Phospholipid composition of methane-utilizing bacteria.

The phospholipids of Methylococcus capsulatus, Methylosinus trichosporium, La Paz, and OBT were examined in relation to their qualitative and quantitative composition. M. capsulatus exhibited a phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and phosphatidyl-choline. The esterified fatty acids were predominantly C16:0 and C16:1. M. trichosporium, La Paz, and OBT exhibited an essentially identical phospholipid composition consisting of phosphatidylmonomethylethanolamine, phosphatidyl-dimethylethanolamine, phosphatidylcholine, and phosphatidylglycerol. Only trace amounts (less than 1%) of cardiolipin were found in these organisms. The major esterified fatty acid in these organisms was C18:1 (87 to 90%). The monounsaturated fatty acids from all four organisms consisted of both cis and trans isomers, each of which contained delta8, delta9, delta10, and delta11 double-bond positional isomers.     

The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath)

Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.     

Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis

A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta 8, delta 10, and delta 11. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol [11.0% 4 alpha-methyl-cholesta-8(14),24-dien-3 beta-ol] was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3 beta-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4% for total tissue, -60.6 and -62.4% for total lipids, -60.2 and-63.9% for phospholipid fatty acids, and -71.8 and 73.8% for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria (R.E. Summons, L.L. Jahnke, and Z. Roksandic, Geochim. Cosmochim. Acta 58: 2853-2863, 1994) further supporting the conversion of the bacteria methylsterol pool.     

Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis.

A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta 8, delta 10, and delta 11. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol [11.0% 4 alpha-methyl-cholesta-8(14),24-dien-3 beta-ol] was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3 beta-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4% for total tissue, -60.6 and -62.4% for total lipids, -60.2 and-63.9% for phospholipid fatty acids, and -71.8 and 73.8% for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria (R.E. Summons, L.L. Jahnke, and Z. Roksandic, Geochim. Cosmochim. Acta 58: 2853-2863, 1994) further supporting the conversion of the bacteria methylsterol pool.     

The effect of dietary sterol on the activity of fatty acid desaturases isolated from Tetrahymena setosa

Tetrahymena setosa has a nutritional requirement for micro amounts of sterol, a requirement which is also satisfied by relatively large amounts of either intact phospholipids or a mixture of unsaturated fatty acids normally found in these ciliates. Three microsomal fatty acyl-CoA desaturases have been isolated from T. setosa and partially characterized. These enzymes which can account for the formation of the majority of the ciliate's unsaturated fatty acids, include: a delta 9, a delta 12 and a delta 6 desaturase which catalyze the transformation of stearoyl-CoA to oleic acid, of oleoyl-CoA to linoleic acid and of linoleoyl-CoA to gamma-linolenic acid, respectively. The stearoyl CoA desaturase required NAD (or NADP), ATP and free CoA; the delta 6 and delta 12 desaturases required NADP, but not ATP or CoA. Cellular levels of the three desaturases were highest in mid-logarithmic phase cells and lowest in stationary phase cells. In order to determine if there was a relationship between the sterol requirement and the ability of the organism to desaturate, T. setosa was grown in a synthetic medium supplemented with either cholesterol or a phospholipid which permits growth in the absence of cholesterol, or with both phospholipid and cholesterol. Cells grown with phospholipid alone had only half as much stearoyl-CoA and oleoyl-CoA desaturase activity as cells of identical culture age grown either on cholesterol alone or on cholesterol plus phospholipid.     

Rhodopseudomonas acidophila strain 10050 contains photosynthetic LH2 antenna complexes that are not enriched with phosphatidylglycerol,and the phospholipids have a fatty acyl composition that is unusual for purple non-sulfur bacteria

