Vol. 134, No. 2JOUJRNAL OF BACTERIOLOGY, May 1978, p. 636-6440021-9193/78/0134-0636$02.00/0Copyright © 1978 American Society for Microbiology

Intracytoplasmic Membrane, Phospholipid, and SterolContent of Methylobacterium organophilum Cells Grown

Under Different ConditionsT. E. PATTt AND R. S. HANSON*

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 8 February 1978

Intracytoplasmic membranes were present in Methylobacterium organophilumwhen cells were grown with methane, but not methanol or glucose, as the solecarbon and energy source. Cells grown with methane as the carbon and energysource and low levels of dissolved oxygen had the greatest amount of intracyto-plasmic membrane. Cells grown with increased levels of dissolved oxygen had lessintracytoplasmic membrane. The amount of total lipid correlated with the amountof membrane material observed in thin sections. The individual phospholipidsvaried in amount, but the same four were present in M. organophilum grownwith different substrates and oxygen levels. Phosphatidyl choline was present asa major component of the phospholipids. Sterols were present, and they differedfrom those in the type I methylotroph Methylococcus capsulatus. The relativeamounts of different sterols and squalene changed with the substrate providedfor growth. The greatest amounts of sterols were found in methane-grown cellsgrown at low levels of dissolved oxygen. None of the unusual or usual membranecomponents assayed was uniquely present in the intracytoplasmic membranes.

Until recently, only three or four well-definedbacterial species capable of growing on methaneas a carbon and energy source had been isolated(30, 33). In 1970, Whittenbury et al. (47), re-ported the isolation of over 100 strains of meth-ane-utilizing bacteria. The isolates of Whitten-bury and colleagues, as well as the previousisolates, have all had an obligate requirementfor methane, methanol, or dimethylether as thegrowth substrate. All of these methane-utilizingbacteria so far examined possess elaborate inter-nal membrane structures when grown on meth-ane (9, 10, 29, 31, 38, 39, 46). When methaneutilizers are grown on methanol these internalmembranes are also present, but to a lesserextent (10, 38). Methylotrophs that are unableto utilize methane have been reported to bedevoid of intracytoplasmic membranes (30),with the exception of Hyphomicrobium species(8).The internal membranes are divided into two

types based on morphological features. Type Imembranes appear as bundles of dish-shapedmembrane vesicles, and type II consist of a

system of paired membranes usually concen-trated around the periphery of the cells (9).These intracytoplasmic membranes ofthe meth-ane oxidizers are similar in appearance in thin

t Present address: The Upjohn Company, Kalamazoo, MI49001.

sections to those found in the photosyntheticbacteria (22), the ammonia and nitrite oxidizers(21, 28), a methanogen (49), and the blue-greenalgae (15).The lipids of several of these bacteria contain-

ing intracytoplasmic membranes have beenidentified. Some are not commonly found inbacteria. Phosphatidyl choline, squalene, andsterols have been detected (5, 16).The isolation of the first well-documented

pure cultures of facultatively methylotrophic or-

ganisms that use methane as a substrate forgrowth has recently been reported (25). One ofthese facultative methane utilizers has sincebeen characterized, and the name Methylobac-terium organophilum has been proposed (26).When grown on methane, M. organophilum pos-sesses type II intracytoplasmic membranes iden-tical to those in the obligate methane-oxidizingbacteria (9, 25). These intracytoplasmic mem-branes could not be detected when M. organo-philum was grown on methanol or glucose (26,27).

MATERIALS AND METHODSMedia and growth conditions. The media used

were developed to optimize the growth yield after a

determination of cellular metal content of M. organ-

ophilum indicated that cell yields on methane mightbe limited by copper and iron (27). The mineral saltsmedium contained (per liter of distilled water): KNO:I,

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COMPOSITION OF M. ORGANOPHILUM CELLS 637

