Lipid composition and lipid metabolism of Spiroplasma citri.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth of S. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S citri was shown to grow best on cholesterol and beta-sitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.     

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium

Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa₃ as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa₃ and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.     

Nutritional requirements of anaerobic coryneforms.

The nutritional requirements of three species of anaerobic coryneforms and their serotypes (Propionibacterium acnes types I and II, P. avidum types I and II, and P. granulosum) were determined. Strains of P. avidum would consistently grow to a transmittance of 1 to 3% at 560 nm in a basal salts medium supplemented with glucose, pantothenate, biotin, thiamine, and 12 amino acids (alanine, arginine, cysteine, glutamine, glycine, histidine, isoleucine, methionine, phenylalanine, serine, tyrosine, and tryptophan). Strains of P. acnes and P. granulosum, however, failed to grow in this medium unless six additional amino acids were present (asparagine, leucine, lysine, proline, threonine, and valine). All three species grew equally well whether the 18 amino acids were supplied in the form of a casein hydrolysate supplemented with tryptophan or were added separately. Nicotinamide enhanced growth of P. acnes but had no effect on growth of P. avidum and P. granulosum. Other nutrients which were not absolute requirements, but which significantly improved growth of these species, included the purines guanine and/or adenine, Tween 80, which served as a source of oleic acid, sodium L-lactate, alpha-ketoglutarate, and pyruvate. Strains (86) comprising all five groups grew well in the defined medium, except four strains of P. acnes type II (29 tested), which failed to grow unless heme and vitamin K were added to the medium. One strain of P. granulosum (22 tested) failed to grow in any defined medium, suggesting an additional growth factor requirement.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

Liposomes replace serum for cultivation of fermenting mycoplasmas

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.     

Liposomes replace serum for cultivation of fermenting mycoplasmas.

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.     

Comparison of glycoproteins from ten human Mycoplasma species.

Multiple bands of glycoprotein, rare in procaryotes, were detected in ten human Mycoplasma species by staining with periodic acid-Schiff (PAS) reagent after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A major contaminant formed in Hayflick medium (H medium), corresponding to an apparent molecular weight of about 80 kD, was eliminated by using the organisms grown in PPLO broth supplemented with PPLO serum fraction (P medium), except that M. genitalium and M. pneumoniae were grown in H medium as monolayers on the glass surface. The comparison of glycoproteins among ten human Mycoplasma species indicated that their profiles were shown to be species-specific. However, those of M. buccale and M. faucium were very similar, and M. pneumoniae and M. genitalium seemed to be related.     

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

An Analysis of the Nutritional Requirements for Fatty Acids of Paramecium aurelia

SYNOPSIS. Axenically cultivated Paramecium aurelia , stock 299, required a fatty acid for growth. This need was satisfied by oleic acid and oleic acid-containing lipids. These included: TEM-4T (tartaric acid esters of tallow monoglycerides), certain phospholipids (crude as well as highly purified preparations), Tween 80, 85, Span 80 and glyceryl monoleate. High concentrations of oleic acid in the medium inhibited growth. This inhibition was partially released or annulled by certain mixtures of "non essential" fatty acids or by increasing the stigmasterol content of the growth medium. Definite but non-stoichiometric levels of oleic acid and sterol were required for optimal growth. Tween 60, a non-ionic emulsifier similar in its surfactant properties to Tween 80, stimulated growth in the presence of suboptimal amounts of oleic acid but failed itself to replace oleic acid as a growth requirement.     

Cultivation of mycoplasmas in a modified tissue culture medium.

A new medium, which contained a chemically defined tissue culture base ("medium 199"), was developed for the cultivation of mycoplasmas. When supplemented with albumin, glucose, serum, and yeast extract, the new medium adequately supported the growth of Mycoplasma and Acholeplasma species.     

Cultivation of mycoplasmas in a modified tissue culture medium.

A new medium, which contained a chemically defined tissue culture base ("medium 199"), was developed for the cultivation of mycoplasmas. When supplemented with albumin, glucose, serum, and yeast extract, the new medium adequately supported the growth of Mycoplasma and Acholeplasma species.     

Growth and cholesterol oxidation by Mycobacterium species in Tween 80 medium.

Mycobacterium strain DP was isolated from marine coastal sediment and tested for its ability to oxidize cholesterol in Tween 80-cholesterol (2.59 mM) medium. Strain DP degraded cholesterol to 4-cholesten-3-one (cholestenone), 4-androsten-3,17-dione (AD), 1,4-androstadien-3,17-dione (ADD), testosterone, and 1-dehydrotestosterone (DHT). Cholesterol disappeared in about 4 days. Cholestenone, AD, testosterone, and DHT accumulations were transient with peak concentrations of 300, 600, 30 to 40, and 21 microM. ADD production peaked after 6 days with a concentration of 1,100 microM. Peak ADD concentrations and production rates compared well with those reported for strain NRRL B3683 on cyclodextrin medium. Tween 80 medium was superior to finely dispersed cholesterol particles for both strains. In comparison, NRRL B3683 (patented for its ability to accumulate AD and ADD) on Tween 80 medium transiently accumulated more AD (approximately 1,000 microM) than did strain DP, but ADD accumulations (200 microM) were significantly lower than those for strain DP. Strain DP could be adapted to grow on ADD, which was initially inhibitory at 3.25 mM. ADD-adapted strain DP cultures produced approximately four times as much DHT from ADD than unadapted cultures did from cholesterol, showing that additional manipulation might enhance testosterone production. We believe that ADD toxicity might account for the low ADD accumulations by NRRL B3683 in Tween 80 medium.     