The phospholipid composition of Rhodopseudomonas acidophila strain 10050 grown aerobically or anaerobically in the light was determined. The major phospholipids present in the aerobic cells were phosphatidylethanolamine (PE; 54%), phosphatidylglycerol (PG; 24%) and cardiolipin (diphosphatidylglycerol, DPG) (14%), together with phosphatidylcholine (PC; 5%). On moving the cells to anaerobic photosynthetic growth in the light PE remained the major phospholipid (37-49%), but there was a major change in the proportion of PC, which increased to 31-33%, and corresponding reductions in the contents of PG to 11-16% and DPG to 4-5%. The fatty acid composition of the phospholipids was unusual, compared with other purple non-sulfur photosynthetic bacteria, in that it contained 16:0 (29%), 17:1 (20%) and 19:1 (9%) plus several mainly unsaturated 2-OH fatty acids (9% total) as major components, when grown aerobically in the dark. In contrast when grown photosynthetically under anaerobic conditions there was <2% 17:1 or 19:1 present, while the amounts of 16:1 and 18:1 increased, and 16:0 decreased. The phospholipid composition of the purified light-harvesting complex 2 (LH2) complex was PE (43%), PC (42%) and DPG (15%). Unexpectedly, there was no PG associated with the purified LH2. These findings contrast with previous studies on several other photosynthetic bacteria, which had shown an increase in PG upon photosynthetic growth [Biochem. J. 181 (1979) 339]. The prior hypothesis that phosphatidylglycerol has some specific role to play in the function of light-harvesting complexes cannot be true for Rps. acidophila. It is suggested that specific integral membrane proteins may strongly influence the phospholipid content of the host membranes into which they are inserted.     

The lipid composition and permeability to the triazole antifungal antibiotic ICI 153066 of serum-grown mycelial cultures of Candida albicans

The total lipid content of Candida albicans (serotype A: NCPF 3153) exponential-phase mycelial cultures grown in tissue-culture medium 199 (containing 10%, v/v, foetal calf serum) was 29.8 +/- 8 mg (g dry weight)-1 (mean +/- SD). The weight ratios of phospholipid to neutral lipid and phospholipid to non-esterified sterol were 2.6 +/- 0.4 and 24.9 +/- 0.5, respectively. The major phospholipid was phosphatidylcholine with smaller amounts of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and diphosphatidylglycerol; the most abundant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. The major neutral lipids comprised esterified sterol, triacylglycerol and non-esterified fatty acid with a smaller amount of non-esterified sterol. The fatty acid compositions of the three fatty-acid-containing neutral lipids were distinct from each other and the phospholipids. Comparison with previous data on yeast cultures of C. albicans A grown in glucose broth shows that mycelial cultures have a larger lipid content, lower phospholipid to neutral lipid ratio and higher phospholipid to non-esterified sterol ratio. We now show that mycelial cultures were more permeable to a [14C]triazole antifungal antibiotic compared with exponentially growing yeast cultures of several azole-sensitive strains. Taken together these data are consistent with there being a relationship between the phospholipid/non-esterified sterol ratio of a culture and its ability to accumulate a triazole.     

The lipid composition of Acer pseudoplatanus cells grown in culture

Cells of Acer pseudoplatanus were grown in batch suspension culture for 22 days. The cultures were initiated at high cell density of 2 × 10⁵ cells per ml of culture. Growth was characterised by a short lag phase, an exponential phase of rapid cell division and growth, and finally a stationary phase. Quantitative but not qualitative changes were observed in total lipid content, fatty acids and phospholipids at different stages of growth. Total lipids, phospholipids and fatty acids showed maximum concentrations in 12 day old cells. The major phospholipids isolated were phosphatidylcholine and phosphatidylethanolamine with minor amounts of phosphatidic acid and lysophosphatides. Other lipid components present were mono- and digalactosyl diglycerides, cerebrosides, sterol glucosides, free fatty acids and esterified sterol glucosides. The major constituent fatty acids were myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3). During exponential cell growth the proportion of 16:0, 18:2 and 18:3 constituted nearly 90% of the total fatty acids. Triglycerides were the major repository of myristic acid (14:0) with substantial amounts of palmitic acid (16:0), whereas phospholipids contained 16:0, 18:2 and 18:3 in high amounts.     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The air content in three types of propagation media, Jiffy-7 and Jiffy-9 which are Sphagnum peat and Grodan which is rockwool, were investigated when they were held at moisture tensions of 0,6 and 12 cm measured from the base of the media. At 0 cm tension the air content (vol. %) was highest in Jiffy-9 and lowest in Jiffy-7. At 12 cm tension the air content was higher in Grodan than in Jiffy-9 and Jiffy-7. Oxygen diffusion coefficients (ODC) and oxygen diffusion rates (ODR) were measured at the different air contents. At air contents below 20 vol. % ODC was about the same for Jiffy-9 and Grodan but at air contents above 20 vol.% it was larger for Jiffy-9 than for Grodan. The oxygen diffusion rate was measured at 0, 4 and 8 cm moisture tension. At all tensions it was approximately 20% higher in Jiffy-9 than in Grodan and Jiffy-7. The ODR in Jiffy-7 and Grodan were affected equally at the same tension, although Grodan contained more air. Report no 253     

Speculations on the evolution of sterol structure and function.

