Nutritional control of differentiation (sclerotization) of the myxomycete Physarum flavicomum.

During differentiation (sclerotization) of the Myxomycete Physarum flavicomum, the acellular phasmodium converts into numerous dormant cells surrounded by cell walls. This work establishes that a condition of nutrient imbalance triggers the differentiation process. Specifically, the unavailability of an adequate spectrum of amino acids in the medium initiates the metabolic and morphological alterations characteristic of the sclerotizing plasmodium. In the absence of extracellular amino acids, the cellular pool of amino acids and cellular protein were catabolized as differentiation proceeded. The pattern of amino acids in the cellular pool also changed during differentiation, as the content of pool amino acids was reduced at least 75 percent. The decrease in protein content was negligible after 12 h incubation but was about 40 percent at 48 h when differentiation was complete. However, in the presence of extracellular amino acids, protein degradation, amino acid pool depletion, and differentiation were all inhibited. Ammonium ions (12.4 mM) similarly delayed differentiation. Differentiation, amino acid pool depletion, and the degradation of cellular protein readily occurred in the presence of an extracellular supply of dextrose, which stimulated cell wall formation. The effect of dimethyl sulfoxide, cyclic 3'-5'-adenosine monophosphate, glutathione, diamide, and other compounds on the differentiation process are reported also.     

Carbohydrate metabolism during differentiation (Sclerotization) of the myxomycete Physarum flavicomum

Summary The metabolism of carbohydrates during differentiation (sclerotization) of Physarum flavicomum was studied using the radiorespirometric technique. After about 36 h in a sclerotization (starvation) medium the metabolism declined to a level characteristic of the dormant state. Sclerotia incubated in complete growth medium quickly reverted to a metabolically active state and by 9 h they regained about 50% of their metabolic potential.Sclerotia metabolize carbohydrates by the Embden-Meyerhof-Parnas (EMP)-tricarboxylic acid and the pentose phosphate (PP) pathways. Compared to growing plasmodia the activity of the EMP is reduced to a greater extent than the PP in sclerotia. Also, EMP-produced triose phosphates are not well equilibrated: there is a greater yield of ¹⁴CO₂ from the C-4 of glucose than from the C-3; the C-3 is incorporated into the lipid fraction to a greater extent than the C-4.The metabolism of carbohydrates by sclerotia is stimulated by cyclic-3-5-adenosine monophosphate.     

Growth of the haploid phase of the myxomycete Physarum flavicomum in defined minimal medium.

The haploid phase (myxamoebae-swarm cells) of the myxomycete Physarum flavicomum grew readily in chemical defined liquid media. The minimal medium contained salts, glucose, biotin, thiamine, hematin, glycine, L-arginine and L-methionine. Cell yields of 1.4 times 10(7) cells/ml were obtained in this medium in aerobic shake culture. These cells consumed about 35 muliters of oxygen/mg protein-hr in the minimal medium. The morphology of cells maintained in this medium appeared to be "normal". L-valine replaced either glycine or L-methionine in the minimal medium but the growth rates and cell yields were reduced. Growth rates increased in media containing four, seven, or fourteen amino acids.     

Growth of the haploid phase of the myxomycete Physarum flavicomum in defined minimal medium

The haploid phase (myxamoebae-swarm cells) of the myxomycete Physarum flavicomum grew readily in chemically defined liquid media. The minimal medium contained salts, glucose, biotin, thiamine, hematin, glycine, l-arginine and l-methionine. Cell yields of 1.4x10⁷ cells/ml were obtained in this medium in aerobic shake culture. These cells consumed about 35 μliters of oxygen/mg protein·hr in the minimal medium. The morphology of cells maintained in this medium appeared to be “normal”. l-valine replaced either glycine or l-methionine in the minimal medium but the growth rates and cell yields were reduced. Growth rates increased in media containing four, seven, or fourteen amino acids.     

Methionine metabolism of the myxomycete Physarum polycephalum

Daniel, John W. (University of Wisconsin, Madison), and Karlee Babcock. Methionine metabolism of the myxomycete Physarum polycephalum. J. Bacteriol. 92:1028-1035. 1966.-Previous studies have shown that Physarum polycephalum requires exogenous methionine for growth, but not cysteine, folic acid, or vitamin B(12). Methionine can also serve as the sole source of sulfur for all cellular requirements, without limiting the growth rate. S-methyl-l-cysteine, 2-hydroxy-4-methiol butyric acid, S-adenosyl-l-methionine, and methionine peptides were the only compounds supporting growth, when substituted for methionine. Other methionine analogues, methyl donors in combination with homocysteine, and intermediates of the cystathionine pathway were not active. Ethionine and S-ethyl cysteine were good methionine antagonists. This myxomycete is apparently unable to synthesize the methyl or S-methyl group, although it still appears able to transmethylate, at least from S-methyl cysteine, and probably from S-adenosyl methionine, which can also serve as a source of adenine.     