1 g; MgSO4 7H20, 0.2 g; CaCl2 2H20, 0.0265 g;Na2HPO4 7H20, 0.437 g; NaH2PO4 H20, 0.07 g;FeSO4 7H20, 50 mg; CuCl2 2H20, 13 mg; H3BO3, 10Lg; MnSO4 5H20, 10 1Lg; ZnSO4 7H20, 70 1Lg; andMoO3, 10 ,ug. The pH was adjusted to 6.8. Glucose,0.5% (wt/vol), and methanol, 0.5% (vol/vol), wereadded as indicated. Methane-grown cells were grownas previously described (25). Cells were grown withcontrolled levels of dissolved oxygen in a New Bruns-wick 14-liter fermentor fitted with a New Brunswickoxygen probe and dissolved oxygen controller to main-tain the desired level of dissolved oxygen. The oxygencontroller was set at 75% of saturation for culturesgrown at high levels of dissolved oxygen. The rate ofagitation was controlled to keep the dissolved oxygenconcentration in a range of 40 to 75% of saturation.The dissolved oxygen concentration was 75% of satu-ration after inoculation and decreased to 40% at highcell densities. For growth under low levels of dissolvedoxygen, the oxygen levels at the start of growth were8 to 10% of saturation and decreased to a reading of0% during exponential growth. Cells were harvested asthey entered the stationary phase of the growth cycle.

Microscopy. The procedures for fixation, embed-ding, and staining ofthin sections have been previouslydescribed (26).

Lipid extraction. Lipids were extracted by theprocedure of Bligh and Dyer (6). The homogenizationstep in their procedure was omitted because the cellswere readily lysed on suspension in the extractingsolvents.

Thick cell pastes contained 0.8 g of water per g (wetweight). Lyophilized cells were suspended in water ata ratio of 1 g of cells (dry weight) to 4 ml of waterbefore the appropriate amounts of methanol and chlo-roform were added. Freshly redistilled solvents wereused for all work. Only glass and Teflon materials werein contact with the solvents.

Nonlipid contaminants in this extract were removedby partition chromatography on Sephadex G25 asdescribed by Wuthier (48).Column chromatography. A slurry of 12 g of

acid-treated Florisil (Fisher Scientific, Pittsburgh, Pa.)in chloroform was poured into a 1-cm-diameter chro-matography tube. The washed, total extractable lipids(ca. 150 to 200 mg) were applied to the column inchloroform. The elution solvents were added at amaximum flow rate of 1 ml/min in the following order:60 ml of chloroform to elute the neutral lipids, 60 mlof chloroform-acetone (1:1, vol/vol) followed by 60 mlof acetone to elute the glycolipids, and 100 ml ofmethanol to elute the phospholipids (18). The glyco-lipid fraction was assayed for total phosphates todetermine whether any phospholipids were elutedwith the glycolipids. None could be detected.

Fractionation of the neutral lipid fraction was per-formed on a 2-g acid-treated Florisil column (1-cmdiameter) as described by Dittmer and Wells (12). Thehydrocarbon and sterol fractions were assayed by gaschromatography.Gas chromatography. Sterol and squalene sam-

ples were analyzed in a Packard-Becker model 421 gaschromatograph equipped with a hydrogen flame de-tector. The column used for most analyses was a glasstube (6 ft [ca. 1.83 m] by 2 mm) packed with 3% SP-

2100 on 80/100 Supelcoport (Supelco, Inc., Bellefort,Pa.). The column temperature was 235°C, the detectorwas at 270°C, and the injection port was set at 275°C.Carrier gas flow (nitrogen) was 25 ml/min. Silyl etherderivatives were prepared as previously described (26).Acetate derivatives of sterols were generated by mix-ing 1 volume of sterol sample dissolved in pyridinewith 5 volumes of acetic anhydride. This mixture washeated at 60°C for 1 h in a sealed container, dried ina stream of nitrogen, and suspended in pyridine.

Fractionation of the phospholipid fraction. Sil-ica gel G plates (Analtech, Inc., Newark, Del.) weredeveloped in acetone, heat activated for 30 min at110°C, cooled in a desiccator, and used the same day.The chromatography tanks were lined with filter pa-per wetted with the freshly prepared chromatographysolvent system to be used. A two-dimensional systemwas used. The solvents were chloroform-methanol-water (65:25:4, vol/vol/vol) and chloroform-methanol-7 N ammonia (60:35:5, vol/vol/vol) (2).The lipids were eluted from the silica gel plates for

the phosphate assays and for the hydrolysis to water-soluble products. The lipids were first visualized withiodine vapors, then sprayed with water to make trans-fer easier, scraped with a razor blade, and placed in aPasteur pipette that had a plug of glass wool in theconstricted end. The lipids were eluted from the silicicacid with 2 ml of chloroform-methanol (1:1, vol/vol),followed by 2 ml of methanol (2). The solvents wereremoved under a stream of nitrogen gas at a temper-ature under 40°C.