The essential oxygen requirement for sterol biosynthesis dates this molecule as a relative latecomer in cellular evolution. Structural details of the cholesterol molecule and related sterols can be rationalized in terms of optimal hydrophobic interactions between the planar sterol ring system and phospholipid acyl chains in the membrane bilayer. The prediction that the cholesterol precursor lanosterol (4,4',14 trimethyl cholastadienol) is incompetent for membrane function is verified by in vivo experiments with eucaryotic sterol auxotrophs and microviscosity measurements of sterol-containing artificial membranes. For procaryotic cells the sterol specificity is very much broader. Methylococcus capsulatus produces 4,4-dimethyl- and 4-monomethyl sterols, but not sterols of the cholesterol type. Similarly lanosterol and its partially demethylated derivatives satisfy the sterol requirement of Mycoplasma capricolum. A more primitive but unspecified role of cyclized squalene derivatives is therefore postulated for procaryotic membranes. The finding that cholesterylmethyl ether satisfies the sterol requirement of certain microbial systems is at variance with current views on the role played by the sterol hydroxyl group in membrane organization and function.     

Modulation of yeast plasma membrane composition of a yeast sterol auxotroph as a function of exogenous sterol

Plasma membranes isolated from a yeast sterol auxotroph (RD5-R) grown on 1, 5, and 15 micrograms ml-1 exogenous concentrations of sterol showed no discontinuity in plots of steady-state fluorescence anisotropy. Liposomes constructed from phospholipid and sterol extracted from RD5-R grown on different sterols indicated that exogenously supplied sterol modulated cellular phospholipids such that lipid-phase transitions were avoided. Liposomes derived from sterol and phospholipid extracted from the same culture exhibited no lipid-phase transitions. However, when phospholipid extracted from a culture grown on a specific sterol was mixed with sterol extracted from a heterologous culture grown on a different sterol to form liposomes, discontinuities were detected in the anisotropy measurements of the liposomes produced. Quantitative analyses revealed that the exogenously supplied sterol coordinately regulated specific phospholipid species, fatty acid composition, and sterol to phospholipid ratios in yeast auxotrophs.     

Identification of the PufQ protein in membranes of Rhodobacter capsulatus.

The PufQ protein has been detected in vivo for the first time by Western blot (immunoblot) analyses of the chromatophore membranes of Rhodobacter capsulatus. The PufQ protein was not visible in Western blots of membranes of a mutant (delta RC6) lacking the puf operon but appeared in membranes of the same mutant to which the pufQ gene had been added in trans. It was also detected in elevated amounts in a mutant (CB1200) defective in two bch genes and unable, therefore, to make bacteriochlorophyll. The extremely hydrophobic nature of the PufQ protein was also apparent in these studies since it was not extracted from chromatophores by 3% (wt/vol) n-octyl-beta-D-glucopyranoside, a procedure which solubilized the reaction center and light-harvesting complexes. During adaptation of R. capsulatus from aerobic to semiaerobic growth conditions (during which time the synthesis of bacteriochlorophyll was induced), the PufQ protein was observed to increase to the level of detection in the developing chromatophore fraction approximately 3 h after the start of the adaptation. The enzyme, S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase, also increased in amount in the developing chromatophore fraction but was present in a cell membrane fraction at the start of the adaptation as well.     

Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.

The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.     

