Phosphatidylinositol synthase of Tetrahymena: inositol isomers as substrates in phosphatidylinositol biosynthesis and headgroup exchange reactions.

Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo-inositol and scyllo-inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non-myo-inositols in Tetrahymena and the potential for the production of novel, non-myo-inositol-containing second messengers.

Subcellular distribution of Plp-40, a lipoprotein in a serotype A strain of Pasteurella multocida.

A 40-kDa lipoprotein (Plp-40) is expressed by serotype A strains of Pasteurella multocida in amounts which correlate with the amount of capsular material present. We hypothesized that Plp-40 is exposed at the outer surface of the outer membrane (OM) of the cell and is associated with the serotype A exopolysaccharide material. The objectives of the present study were to confirm the lipoprotein nature of Plp-40 and to determine its subcellular location. Plp-40 maturation was shown to be sensitive to globomycin, thereby confirming it to be a bacterial lipoprotein. Plp-40 was shown to be present in the OM fractions of P. multocida obtained by both sarkosyl extraction and sucrose density gradient centrifugation, as well as in capsule fractions obtained by either hyaluronidase treatment or warm buffer extraction. [(3)H]palmitic acid-labeled Plp-40 could be removed from the surface of whole cells by exposure to proteinase K. Autoradiography of (125)I-labeled cell surface proteins exhibited a 40-kDa band that was prominent in capsulated strains and greatly diminished in a noncapsulated strain. These results support the hypothesis that Plp-40 is a lipid-modified OM protein, which is exposed on the outer cell surface and is likely associated with serotype A extracellular polysaccharide.

Phospholipids of the differentiating species of Tetrahymena.

The whole-cell phospholipid composition of the six known polymorphic species of Tetrahymena has been examined by [(3)H]acetate and [(3)H]myristic acid radiolabeling, and by gas-liquid chromatography of total phospholipid-bound fatty acids. Five of the polymorphic species contained similar phospholipid profiles following radiolabeling in that phosphatidylethanolamine (PE) was the predominant phospholipid; however, in cells of Tetrahymena patula LFF, aminoethylphosphonolipid was present in amounts nearly equal to PE. Tetrahymena patula LFF contained an unusually large percentage of sphingolipid (16.2% by [(3)H]acetate radiolabeling). Substantial differences were found in the fatty acid profiles of the polymorphic species, which included the degree of fatty acid unsaturation and relative weight percentages of odd-chain fatty acids. Tetrahymena vorax contained a low ratio of unsaturated C(18) fatty acids to saturated C(18) fatty acids as compared with all other species examined. The differentiating species generally contained a lesser percentage of monoenoic fatty acids and a lower ratio of unsaturated C(16) fatty acids to saturated C(16) fatty acids as compared with the two monomorphic species examined.

Sodium-dependent transport of [3H](1D)chiro-inositol by Tetrahymena.

The transport characteristics of (1D)chiro-inositol by the ciliate Tetrahymena were examined in competition studies employing [3H](1D)chiro-inositol. (1D)chiro-Inositol transport was competed by unlabeled (1D)chiro-inositol, myo-inositol, scyllo-inositol, and D-glucose in a concentration-dependent manner. Conversely, (1D)chiro-inositol competed for [3H]myo- and [3H]scyllo-inositol transport. Lineweaver-Burke analysis of the competition data indicated a Km of 10.3 mM and a Bmax of 4.7 nmol/min/mg for (1D)chiro-inositol. Transport of (1D)chiro-inositol was inhibited by cytochalasin B, an inhibitor of facilitated glucose transporters, and phlorizin, an inhibitor of sodium-dependent transporters. Removal of sodium from the radiolabeling buffer also inhibited uptake. The presence of 0.64 mM calcium or magnesium ions exerted negligible effects on transport, although potassium was inhibitory. [3H](1D)chiro-Inositol was shown to be incorporated into Tetrahymena phosphoinositides.

Identification of the inositol isomers present in Tetrahymena.

The inositol isomer composition of phosphoinositides, polyphosphoinositols, phosphatidylinositol-linked glycans, and glycosyl phosphatidylinositol-anchored proteins of logarithmic phase Tetrahymena vorax was determined by GC-MS analysis of trimethylsilylimadazole derivatives. The most abundant inositol found was the myo-isomer; however, appreciable percentages of scylloinositol were present in the free inositol pool, phosphatidylinositol-linked glycan fraction, and glycosyl phosphatidylinositol-anchored protein fraction. Trace quantities of chiro- and neo-inositols also were present.

Prevalence of a novel capsule-associated lipoprotein among pasteurellaceae pathogenic in animals.

Capsular serotype A strains of Pasteurella multocida of avian origin express a 40-kDa lipoprotein (Plp-40) thought to attach the extracellular polysaccharide to the cell surface. The objective of the present study was to assess the prevalence of Plp-40 in P. multocida strains of disparate serotypes and host origins, as well as other pathogenic members of the family Pasteurellaceae. Exponential-phase reference and clinical isolates were radiolabeled with [3H]-palmitate, lysed to obtain whole-cell protein fractions, and analyzed using SDS-PAGE and fluorography to assess lipoprotein content. The ability to produce Plp-40 was found to be conserved among certain P. multocida reference and clinical strains of different host origins including avian, human, porcine, bovine, feline, canine, ovine, and cervine, but not rabbit. Production of a 40-kDa lipoprotein was exhibited by all clinical isolates of Pasteurella aerogenes, Pasteurella pneumotropica, Actinobacillus suis, Actinobacillus suis-like organism, and Actinobacillus pleuropneumoniae examined, but not Pasteurella (Mannheimia) haemolytica, Actinobacillus lignieresii, or Haemophilus spp. These data suggest that, while not all Pasteurellaceae are able to produce a 40-kDa lipoprotein under the present experimental conditions, expression is somewhat conserved among diverse isolates of disparate host origins.

Phenotype switching in polymorphic Tetrahymena: a single-cell Jekyll and Hyde.

For nearly half a century, phenotype switching in the group of polymorphic species of the ciliate genus Tetrahymena has been the subject of investigations of the underlying mechanisms, the accompanying biochemical and structural changes, and the evolution of polymorphic survival strategy. Beginning with the pioneering systematic studies by Furgason in 1940 of hymenostome ciliates, the experimental approach rapidly expanded to include investigations of growth, nutrition, physiology, morphology, and morphogenesis in the polymorphic species. Recently, with progress in elucidation of the novel signaling ligand and identification of elements of the subsequent signal transduction cascade, in addition to the growing catalog of intracellular events associated with differentiation in these unicellular eukaryotes, we have begun to address the mechanistic basis of polymorphism. This review summarizes and integrates the history and recent discoveries concerning Tetrahymena polymorphic cells. We are now poised to answer fundamental questions about this interesting pathway of cell differentiation.