Mild alkaline hydrolysis. Phospholipids were de-acylated using the procedure of Kates (18). Chloro-form, methanol, and 1.25 N NaOH (1:4:4.5,vol/vol/vol) were added to partition the deacylatedphospholipids and the fatty acids. The methanol-wa-ter phase was neutralized with BioRad AG 50Wx8(H+) resin until it was neutral to slightly acidic toindicator paper. The supernatant fraction was thenremoved from the cation exchange resin. A drop ortwo of methanolic NH40H (10 ml of concentratedNH40H diluted to 100 ml with methanol) was addedto make the supernatant slightly alkaline. The super-natant was concentrated almost to dryness and thendissolved in methanol-water (10:9, vol/vol).The products of mild alkaline hydrolysis (deacyla-

tion) of the acyl phosphatides were water-soluble glyc-erol phosphate esters and a chloroform-soluble frac-tion of the fatty acids. The glycerol phosphate esterswere separated by paper chromatography ofWhatmanno. 1 paper using a solvent system of water-saturatedphenol-ethanol-glacial acetic acid (50:5:6, vol/vol/vol)(18).Acid hydrolysis. Phospholipids that had been sep-

arated by thin-layer chromatography were also sub-jected to acid hydrolysis. The conditions were 100°Cfor 4 h in 1 N- HCI in sealed glass ampoules. Thehydrolysates were extracted with hexane. The aqueousphase was collected and evaporated to dryness. Thefree bases were dissolved in water and were separatedby descending chromatography on Whatman no. 1paper in the solvent system used for the mild alkalinehydrolysis products (12).

Sterol isolation by digitonin precipitation. M.organophilum cells were saponified in a solution of

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50% methanol, 10% KOH, and 0.5% pyrogallol byrefluxing for 20 h (23). The nonsaponifiable fractionwas extracted into hexane. The hexane extract waswashed with water before drying in a rotary evapora-tor. The vacuum was broken by adding an atmosphereof nitrogen gas. At all stages a nitrogen atmospherewas used to prevent oxidation of sterols. The sterolswere dissolved in a known volume of absolute ethanol-acetone (1:1, vol/vol) and precipitated with an equalvolume of 0.5% digitonin in 50% ethanol (wt/vol). Theprecipitate was allowed to form overnight at 4°C, afterwhich time it was washed twice with acetone-diethylether (1:1, vol/vol). The acetone-diethyl ether wasdecanted, and final traces of solvents were removed ina stream of nitrogen. Dimethylsulfoxide (5 ml) wasadded, and the digitonide was heated for 30 min in anautoclave at 121°C to hydrolyze the digitonide (1).The free sterols were extracted into hexane, and thehexane was washed with water to remove the dimethylsulfoxide. The hexane and traces of water were re-moved under vacuum. The sterol residue was sus-pended in chloroform.

Analytical procedures. The following procedureswere used for the detection of lipids on thin-layerchromatography plates: iodine vapors, a saturated so-lution of K2Cr2O7 in 70% sulfuric acid followed bycharring at 180°C, or 0.05% Rhodamine 6G in 95%ethanol under UV light (36, 37). The iodine vaporswere also used for detection on the paper chromato-grams of mild alkaline and acid hydrolysis products ofphospholipids.Amino groups of phospholipids and their water-

soluble products were detected with ninhydrin spray(37).

For the detection of all phospholipids on silica gelplates, the molybdenum blue spray of Dittmer andLester was used (11).

Choline-containing phospholipids and water-solu-ble products containing choline were visualized by theDragendorff test (37).

Vicinal glycol groups were detected with the perio-date-Schiff reagent (37).

Water-soluble deacylated products of phospholipidson paper were detected by the sulfosalicylic acid-ferricchloride procedure as described by Vorbeck and Mar-inetti (43).