Fatty acid composition of diacyl, alkylacyl, and alkenylacyl phospholipids of control and arachidonate-depleted rat polymorphonuclear leukocytes

Phospholipid fatty acid composition and phospholipid subclass distribution of control and arachidonate-depleted rat polymorphonuclear leukocytes (PMN) were compared. The 20:4-depleted PMN contained significantly higher amounts of 16:1, 18:1 and 20:3 (delta 5,8,11) and lower amounts of 18:2 and 20:4 than the phospholipids from control cells. Choline-containing glycerophospholipids (CGP) were the major phospholipids of both control and 20:4-depleted cells representing 34% and 37% of the total phospholipids, respectively. Significant amounts of ethanolamine-containing glycerophospholipids (EGP) (29% and 30%) and sphingolipids (20% and 18%) were also present in both cell types. Serine-containing glycerophospholipids (SGP) together with inositol-containing glycerophospholipids (IGP) constituted 16% and 13% of the phospholipids in control and 20:4-depleted cells, respectively. CGP from control cells had significantly higher amounts of 16:0 and 18:2 and lower amounts of 18:0 and 20:4 than EGP, whereas CGP from 20:4-depleted cells has higher amounts of 16:0 and 16:1 and lower amounts of 20:3 than EGP. Analysis of the subclass composition of CGP and EGP revealed that both control and 20:4-depleted cells contained significantly large amounts of alkylacyl-GPC and alkenylacyl-GPE. Small amounts of alkylacyl-GPE and alkenylacyl-GPC were also observed. The predominant fatty acyl residues found in the 1,2-diacyl-GPC, alkylacyl-GPC of control cells were 16:0, 18:0, 18:1, 18:2, and 20:4, while those of 20:4-depleted cells were 16:0, 16:1, 18:0, 18:1, and 20:3. More than 60% of CGP-bound 20:4 of control cells and about 70% of the CGP-bound 20:3 of 20:4-depleted cells were found in their alkylacyl species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of copper on membrane lipids and on methane monooxygenase activity of Methylococcus capsulatus (Bath)

The effect of copper supplementation on growth, methane monooxygenase activity and lipid composition of Methylococcus capsulatus (Bath) was studied. Copper increased biomass yield, methane monooxygenase activity and phospholipid content from 7.7 to 10.2% of dry weight. Cells from copper-deficient and copper supplemented cultures contained the same major fatty acids but in the presence of copper only the contents of C16:0 and the three C16:1 isomers were increased while the contents of C14:0 and cyclic C17:0 remained unchanged. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol and cardiolipin were analysed amongst the polar lipids. PE was the main component (about 60 mol-%) but the most notable copper-induced increment occurred in the proportion of PC, from about 10 to 16 mol-%. Concomitantly with this increment the fatty acids of PC were changed so that the mol-% of C16: 1 isomers were increased at the expense of other acids. Similar trends were seen also in the fatty acid compositions of other polar lipid fractions. It is therefore concluded that phosphatidylcholine would be one of the key factors when the role of membranous lipids in methane monooxygenase activity is to be considered.     

The Effect of Dietary Sterol on the Activity of Fatty Acid Desaturases Isolated from Tetrahymena setosa

Tetrahymena setosa has a nutritional requirement for micro amounts of sterol, a requirement which is also satisfied by relatively large amounts of either intact phospholipids or a mixture of unsaturated fatty acids normally found in these ciliates. Three microsomal fatty acyl-CoA desaturases have been isolated from T. setosa and partially characterized. These enzymes which can account for the formation of the majority of the ciliate's unsaturated fatty acids, include: a Δ9, a Δ12 and a Δ6 desaturase which catalyze the transformation of stearoyl-CoA to oleic acid, of oleoyl-CoA to linoleic acid and of linoleoyl-CoA to ?-linolenic acid, respectively. The stearoyl CoA desaturase required NAD (or NADP), ATP and free CoA; the Δ6 and Δ12 desaturases required NADP, but not ATP or CoA. Cellular levels of the three desaturases were highest in mid-logarithmic phase cells and lowest in stationary phase cells. In order to determine if there was a relationship between the sterol requirement and the ability of the organism to desaturate, T. setosa was grown in a synthetic medium supplemented with either cholesterol or a phospholipid which permits growth in the absence of cholesterol, or with both phospholipid and cholesterol. Cells grown with phospholipid alone had only half as much stearoyl-CoA and oleoyl-CoA desaturase activity as cells of identical culture age grown either on cholesterol alone or on cholesterol plus phospholipid.     

