A complex of iron and nucleic acid catabolites is a signal that triggers differentiation in a freshwater protozoan.

The polymorphic ciliated protozoan Tetrahymena vorax can undergo differentiation from the microstomal form, which normally feeds on bacteria and other particulate matter, into the macrostomal cell type, which is capable of ingesting prey ciliates. The process is triggered by exposure of the microstome to an inducer contained in stomatin, an exudate of the prey. To establish the identity of the signal, stomatin was fractionated by combinations of cation exchange, HPLC, and TLC, and the fractions were assayed for biological activity. Although no single active fraction of purified inducer was obtained, all fractions with activity contained ferrous iron and the nucleic acid catabolites hypoxanthine (6-oxypurine) and uracil (2, 4-dioxopyrimidine), probably in a chelated form. The activity of synthetic complexes containing these three components is equivalent to stomatin. These results indicate a role for ferrous iron and its potential in chelated form to signal differentiation in certain protozoa and, perhaps, in other organisms as well.

Derivation of extracellular polysaccharide-deficient variants from a serotype A strain of Pasteurella multocida.

The production of serotype A extracellular polysaccharide is thought to be associated with expression of an approximately 40-kDa lipoprotein (P1p-40) present on the outer surface of Pasteurella multocida strains of avian origin. The tendency of certain strains to undergo colonial dissociation concomitantly with serial passaging on laboratory growth media was exploited to derive two variant strains exhibiting the capsule-deficient phenotype from a heavily capsulated parental strain. Assessments of colonial consistency, iridescence, gentian violet binding, and hyaluronidase sensitivity were consistent with cellular observations indicating little or no capsulation of derivative strains. Fluorographic analysis of electrophoretically resolved cellular lipoproteins labeled with [3H]-palmitate revealed capsular loss occurred with a concomitant diminution of P1p-40 production in the variant strains. In contrast, a phenotypically stable strain that did not undergo colonial dissociation under identical conditions exhibited no decrease in P1p-40 content. This work provides a model system for investigating the role of extracellular polysaccharide in the cell surface physiology and pathogenicity of P. multocida. The present results strongly support the notion that P1p-40 is associated with serotype A capsular material and suggest coordinate regulation of their biosynthesis.

Sodium-dependent uptake of [3H]scyllo-inositol by Tetrahymena: incorporation into phosphatidylinositol, phosphatidylinositol-linked glycans, and polyphosphoinositols.

[3H]Scyllo-inositol was taken up by Tetrahymena cells through a sodium-dependent pathway wherein unlabeled scyllo- and myo-inositol competed for uptake. d-Glucose was a competitor of [3H]myo-inositol uptake, but did not appear to compete for [3H]scyllo-inositol uptake. Transport of [3H]scyllo- and [3H]myo-inositol was inhibited when sodium was removed from the labeling buffer and by phlorizin, an inhibitor of sodium-dependent transporters. Cytochalasin B, an inhibitor of facilitated glucose transporters, had no significant effect on inositol transport. Internalized [3H]scyllo-inositol was readily incorporated intact into phosphatidylinositol, phosphatidylinositol-linked glycans, and polyphosphoinositols. Distribution of [3H]scyllo- and [3H]myo-inositol radioactivity into individual polyphosphoinositols was found to differ.

Evidence for early signaling events in stomatin-induced differentiation of Tetrahymena vorax.

The mechanism of stomatin-induced differentiation of Tetrahymena vorax was investigated by in vivo protease degradation of cell surface proteins, the direct measurement of products formed from the activation of phospholipase C, and the use of an array of signal transduction inhibitors/activators. The data indicate that a surface-exposed protein is required for stomatin to signal the cells to differentiate and that the cells are committed to the differentiation pathway within two hours after exposure to stomatin. Analysis of radiolabeled polyphosphoinositols and inositol lipids from control and stomatin-treated populations in the presence of 10 mM LiCl were consistent with a rapid activation of phospholipase C. Within five min following addition of stomatin, this resulted in an increase in polyphosphoinositols and a concomitant decrease in the relative amounts of phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate.

Relationship between serotype A encapsulation and a 40-kDa lipoprotein in Pasteurella multocida.