Phosphate determinations were carried out in acid-washed glassware by the procedure of Ames and Du-bin (2). Pyrex brand test tubes (13 by 100 mm) wereused. The standard and samples were diluted to therange of 0 to 5 Ag of phosphate and ashed withMg(NO3)2 6H20. Blanks of silica gel from areas ofplates without any phosphatides were used for phos-phate blanks.The Liebermann-Burchard assay for sterols was

performed as described by Stadtman (40). The Lie-bermann-Burchard positive color was measured at 625nm.

Poly-fi-hydroxybutyric acid was determined by themethod of Law and Slepecky (19), using 3-hydroxy-butyric acid as a standard.

Materials. Phosphatidyl ethanolamine was pur-chased from Nutritional Biochemicals Corp. (Cleve-land, Ohio), and phosphatidyl inositol was a productof Mann Research Laboratory (New York, N.Y.). Ly-

sophosphatides of phosphatidyl choline and phospha-tidyl ethanolamine were prepared using phospholipaseA (Sigma, bee venom) according to the procedures ofMarinetti et al. (20). The other lipid standards wereproducts of Sigma Chemical Co. (St. Louis, Mo.).

Zymosterol acetate was a generous gift from L. W.Parks (Oregon State University, Corvallis).

RESULTSFine structure of methane-grown M. or-

ganophilunL In thin sections of M. organo-philum grown on one-carbon or more complexsubstrates, a typical gram-negative cell envelopeis observed (25-27). M. organophilum cellsgrown with methane as a carbon and energysource contain intracytoplasmic membranestypical of those observed in all of the obligatemethane-oxidizing bacteria examined (25). Theintracytoplasmic membranes of M. organo-philum are type II as defined by Davies andWhittenbury (9). The pathway of one-carbonassimilation is also consistent with the designa-tion of this organism as a type II methylotroph(25). We observed apparent variations in theamount of intracytoplasmic membranes in dif-ferent preparations prepared for thin sectioning.This variation could not be correlated with thegrowth phase that the cultures were in whenthey were suspended in the fixation media.The highest methane oxidation activity in the

water column of Lake Mendota occurs in sam-ples taken at the thermocline after summerstratification (25). The dissolved oxygen level atthe depth where maximum methane oxidationoccurs is 10 to 20% of that observed by equili-bration of lake water with one atmosphere of air(26). This suggests that the methane-oxidizingbacteria might prefer low levels of dissolvedoxygen. Several growth studies have confirmedthis observation. In Azotobacter vinelandii, thedissolved oxygen concentration has been impli-cated in regulating the amount of internal mem-brane structure present (24). By regulating thedissolved oxygen level of M. organophilum cul-tures growing on methane, it appears, one canregulate the amount of intracytoplasmic mem-brane material present within the cells. Figure1A is a thin section of M. organophilum grownon methane with the level of dissolved oxygenapproaching 0%. Typically, we found three tofour pairs of intracytoplasmic membranes incells ofM. organophilum grown at low dissolvedoxygen levels. Figure 1B is a thin section of M.organophilum grown with methane at relativelyhigh levels of dissolved oxygen. We typically sawone, and sometimes two, pairs of intracyto-plasmic membranes around the periphery ofM.organophilum under the conditions of high dis-solved oxygen levels.

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COMPOSITION OF M. ORGANOPHILUM CELLS

A * a

FIG. 1. Electron micrographs of thin sections ofM. organophilum grown on a liquid mineral salts mediumwith methane as the sole source of carbon and energy. (A) The dissolved oxygen concentration of the mediumwas maintained below 5% ofsaturation. (B) The dissolved oxygen concentration ofthe medium was maintainedbetween 40 and 75% of saturation (see the text).