Flow cytometric determination of nuclear DNA content during differentiation (spherulation and germination) of the myxomycete Physarum polycephalum

Summary Using flow cytometry, spherulating nuclei of Physarum isolated at the beginning of spherule wall formation were found to exhibit a DNA content corresponding to the G₂ phase of the cell cycle, although 8% lower. Before the first mitosis after spherule germination, a very slight incorporation of ³H thymidine into DNA was observed that was too weak to correspond to S phase, strongly suggesting that nuclei are stopped in G₂ phase inside the spherules. The lower value of nuclear DNA content found using flow cytometry of germinating spherules may not be related to DNA quantity, but may be due to a difference in chromatin organization during growth or spherulation, resulting in interference with the staining.     

Amino acid pool and protein turnover during differentiation (spherulation) ofPhysarum polycephalum

The composition of the amino acid pool during spherulation was determined. It changes in size and in composition, the concentration of each amino acid behaving individually. The first response to the onset of spherulation either by starvation or osmotic shock (0.5 M mannitol) always is a decrease of the pool's size, which during further starvation expands for a short period and then decreases again. During development induces by mannitol in the presence of external amino acids, the pool size increases continuously after the initial depletion.As shown by radioactive labeling, amino acids were actively released from the plasmodium into a medium containing amino acids, but retained by the microplasmodia in an amino acid-free medium. The kinetics of the uptake of radioactive amino acids from the medium is biphasic, indicating the existence of multiple pools. Even after a labeling period of 8 h the amino acid pool is not yet in equilibrium with the medium. The possibility of a compartimentation of the pool was confirmed by density labeling of two different enzymes.Whereas the turnover of total protein is only very low during growth, it is rather high in spherulating microplasmodia. At least 70% of the originally existing protein is degraded during this development, while, simultaneously, at least 50% of the protein present after 24 h starvation is newly synthesized during that period.     

Intracellular polyamine patterns during encystment of Physarum flavicomum

In Physarum flavicomum Berk., growing amoebae convert to dormant cysts under conditions of nutrient imbalance. Exogenous adenine inhibits the process and the cells produce an elevated intracellular concentration of S-adenosylmethionine. Evidence indicates that the increased level of S-adenosylmethionine is responsible for the disruption of the normal developmental process. One of the biological functions of S-adenosylmethionine is in polyamine synthesis and it is known that a well-controlled intracellular concentration of polyamines is essential for normal cell growth and differentiation. In this study, high-performance liquid chromatography was used to determine the intracellular polyamine patterns in growing cells, adenine-treated and normal encysting cells, and dormant cysts. Putrescine and spermidine were the most abundant polyamines found in the cells; growing cells had the highest level, adenine-treated cells had a 1.5 to 2.0 times higher level than normal encysting cells, while cysts had the lowest (only 3 and 12% of that of growing cells). Cadaverine and N1-acetylspermidine were found in all the cells and their levels decreased during encystment. Acetylputrescine was found in growing cells only and acetylspermine was found in all cells except cysts. Acetylcadaverine, N8-acetylspermidine, 1,3-diaminopropane, and spermine were not detected in any of the cells.     

Fluctuations in S-adenosyl-L-methionine (AdoMet) during mitotic cycle of the myxomycete Physarum polycephalum

A sensitive radioisotope dilution method was used to measure the S-adenosyl-L-methionine (AdoMet) content in macroplasmodia of the slime mold Physarum polycephalum during the mitotic cycle. The AdoMet pool had two maxima, one during mitosis, the other in the middle of G2 phase.     