Eleven serotype A encapsulated and nonencapsulated strains of Pasteurella multocida were examined with regard to lipoprotein content. Relative amounts of an approximately 40-kDa lipoprotein (Plp-40) were found to correlate directly with the degree of encapsulation in that heavily encapsulated strains exhibited the greatest amounts, while nonencapsulated strains possessed little or no Plp-40.

Polysaccharide inducible outer membrane proteins of Bacteroides xylanolyticus X5-1.

Little is known about how bacteria degrade structural polysaccharides or the regulatory systems that control this degradation. Bacteroides xylanolyticus X5-1 is a Gram-negative, anaerobic bacterium that can grow on structural polysaccharides such as xylan and pectin. In order to determine the response of this organism to specific substrates,B. xylanolyticus was grown on a variety of mono-, di-, and polysaccharides. Electrophoretic analysis revealed no distinct differences in the polypeptide profile of the inner membrane enrichments of cells grown on different carbohydrates. However, distinct differences in protein composition of outer membrane enrichments were clearly observed. The profiles from cells grown on starch, xylan, and pectin were distinct from each other and their respective monosaccharide. In addition, cells initially grown on xylan did not alter their total membrane protein composition after three generations of growth in medium containing xylan and glucose. Thus,B. xylanolyticus X5-1 altered its outer membrane protein composition in response to specific polysaccharide substrates, but analysis of this specific response revealed no evidence that glucose was preferred over xylan as a substrate.

Effects of dichloroisoproterenol on macromolecular synthesis and differentiation in Tetrahymena vorax.

The effect of dichloroisoproterenol on macrostomal cell formation in Tetrahymena vorax was examined and the drug was found to be 50% inhibitory at a concentration of 88 microM. Cellular uptake and incorporation of a variety of radiolabelled precursors was monitored in the presence of dichloroisoproterenol. The results demonstrate a strong, concentration-dependent inhibitory effect on RNA and protein biosynthesis, with a lesser inhibition observed for lipid biosynthesis. These data indicate that dichloroisoproterenol's reported effects on vacuole formation and processing in Tetrahymena are nonspecific with regard to phagocytic processes, but result from a general suppression of macromolecular synthesis.

Cytodifferentiation in Tetrahymena vorax is linked to glycosyl-phosphatidylinositol-anchored protein assembly.

The role of glycosyl-PtdIns (GPI)-anchored proteins in the cytodifferentiation of Tetrahymena vorax was examined. Labelling of cells with [3H]myristate or [3H]palmitate followed by electrophoresis showed an array of proteins carrying covalently bound lipids. Electrophoresis of protein from cells labelled with the GPI-anchor components [3H]Ins and [14C]ethanolamine revealed three polypeptides on fluorograms which have apparent molecular masses of approx. 28, 50 and 82 kDa. Labelled lipid associated with these polypeptides was susceptible to release by in vitro exposure to Bacillus thuringiensis PtdIns-specific phospholipase C (PI-PLC). Using labelled fatty acids, cells induced to differentiate showed altered GPI-anchored protein-labelling patterns in comparison with undifferentiated control cells, with a heavily labelled 32 kDa band appearing upon differentiation. Pre-incubation of cells in 10 mM D-mannosamine, an inhibitor of GPI incorporation into protein, resulted in a reduction of the incorporation of label into the three GPI-anchored proteins, nearly complete inhibition of differentiation and a reduction in the rate of digestive vacuole formation. A 50% inhibition of differentiation was obtained using 500 microM mannosamine. The inhibitory impact of D-mannosamine on differentiation could be competitively and completely reversed by the inclusion of D-mannose, but not D-glucose. Neither glucosamine nor tunicamycin inhibited differentiation. Incubation of cells in PI-PLC (5 units/ml) plus the differentiation inducer resulted in an acceleration of differentiation and generally higher percentages of differentiated cells versus controls.

Phosphatidylinositol glycan formation and utilization by the ciliate Tetrahymena mimbres.