Fine structure of non-methane-grown M.organophilunm Intracytoplasmic membraneshave never been observed in cells grown on

methanol, succinate, or glucose. Furthermore,lowering the dissolved oxygen to a level ap-

proaching 0% does not induce intracytoplasmicmembrane formation unless methane is the solecarbon and energy source (25-27). Cultures ofM. organophilum grown in the presence ofmethane and methanol did not possess observ-able intracytoplasmic membranes.Fraction of total extractable lipids. The

total lipid fraction from the Bligh and Dyer (6)extraction procedure was treated to remove any

nonlipid contaminants, as described above. Thetotal lipid fraction, minus poly-,B-hydroxybutyricacid, of M. organophilum was found to vary

with the carbon and energy source and the levelof dissolved oxygen in methane-grown cells (Ta-ble 1).The total extractable lipids of M. organo-

philum were fractionated on an acid-treatedFlorisil column. The fractions were classifiedaccording to the polarity of the eluting solventsinto neutral, glycolipid, and phospholipid frac-tions. The percentage of each of these fractionsfor methanol- and methane-grown M. organo-philum is shown in Table 2. The neutral lipidfraction in methane-grown cells was more than

TABLE 1. Total lipid composition ofM.organophilum grown on different substrates andwith methane at low and high levels of dissolved

oxygen

Growth substrate Total lipid'(%)

Methane-0% 02 saturation ............ 19Methane-30-70% 02 saturation ....... 9Methanol 6Glucose". 6

a Total Bligh and Dyer extracted lipids as a per-centage of the cell dry weight determined gravimetri-cally.

'Glucose-grown cells accumulate poly-f8-hydroxy-butyric acid at approximately 8% of the cell dry weightto give approximately 14% total lipids. No poly-f8-hydroxybutyric acid was detected in early-stationary-phase methane- or methanol-grown cells.

twice that found in methanol-grown cells of M.organophilum.

Fractionation of the phospholipid frac-tion. The phospholipid fractions from the acid-treated Florisil column were separated on silicagel G thin-layer plates into four discrete spotsthat accounted for all the phosphorus applied tothe plate. The phospholipids were identified bycochromatography with authentic standard, spe-cific color reactions, and hydrolysis and identi-

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fication of water-soluble products obtained frommild alkaline and acid hydrolysis. Qualitatively,there were no differences in the phospholipidfractions obtained from M. organophilum grownon various carbon and energy sources.The identities of the four phospholipids from

M. organophilum are listed in Table 3. Theaverage Rf values for these phospholipids wereidentical to the corresponding four authenticstandards. All four compounds were positive tothe molybdenum blue spray, indicating theycontained phosphate, and all were negative to-ward the periodate-Schiff reagent, which indi-cates the absence of vicinal glycol groups. Thephospholipids identified as phosphatidyl etha-nolamine and phosphatidyl serine were ninhy-drin positive, indicating a free amino group. TheDragendorff test for choline-containing com-pounds was positive for the compound identifiedas phosphatidyl choline and weakly positive forthe compound identified as phosphatidyl-N,N-dimethylethanolamine.The four compounds were eluted from the

silica gel plates, deacylated under mild alkalineconditions to yield the glycerol phosphate esters,and subjected to acid hydrolysis to yield the freebases. The products obtained by mild alkalinehydrolysis and acid hydrolysis were identifiedby cochromatographic mobility with standard

TABLE 2. Lipid fractions ofM. organophilumPercentage of total lipid' in growth sub-

strate:Lipid fraction'

Methane + Methane + Methanollow oxygen high oxygen

Neutral 9 9 4Glycolipid 5 3 4Phospholipid 86 88 92

'The amount of each lipid fraction of M. organo-philum was determined gravimetrically after fraction-ation of the total lipid extract on acid-treated Florisil,as described in the text.

b Fractions eluted from acid-treated Florisil.

deacylated phospholipids and standard basesand by specific staining reactions describedabove (Table 4).The quantitation of each of the phospholipids

in M. organophilum was performed after sepa-ration by using an assay for phosphates. Thiswas done for M. organophilum grown on meth-ane with high and low dissolved oxygen levels,methanol, and glucose. Table 5 lists the data forthe determinations of the percentage of thephospholipid phosphorus found in M. organo-philum grown under the four different growthconditions tested. Phosphatidyl choline waspresent in all the cells and, therefore, cannot berelated to the presence of intracytoplasmicmembranes. Phosphatidyl choline was presentin methane-grown cells at approximately 16% ofthe total phospholipids irrespective of the dis-solved oxygen level. M. organophilum, whengrown on methanol or glucose, had significantlymore phosphatidyl choline than methane-growncultures. Phosphatidyl ethanolamine was twiceas high in methane-grown cells as in cells grownon methanol or glucose. Phosphatidyl serine, a

