical conditions (Table 4). When whole cells of V. cholerae 569B ([2-3H]glycerol labelled) treated with activated dextran were lysed with SDS (2% w/v, 2 min, 100 "C) and directly spotted on TLC plates, radioactive material could be recovered from the origin.…”