Ryals et al. (Ryals, P.E., Pak, Y., and Thompson, G. A., Jr., (1991) J. Biol. Chem. 266, 15048-15053) have described and partially characterized phosphatidylinositol glycans (PI glycans) present in Tetrahymena mimbres. We now describe the time course of PI glycan labeling from exogenous [3H]myristate, [14C]glucosamine, and [3H]ethanolamine. Over the first 2-12 h following pulse radioisotope addition a sizeable proportion of the radioactivity associated with the protein pellet remaining after cell delipidation existed as PI glycans. These compounds were distributed throughout the cell, with the largest proportion at 12 h being associated with a fraction containing mitochondria, lysosomes, and peroxisomes. However, by 24 h radioactivity had nearly disappeared from the PI glycans and had become associated with proteins by a process that was almost totally inhibited by cycloheximide or tunicamycin. PI glycans appeared to be incorporated mainly into a protein migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a relatively broad band with an apparent molecular mass of 24-29 kDa. The exact mobility of the protein band within this molecular weight range was dependent upon the growth temperature of the cells. The apparent molecular masses of the principal PI-anchored proteins formed by other closely related Tetrahymena species varied widely, ranging from 22 to 76 kDa. The PI-anchored proteins may belong to a group of surface proteins known as immobilization antigens. Treatment of 24-h-labeled T. mimbres cells with phosphatidylinositol-specific phospholipase C in vivo released labeled proteins from the cells. Some labeled proteins were present even in the medium of control, non-phospholipase-treated cells. Tetrahymena PI glycans appear to accumulate in a metabolic pool from which they are gradually removed for attachment to externally oriented PI-anchored proteins. Tetrahymena is a versatile system well suited for studying the regulation of PI-anchored protein biochemistry.

Phosphatidylinositol-linked glycans and phosphatidylinositol-anchored proteins of Tetrahymena mimbres.

The insoluble residue from Tetrahymena mimbres cells that had been preincubated in vivo for 2 h with [3H]myristic acid and then exhaustively delipidated with organic solvents retained radioactivity, principally in material which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 10-14 kDa. This material was extractable from the delipidated cell residue with organic solvents known to solubilize phosphatidylinositol glycans (PI glycans). The same material could also be labeled with [3H]inositol, [14C]glucosamine, and [3H] ethanolamine. When the delipidated residue of cells labeled for 2 h with [3H]myristate was treated with phosphatidylinositol-specific phospholipase C or nitrous acid, much of the associated radioactivity was released. A similar release was obtained using the putative PI glycan fraction extracted from the cell residue. After further purification by thin layer chromatography, this latter material was hydrolyzed with HCl and shown to contain fatty acids, alkylglyceryl ethers, phosphate, inositol, glucosamine, mannose, and ethanolamine. The findings indicate that T. mimbres contains PI glycans resembling in structure those recently characterized in trypanosomes and mammalian cells. As the time of incubation with the radiotracers enumerated above was increased to 6-24 h, increasing amounts of radioactivity appeared in the 22-27-kDa region of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. This higher molecular weight material is shown in the companion paper (Pak, Y., Ryals, P.E., and Thompson, G.A., Jr. (1991) J. Biol. Chem. 266, 15054-15059) to be released by in vivo phosphatidylinositol-specific phospholipase C treatment. Thus T. mimbres contains a pool of free PI glycans and at least one phosphatidylinositol-anchored protein.

Lipid modification during cytodifferentiation of Tetrahymena vorax. Whole cell phospholipids and triacylglycerols of microstomal and macrostomal phenotypes.

Microstomal cells of the ciliate Tetrahymena vorax V2S can be induced to undergo cytodifferentiation to form an alternate phenotype known as the macrostomal cell; however, sublines of T. vorax exist that respond differently to methods that induce macrostomal cell formation. The phospholipid- and triacylglycerol-bound fatty acid compositions of microstomal and macrostomal cells of a high-transforming subline (designated 3-C) were determined and compared to similar data from cells of a low-transforming subline (designated Ala). Differences in fatty acid composition were found between the two phenotypes as well as between the different sublines. Some change in the distribution of radioactive acetate and lauric acid into phospholipid classes of the different subline was observed, and evidence was also obtained that indicated changes in the relative amounts of the sterol-like pentacyclic triterpenoid tetrahymanol. A limited analysis of the lipid composition of stomatin revealed the presence of small amounts of tetrahymanol, phospholipid and free fatty acid. Stomatin is the naturally produced material obtained from T. pyriformis that triggers differentiation in T. vorax. The existence of a low-transforming subline provides a powerful experimental tool for elucidating the underlying biochemical and molecular mechanisms that control cytodifferentiation in T. vorax and possibly in other eukaryotic cells.