TABLE 4. Water-soluble hydrolysis products fromM. organophilum phospholipids

Phospho- Products (Rf x 100') of:lipidh Alkaline hydrolysis Acid hydrolysis

Phosphatidyl Glycerol-phos- (86) Choline (93)choline phoryl-cho-

linePhosphatidyl Glycerol-phos- (66) Ethanolamine (51)

ethanol- phoryl-etha-amine nolamine

Phosphatidyl- Glycerol-phos- (80) N,N-Dimethyl- (86)N,N-di- phoryl-N,N- ethanolaminemethyl-eth- dimethyl-eth-anolamine anolamine

Phosphatidyl Glycerol-phos- (28) Serine (27)serine phoryl-serine

aRf values in water-saturated phenol-ethanol-acetic acid(50:5:6) as described in the text.

'Hydrolysis products of authentic phospholipids had mo-bilities and color reactions identical to those of the identifiedcompounds.

TABLE 3. Chromatographic properties of the phospholipids ofM. organophilum

Rf value x 100' by treatment: Reaction with specific spray:Phospholipid^ C-M-water C-M-7 N NH40H C-M-HAc-water Ninhydrin Dragendorff

(65:25:4) (60:35:5) (50:25:8:4)

Phosphatidyl choline 29 32 45 - +Phosphatidyl ethanolamine 60 51 88 +Phosphatidyl-N,N-dimethyl- 52 59 69 - +, weakethanolamine

Phosphatidyl serine 17 22 82 +

a C, Chloroform; M, methanol; HAc, acetic acid (vol/vol).'Authentic standard phospholipids had mobilities and color reactions identical to those of the identified

phospholipids.

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COMPOSITION OF M. ORGANOPHILUM CELLS 641

TABLE 5. Relative amounts of each phospholipidclass from M. organophilum

Percentage of total phospholipids' undergrowth conditions:

PhospholipidMethane- Methane- Methanol Glucose

high [02] low [021

Phosphatidyl 15 17 38 44choline

Phosphatidyl 57 46 25 26ethanolamine

Phosphatidyl- 24 31 24 28N,N-dimeth-ylethanol-amine

Phosphatidyl 1 1 9 2serine

Percentage of total phospholipid phosphorus.

precursor of the other phospholipids, was atrelatively low levels except in cells grown on

methanol. In methanol-grown cells, phosphati-dyl serine comprised 9% of the total phospho-lipid fraction.Squalene and sterols from M. organo-

philunL Squalene was detected and measuredin the hydrocarbon fraction from the acid-treated Florisil column. The identification ofsqualene was based on the identical elutionproperties of squalene present in M. organo-

philum and authentic squalene from acid-treated Florisil and from 3% SP2100 liquid phasein a gas-chromatographic column. Squalene wasestimated by comparing peak areas from au-

thentic squalene with the sample from M. or-

ganophilum. Table 6 contains the data for meth-ane- and glucose-grown M. organophilum.Squalene was present at a concentration of 690Ag/g (dry weight) of glucose-grown cells and 27to 50,ug/g (dry weight) of methane-grown cells.

Sterols were also found to be present as a

component of the lipid fraction from M. organ-ophilum. Sterols were found in M. organo-philum grown on methane, methanol, or glucose.The greatest amount was found in cells grownon methane with a very low dissolved oxygen

concentration. There was less membrane in cellsgrown under other conditions. This may in partaccount for the reduced sterol content of cellsgrown on substrates other than methane and athigh dissolved oxygen levels. A gas-chromato-graphic tracing of the acid-treated Florisil col-umn sterol fraction from M. organophilumgrown on methane with a dissolved oxygen con-

centration approaching zero revealed threesterol fractions, labeled A, B, and C. The amountof each was determined. Table 6 lists the datafor these three different sterols found in M.organophilum under all four growth conditions.Sterols A and B were greatest in methane-growncells with the low levels of dissolved oxygen.

TABLE 6. Squalene and sterols in M. organophilumSterol peaks"

Substrate SqualenehA B C

Methane-low O2 50 220 75 13Methane-high 02 27 9 2 27Methanol NT' 6 3 7Glucose 690 24 18 132

a Nanograms per gram (dry weight) of cells, basedon cholesterol response.

b Micrograms per gram (dry weight) of cells." Not tested.