Fatty acid desaturase activities are modulated by phytosterol incorporation in microsomes

The effect of phytosterol-rich diets (3% beta-sitosterol + 2% campesterol) on rat liver microsomal fatty acid desaturases, membrane dynamics and lipid composition was investigated. After a 21 day period, phytosterol was incorporated into microsomes and the membrane fluidity decreased. There were no changes in either the phospholipid composition or in the total sterol content. However, the phytosterol/cholesterol ratio increased. In the animals fed phytosterols, the delta 5-, delta 6- and delta 9-fatty acid desaturases were significantly more active than in control animals. The changes in the lipid fatty acid composition were consistent with those of the desaturase activities. Hence, it is suggested that: (1) dietary phytosterol modulates desaturase activities; (2) phytosterols make the membrane more rigid but do not induce changes in the relative phospholipid composition; (3) delta 9-, delta 5- and delta 6-desaturase activities increase when the membrane becomes more rigid without changes in the phospholipid composition.     

Lipids of the antarctic sea ice diatom Nitzschia cylindrus

The sterol and neutral, glyco- and phospholipid fatty acid profiles of the sea ice diatom Nitzschia cylindrus, isolated from McMurdo Sound, Antarctica, are reported. Two sterols were detected, trans-22-dehydrocholesterol (66% of total sterols) and cholesterol (34%); no sterols containing alkyl groups at the C₂₄ position were present. The major fatty acids in N. cylindrus, 16:1Δ9c, 14:0, 16:0, 20:5Δ5,8,11,14,17 and 20:4Δ5,8,11,14, were typical of previous reports of diatom fatty acids. A number of long-chain monounsaturated fatty acids were also detected, with higher relative proportions present in the phospholipid fraction. GC-MS analysis of the dimethyldisulphide adducts of these monounsaturated components showed that 24: 1Δ13c, 24:1Δ15c, 26:1Δ15c and 26:1Δ17c were the major components. The distribution of these fatty acids suggests that chain elongation of monounsaturated fatty acids was occurring in N. cylindrus. The proposed chain lengthening occurring for N. cylindrus represents, to our knowledge, the first report of possible chain lengthening of monounsaturated fatty acids in microscopic algae. These features, the presence of long-chain monounsaturated fatty acids and the sterol profile, may allow the input of this alga into benthic marine sediments or food webs to be monitored.     

Lipid Content and Cryotolerance of Bakers' Yeast in Frozen Doughs

The relationship between lipid content and tolerance to freezing at −50°C was studied in Saccharomyces cerevisiae grown under batch or fed-batch mode and various aeration and temperature conditions. A higher free-sterol-to-phospholipid ratio as well as higher free sterol and phospholipid contents correlated with the superior cryoresistance in dough or in water of the fed-batch-grown compared with the batch-grown cells. For both growth modes, the presence of excess dissolved oxygen in the culture medium greatly improved yeast cryoresistance and trehalose content (P. Gélinas, G. Fiset, A. LeDuy, and J. Goulet, Appl. Environ. Microbiol. 26:2453-2459, 1989) without significantly changing the lipid profile. Under the batch or fed-batch modes, no correlation was found between the cryotolerance of bakers' yeast and the total cellular lipid content, the total sterol content, the phospholipid unsaturation index, the phosphate or crude protein content, or the yeast cell morphology (volume and roundness).     

Intracytoplasmic membrane, phospholipid, and sterol content of Methylobacterium organophilum cells grown under different conditions.

Intracytoplasmic membranes were present in Methylobacterium organophilum when cells were grown with methane, but not methanol or glucose, as the sole carbon and energy source. Cells grown with methane as the carbon and energy source and low levels of dissolved oxygen had the greatest amount of intracytoplasmic membrane. Cells grown with increased levels of dissolved oxygen had less intracytoplasmic membrane. The amount of total lipid correlated with the amount of membrane material observed in thin sections. The individual phospholipids varied in amount, but the same four were present in M. organophilum grown with different substrates and oxygen levels. Phosphatidyl choline was present as a major component of the phospholipids. Sterols were present, and they differed from those in the type I methylotroph Methylococcus capsulatus. The relative amounts of different sterols and squalene changed with the substrate provided for growth. The greatest amounts of sterols were found in methane-grown cells grown at low levels of dissolved oxygen. None of the unusual or usual membrane components assayed was uniquely present in the intracytoplasmic membranes.