A characterization of ribonucleic acid in the myxomycete Physarum polycephalum

This report deals with the quantitative extraction of total nucleic acid (TNA) containing undegraded RNA from the slime mold Physarum polycephalum. With the use of a three-step phenol extraction technique, approx. 95 % of the nucleic acid optical density and 90 % of the ³H-uridine incorporated radioactivity were routinely recovered in the extracts. With the use of this technique it was shown that (1) the TNA mg dry wt of the mold did not change throughout the mitotic cycle, even though the dry wt doubled; this indicates a continual net synthesis of nucleic acid throughout the cycle; (2) the relative proportions of the various nucleic acid components did not change significantly during the cycle and were found to be DNA, 6 %; rRNA, 82 %; and sRNA, 12 %; (3) RNA molecules with mol wts of 4.1 m and 1.9 m, which exhibit properties of rRNA precursors were found in plasmodia labeled for 20 min with ³H-uridine. Furthermore, there appears to be an RNA fraction, found only in nucleic acid preparations presumably enriched in nuclear RNA components, which is heat-labile, does not enter 2.6 % acrylamide gels during 4 h of electrophoresis, and has a uridine/methyl ratio different from the presumed rRNA precursors and mature rRNA.     

Sterol content of the Myxomycetes Physarum polycephalum and P. flavicomum.

The sterol content of two Myxomycetes, Physarum polycephalum and P. flavicomum has been examined. The sterols of the two species are apparently identical, the two major sterols in each being poriferasterol and 22-dihydroporiferasterol. Threee minor sterols are probably delta5-ergostenol, ergostanol, and poriferastanol. The triterpenoids of the two species differ in that, though lanosterol was identified in both, 22-dihydrolanosterol was indicated only in P. flavicomum. The occurrence of lanosterol together with a typical mixture of plant sterols is somewhat unusual.     

Plasmodial ultrastructure of the myxomycete Physarum polycephalum

When plasmodia of P. polyeephalutu are fixed simultaneously with osmium tetroxide and glutaraldchyde, the cytoplasm is so preserved that a system of microchannels, resembling pinocytosis channels, and numerous discrete vacuoles can be observed. With either fixative alone, the cytoplasm appears to contain large irregular vacuoles or a vacuolar continuum. The microehannels, approximately l µ in diameter, arising as invaginations of the plasma membrane are each surrounded by a sheath of thin filaments which in areas of invagination at the plasmodial surface seems to merge with a well-defined cortex underlying the plasma membrane. Coating the plasma membrane is a structured slime layer continuous with the microchannel contents. The location and structure of the microchannel-cortex system strongly suggests it as the site for localization of contractile function implicated in cyclosis and motility. Mitochondrial structures of special interest are also described.     

Growth of Physarum flavicomum and Physarum rigidum in Chemically Defined Minimal Media

Physarum flavicomum, P. polycephalum, and P. rigidum grew at pH 4.2 in a medium composed of mineral salts, glucose, biotin, thiamine, hematin, and four amino acids. Important differences in pH tolerance were noted among the species. The minimal medium of P. flavicomum and P. polycephalum contained the amino acids methionine, glycine, and arginine, but valine was also required by P. rigidum. Starting with an inoculum of about 0.3 mg of protein per 25 ml of minimal medium, P. flavicomum and P. polycephalum grew to 23 mg and P. rigidum to 12 mg of protein per 25 ml in 3, 2, and 4 weeks, respectively. P. flavicomum and P. polycephalum grew with valine or leucine replacing arginine in the minimal medium but the growth yields and growth rates were decreased. All three species utilized homocysteine thiolactone in the minimal media in place of methionine. Serine adequately replaced glycine for P. rigidum but was inhibitory in the minimal medium of P. flavicomum or P. polycephalum unless homocysteine thiolactone also replaced methionine. Growth rates of all three organisms were increased in the presence of seven amino acids (original four plus leucine, lysine, and isoleucine).     

Amino acid metabolism during flight in tsetse flies.

Experiments carried out using teneral Glossina pallidipes indicate that flight can continue for at least 4 to 7 min after the thoracic proline reserves have fallen to low levels, suggesting that some other energy source is available. Earlier work suggests that alanine formed during flight is transported from the thorax to the abdomen where proline is resynthesized. Injection experiments using ¹⁴C alanine confirm that the transport mechanism does occur, that it is enhanced by flight, and that alanine is more rapidly incorporated into glutamate and proline in the abdomen than in the thorax. An analysis of published work shows that there is evidence for the involvement of residual blood meal amino acids even in the early stages of flight and supports the suggestion that they are of importance in prolonging flight. A decline in amino nitrogen during the early stages of flight is consistent with the action of glutamate dehydrogenase at this time. The poor flight durations in teneral flies may be due both to the low proline levels and to the absence of the residual blood meal. Very high energy consumptions are noted and appear to be related to the abnormally large musculature necessary for the fly's haematophagous and viviparous habits.