Sterol C was found in the greatest amount incells grown on glucose.The criteria used to identify the peaks as

sterols were the elution characteristics on acid-treated Florisil and 3% SP2100, color reactionson thin-layer chromatography plates (27), a Lie-bermann-Burchard positive colorimetric assay,digitonin precipitation, and molecular-weightdetermination by mass spectrometry (molecularweights of approximately 400; unpublisheddata).A comparison has been made of the digitonin-

precipitated sterols from M. organophilum andMethylococcus capsulatus based on relative re-tention times (relative to cholesterol). The ster-ols in Methylococcus capsulatus have been iden-tified as 5a-cholesta-8(9),24-dienol (zymolsterol)and 4a-methyl- and 4,4-dimethyl- derivatives of5a-cholesta-8(9)-en-31?-ol and 5a-cholesta-8(9),24-dien-3,8-ol by Bird et al. (5). None of thesterols in M. organophilum had retention timesidentical to any of the sterols in Methylococcuscapsulatus. The sterol listed as A has beenidentified by gas chromatography-mass spec-trometry data (The Upjohn Co., unpublisheddata) as 5a-cholesta-8,22,24-trien-4,4,14a-trime-thyl-3,B-ol (Lanosta-8,22,24-trien-3,8-ol).

DISCUSSIONIn this paper we report on two factors which

control the amount and composition of the ex-tensive intracytoplasmic membranes in the fac-ultatively methylotrophic bacterium M. organ-ophilum. These intracytoplasmic membranesare similar in appearance to those found in theobligate methylotrophs (9, 10, 29, 38, 39). Smithand Ribbons (38) noted that the presence ofthese intracytoplasmic membranes seems to belimited to those bacteria that utilize specializedenergy sources. It is of interest to determinewhat role, if any, these intracytoplasmic mem-branes play in the utilization of specialized sub-strates such as methane.Our results show that the intracytoplasmic

membrane content of M. organophilum is con-

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642 PATT AND HANSON

trolled by the carbon and energy source as wellas by the concentration of dissolved oxygen incultures grown with methane as the sole carbonand energy source. Electron micrographs of thinsections from methane-grown M. organophilumreveal that there is approximately twice thenumber of layers of intracytoplasmic mem-branes present when the cells are grown at lowdissolved oxygen concentrations as in culturesgrown at much higher levels of dissolved oxygen.When M. organophilum was grown on methanolor glucose as the carbon and energy source, nointracytoplasmic membranes were observed.These observations have been supported by es-timations of the total extractable lipid contentsof cells. On the basis of the data from the totallipid extracts, there is twice as much membranematerial in M. organophilum grown on methanewith an oxygen concentration approaching zeroas there is in M. organophilum grown on meth-ane with a high oxygen concentration. Metha-nol- and glucose-grown cultures of M. organo-philum have lipid contents similar to many othergram-negative bacteria that do not contain in-tracytoplasmic membranes (3, 4). The presenceof intracytoplasmic membranes in methane-grown cells and their absence in cells grown onother substrates suggests that the intracyto-plasmic membranes are associated with meth-ane oxidation.A number of the enzymes involved in methane

oxidation, including methane hydroxylase (13,14, 32, 34, 42), methanol oxidase and methanoldehydrogenase (44), and formaldehyde oxidaseand formaldehyde dehydrogenase, are particu-late (44). The requirement for phospholipase ordetergents for the solubilization of methane hy-droxylase suggests that methane hydroxylase isan integral membrane protein of Methylosinustrichosporium (42). The membrane matrix couldprovide a favorable interface for the accommo-dation of methane and the methane hydroxylasebecause the hydrophobic core of the intracyto-plasmic membrane would maintain a higher con-centration of methane in the vicinity of theenzyme (35). The free energy change (AGo', pH7.0) for the oxidation of methane is more favor-able by 2.6 x 103 cal/mol (ca. 10.9 x 103J) if itexists in a nonpolar region such as the hydro-phobic region ofthe intracytoplasmic membranethan in the water phase of the cell (35). Theintracytoplasmic membrane might also play asimilar role in providing a favorable environ-ment for oxygen to react in the mono-oxygenasereaction catalyzed by methane hydroxylase. It isinteresting that the total amount of sterols pres-ent is greatly reduced in cells grown on methanewith high levels of dissolved oxygen. Theamount of internal membranes in Azotobacter