Chlamydospore Production and Germ-Tube Formation by Auxotrophs of Candida albicans

A prototrophic strain and 21 auxotrophic strains of Candida albicans were assessed for their capacity to produce chlamydospores and germ tubes. All of the mutants were able to produce germ-tubes in human serum but only two mutants produced them in defined medium with L-alpha-amino-n-butyric acid as the sole source of nitrogen. Most auxotrophs were not able to produce chlamydospores on corn meal agar with 1% Tween 80, but they could be induced to do so if the medium was supplemented with their growth requirement(s). Although L-cysteine was able to support the growth of two methionine mutants, it did not support chlamydospore formation when added to corn meal agar with 1% Tween 80. Mutants of C. albicans that do not form chlamydospores could be incorrectly identified in laboratories that rely on chlamydospore formation for identification.     

Autotrophic Growth of Thiobacillus acidophilus in the Presence of a Surface-Active Agent, Tween 80

Cellular protein, pH, dissolved oxygen concentration, and static surface tension were measured during growth of Thiobacillus acidophilus on elemental sulfur in the absence and presence of up to 5,000 mg of Tween 80 per liter. The decrease in pH and the increase in sulfate production were observed to be less accurate measurements of growth when compared with the increase in cellular protein. The doubling time of the bacterium decreased approximately 50% with the addition of 500 mg of Tween 80 per liter. The bacteria did not appear to synthesize any wetting agents as demonstrated by the constant surface tension of the medium during growth. Morphological alterations in the presence of Tween 80 were also observed.     

Determination of ligninase activity in P. chrysosporium pellets with diffuse reflectance spectrophotometry

Diffuse reflectance spectrophotometry was applied for measuring ligninase activity in pellets of Phanerochaete chrysosporium. Enhanced ligninase activity in pellets and in growth medium were detected in cultures supplemented with oleic acid emulsified with Tween 80, Tween 80, hydrophilic (alcoholic) residue of Tween 80 hydrolysate, Tween 20, and (15OE)C_18:1. In cultures with low extracellular ligninase activity, low activity in pellets was also observed. Our results indicate that the stimulatory effect of the tested surfactants cannot be contributed solely to promotion of ligninases through the cell membrane. Correspondence to: D. Letan     

Leptospiral selection, growth, and virulence in synthetic medium

Stalheim, O. H. V. (National Animal Disease Laboratory, Ames, Iowa). Leptospiral selection, growth, and virulence in synthetic medium. J. Bacteriol. 92:946-951. 1966.-The need for protein in leptospiral cultural medium may be circumvented by the use of strains which tolerate the lytic activity of polyoxyethylene sorbitan monooleate (Tween 80), a relatively nonlytic source of essential fatty acids. In an otherwise adequate medium, the primary function of a serum protein (bovine albumin fraction V) in the cultivation of Leptospira pomona was detoxification of fatty acids. Treatment to destroy or block end groups (amino, sulfhydryl, or hydroxyl) did not impair this function, but, after treatment with trypsin, albumin was inactive. Synthetic and derived peptides or polyvinylpyrrolidone did not substitute for albumin. L. pomona grew in medium with surface tension values of 44 to 58 dynes/cm(2); after growth, the values were increased slightly (5 to 8). The growth responses did not correlate with the surface tension of the medium, but they were in proportion to the concentration of Tween 80. Of six strains of L. pomona, five were transferred from medium containing rabbit serum and were subcultured in Tween synthetic medium (TSM) containing low, nonlytic concentrations (0.002%) of Tween 80. The poor antigenicity of L. pomona in carbon-limited TSM was associated with a deficiency of those carbonaceous cellular components which were extractable with 50% ethyl alcohol. After as few as four subcultures in TSM, L. pomona tolerated higher concentrations of Tween 80 (0.06% was optimal; MTSM). If grown on a shaker, the rate and amount of growth and the antigenicity of L. pomona in MTSM equaled that in medium supplemented with rabbit serum. After cultivation in MTSM, all of the five strains were avirulent when administered to hamsters, guinea pigs, and swine. They were still avirulent after three subcultures in complex media or after two serial passages in hamsters.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp.

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Control of membrane lipids in Mycoplasma gallisepticum: effect on lipid order.

Adaptation of Mycoplasma gallisepticum, a sterol-requiring Mycoplasma sp., to growth in a serum-free medium supplemented with cholesterol in decreasing concentrations and with various saturated or unsaturated fatty acids enabled us to control both the cholesterol levels and the membrane fatty acid composition. An estimate of the membrane physical state from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane lipids of native M. gallisepticum were highly ordered. Elongation of the saturated fatty acid chains from 14 to 18 carbon atoms caused only a small increase in the membrane lipid ordering, whereas the introduction of a cis double bond reduced it significantly. Lipid-phase transitions were observed in low-cholesterol-adapted organisms, whose membrane lipids were still highly ordered at the growth temperature.