Fatty Acid Acylated Proteins of the Halotolerant Alga Dunaliella salina.

The unicellular, wall-less alga Dunaliella salina has been shown to contain an array of proteins modified by the covalent attachment of fatty acids. Myristic acid (14:0) comprised approximately 80% by weight of the protein-linked acyl groups in samples derived from cells cultured in medium containing 1.7 molar NaCl and 93% in samples from cells grown in medium containing 3.0 molar NaCl. Palmitic and stearic acids accounted for most of the remaining protein-bound acyl chains. Approximately 0.2% of the incorporated radioactivity was estimated to be in linkage with protein. The bulk of acyl chains (about 99%) were resistant to cleavage by alkali, indicating a preponderance of amide bonding. The sodium dodecyl sulfate-polyacrylamide electrophoresis labeling pattern of proteins from [(3)H]myristic-labeled cells was significantly different from that of proteins from cells exposed to [(3)H]palmitate. The appearance of radioactivity in certain proteins was also influenced by the salinity of the culture medium. Thus growth in moderate (1.7 molar) salt favored the acylation of a 48-kilodalton polypeptide whereas in high (3.0 molar) salt, a 17-kilodalton polypeptide was more heavily labeled.

Protein acylation in Tetrahymena.

Examination of exhaustively delipidated Tetrahymena mimbres cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of several protein bands containing covalently linked fatty acids. Palmitic (16:0) and stearic (18:0) acids together accounted for approximately 90% of the protein-linked acyl chains, with myristic acid (14:0) comprising most of the remainder. Each of these three fatty acids was present mainly in alkali-stable linkage, indicating that unlike most other systems examined, fatty acids are attached to proteins of Tetrahymena principally by amide bonds. Smaller proportions of the acyl chains were susceptible to release by hydroxylaminolysis or by alkaline hydrolysis as would be expected from an ester linkage. The protein-bound acyl chains accounted for 0.3% of the cells' total fatty acids. They closely resembled in composition the highly saturated free fatty acid pool but not the vast pool of glycerolipid-associated fatty acids, which were mainly unsaturated. Cells subjected to thermal stress by rapid chilling from 39 to 15 degrees C responded by sharply increasing the ratio of palmitate to stearate in covalent association with proteins.

Alterations of the composition and size of the free fatty acid pool of Tetrahymena responding to low-temperature stress.

Cells of Tetrahymena mimbres (formerly T. pyriformis NT-1) in midlogarithmic growth under isothermal conditions (at 39 degrees C) contained a very small, compositionally discrete pool of free fatty acids, principally (60.6% of the total free fatty acid mass) palmitic and stearic acids. The composition, degree of unsaturation, and size of this free fatty acid pool were rapidly (15 min or less) altered in response to chilling. During the acclimation period following chilling to 15 degrees C, the size of the free fatty acid pool increased from a mean value of 15.5 nmol free fatty acid/mumol lipid phosphorus in 39 degrees C cells to 24.2 nmol free fatty acid/mumol lipid phosphorus. The degree of free fatty acid saturation rapidly increased over the initial hour following the onset of hypothermal conditions, but by 24 h the unsaturated free fatty acid/saturated free fatty acid ratio was 0.35 (equivalent to a 2.7-fold increase in unsaturation relative to 39 degrees C controls (unsaturated/saturated ratio = 0.13) and 4.4-fold greater than cells acclimated for 1 h (unsaturated/saturated ratio = 0.08)). By 24 h the percentage of palmitic and stearic acids had decreased to 45.6%. Similar, and in some instances more pronounced, changes were observed to occur in triacylglycerol-bound fatty acids. Modulation of steady-state free fatty acid composition could also be achieved by the addition of exogenous fatty acids to the growth medium. The ability to manipulate the level of intracellular free fatty acids should prove to be a valuable experimental tool in determining how specific fatty acids regulate various lipid-modifying enzymes.