vinelandii has been shown to be regulated bythe levels of dissolved oxygen (24). More mem-branes were synthesized when oxygen was lim-iting.The phospholipid composition was analyzed

to determine whether there were any majordifferences in the cells grown under differentconditions. Different phospholipids in cells withintracytoplasmic membranes would indicate acompositional difference between the plasmamembrane and the intracytoplasmic membrane.The data indicate that there may be quantitativedifferences in the phospholipids, but there donot seem to be any qualitative differences thatcan be used as markers for membrane purifica-tion attempts. Phosphatidyl choline was presentunder all growth conditions and, therefore, can-not be uniquely present in the intracytoplasmicmembranes. Unusually high levels of phospha-tidyl-N,N-dimethylethanolamine are present inM. organophilum, as is also the case for themethanol-utilizing Hyphomicrobium species (6).The other major phospholipid found under allgrowth conditions was phosphatidyl ethanol-amine, a common constituent of bacterial phos-pholipids (4) and a precursor of phosphatidyl-N,N-dimethylethanolamine and choline.Growth of the cells on methane results in adecrease in the ratio of phosphatidyl choline tophosphatidyl ethanolamine. A change in thisratio cannot be correlated with the inclusion ofa specific sterol in the membranes because theratio of these phospholipids does not changesignificantly when cells are grown with methaneand high levels of oxygen. The total amount ofsterols or of any single sterol fluctuates inde-pendently of the amount of any phospholipid. Itis possible that the inclusion of specific proteinsinto a phospholipid bilayer is affected bychanges in the phospholipid composition. Themethane hydroxylase proteins are present onlyin cells grown on methane (27), a condition thatcauses a large increase in the total phosphatidylethanolamine content of the membranes. Thephospholipid composition of M. organophilumdiffers from that reported for the obligate meth-ane-oxidizing bacterium Methylosinus tricho-sporium (45). The phospholipids of M. tricho-sporium consist of 57.3% phosphatidyl glycerol,37.5% phosphatidyl ethanolamine, 4.2% phos-phatidyl serine, 0.6% phosphatidyl choline, and0.4% diphosphatidyl glycerol. The phospholipidswere present in the same percentages whetherthey were extracted from whole cells or frompurified membrane fractions. Any generaliza-tions concerning the phospholipids of methane-oxidizing bacteria will have to wait until othermethane-oxidizing bacteria are studied.

Sterols have recently been isolated and char-

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COMPOSITION OF M. ORGANOPHILUM CELLS 643

acterized from Methylococcus capsulatus, a

type I obligate methane-oxidizing bacterium (5).The sterols present in membranes are believedto play a structural role by condensing the mem-branes, and sterols also make membranes more

rigid without making them solid (7, 17). It hasalso been suggested that sterols donate hydrogenbonds to the carbonyl groups of phospholipidsin hydrogen-bonding regions of membranes.Brockerhoff (7) suggested that the hydrogen-bonding properties of /8-hydroxy sterols reducethe molecular area of phospholipids, condensemembranes, and reduce their permeability toions, water, and several polar substrates. In thisstudy we demonstrate the presence of sterols ina second methane-oxidizing bacterium, M. or-

ganophilum. The ability of digitonin to precipi-tate these sterols indicates the presence of a 3-hydroxy group in the 3-configuration (41). Al-though the sterols may play some significantrole in the intracytoplasmic membranes ofmeth-ane-oxidizing bacteria, the sterols are not con-

fined to the intracytoplasmic membrane mate-rial.

ACKNOWLEDGMENTSThis research was supported by the College of Agricultural

and Life Sciences, University of Wisconsin, Madison, and bygrants from the National Science Foundation (BMS75-14012)and the University of Wisconsin Graduate School ResearchCommittee. T. E. Patt was supported by Public Health Servicetraining grant 5-T01-GM00686-15 from the National Instituteof General Medical Sciences.We thank Thomas Miller of the Upjohn Company for his

gas-liquid chromatography-mass spectrometry identificationof the sterol present in M. organophilum.

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