JOURNAL OF BACRIOLOGY, Feb. 1972, p. 916-921 Vol. 109, No. 2Copyright 0 1972 American Society for Microbiology Printed in U.SA.

New Obligate MethylotrophJEAN STOKES DAHL,' R. J. MEHTA,' AND DEREK S. HOARE'

Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712

Received for publication 30 August 1971

A new and unique obligate methylotroph was isolated from enrichment cul-tures with methanol as the sole source of carbon and energy. The organismgrows only on methanol and methylamine and not on methane. It does nothave a complex intracellular membrane system. "IC-acetate was assimilated bygrowing cultures and cell suspensions but was incorporated into only a limitednumber of cell constituents. "4C-acetate incorporation was strictly dependenton the oxidation of methanol or methylamine as a source of energy. Extractshad relatively low levels of enzymes of the tricarboxylic acid cycle, and a-keto-glutarate dehydrogenase was not detected. Comparisons were made with a fa-cultative methylotroph isolated from the same enrichment cultures. The newobligate methylotroph contained hexose phosphate synthetase, a key enzyme inthe ribose phosphate cycle of methyl metabolism.

Many organisms grow on reduced C, com-pounds as the sole source of carbon and energy(24). These include both obligate and faculta-tive methylotrophs. Obligate methylotrophsgrow only on methane or methanol (6). Theyresemble obligate lithotrophs in their strictdependence on a specific energy source. Theseorganisms use one of two pathways to assimi-late C, compounds, either the serine pathway(8) or the ribose phosphate cycle (13, 14). Fa-cultative methylotrophs do not grow on meth-ane, but can use methanol, methylamine, for-mate, or other organic compounds as their solecarbon and energy source (26). They use theserine pathway for C, assimilation (26).We set up a number of enrichment cultures

for methanol-oxidizing bacteria in order to iso-late pure cultures of methylotrophs for bio-chemical studies. This paper describes the iso-lation and characterization of a new andunique obligate methylotroph which growsonly on methanol or methylamine as the solesource of carbon and energy. It contains keyreactions of the ribose phosphate cycle.

MATERIALS AND METHODSEnrichment and isolation. The basal salts solu-

tion of Foster and Davis (6) containing 0.1 M meth-anol was used for enrichment cultures. Flasks (250ml) containing 50 ml of medium were inoculated

I Present address: Universit9t Zurich, Institut fUr alleg-meine Botanik, Kunstlergasse 16, 8006 Zurich, Switzerland.

2Present address: Smith, Kline and French Laboratories,Philadelphia, Pa. 19101.

3Deceased 16 May 1971.

with soil samples (Austin, Texas) and incubated for2 days on a rotary shaker at 30 C. Portions (1 ml) ofthe turbid suspensions were transferred to freshmedium (50 ml) and incubated in the same manner.After three serial transfers in liquid medium, inoculawere streaked on the same medium with 2% (w/v)agar. Within 3 days, three colony types appeared:pink colonies, yellow mucoid colonies, and nonpig-mented mucoid colonies. The pink colonies werepredominant. Cultures of each colony type were pu-rified by repeated streaking from isolated colonies.The pink organism was characterized as a facultativemethylotroph by testing growth on a variety of sub-strates. It resembled in many respects, but was notidentical to, the pink, methanol-oxidizing bacteriumPseudomoras M27 (1). Our pink facultative methy-lotroph was used for comparative studies in some ofthe experiments described in this report. A nonpig-mented organism (W1) was isolated from one of thenonpigmented mucoid colonies. This orgnism isdiscussed in detail.

Stock cultures of Wl were maintained on basalsalts medium with 0.1 M methanol and 2% (w/v) agrin petri plates. They were transferred every 2months and stored at 4 C.

Batch cultures were grown in 2-liter flasks con-taining 1 liter of basal salts medium with either 0.1M methanol or 0.02 M methylamine as the carbonsource. A 0.5% inoculum was used. Flasks were incu-bated on a rotary shaker at 30 C for 3 days. Har-vested cells were washed twice with 0.05 M potas-sium phosphate buffer, pH 7.0, and used immedi-ately or stored at 4 C.Chemicals. Methanol was purchased from

Eastman Kodak Co., Rochester, N.Y., and methyl-amine hydrochloride (reagent grade) from FisherScientific Co., Fair Lawn, N.J. Sodium acetate-1-14C(specific activity, 25 mCi/mmole) was purchased

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from Calatomic, Los Angeles, Cal. D-Ribose-5-phos-phate was obtained from Sigma Chemical Co., St.Louis, Mo. Formaldehyde was generated from para-formaldehyde (Eastman Kodak Co., Rochester,N.Y.).Growth experiments. Side-arm flasks (250 ml)

containing 50 ml of basal salts medium and the ap-propriate carbon source were inoculated with 1 ml ofa methanol-grown culture and incubated in thenormal manner. Growth substrates were added to afinal concentration of 0.02 M, except methanol (0.1M). Growth on methane was tested in liquid mediumunder an atmosphere of methane-air (1:1, v/v). Op-timal pH and temperature for growth were estab-lished with methanol as the carbon source. Growthwas followed in a Klett-Summerson colorimeter at600 nm.

Electron microscopy. Specimens were fixed in1% osmium tetroxide in Kellenberger buffer, pH 6.1,(10) for 3 hr at room temperature. After fixation,specimens were soaked in two 15-min changes of0.5% aqueous uranyl acetate. Specimens were dehy-drated in a graded alcohol series, followed by twochanges of acetone. The cells were embedded in aplastic mixture consisting of 70% dodecenyl succinicanhydride, 20% Araldite 6005, and 10% Epon 812with one drop of accelerator DMP-30 (Rohm & HaasCo., Philadelphia, Pa.) added per ml of plastic used.Sections were cut on a Sorvall Porter-Blum MT-2microtome with a diamond knife. Sections werestained with Reynold's lead citrate (24).

Specimens were viewed with a Hitachi HS-7Selectron microscope. Micrographs were taken onKodak contrast process Ortho film.

Oxidation of organic compounds by cell sus-pensions. Warburg flasks contained, in 3.0 ml: cells(0.5 to 4 mg of protein) grown on methanol or meth-ylamine; 150 gmoles of potassium phosphate buffer,pH 7.0; and 20 Mmoles of substrate. The center wellcontained 0.1 ml of 20% (w/v) KOH. Oxygen uptakewas followed at 30 C under air. The protein concen-tration of whole cells was determined after alkalinedigestion by the procedure of Lowry et al. (19).

Assimilation of acetate-l-'4C by cell suspen-sions. For following the time course of acetate incor-poration, Warburg flasks contained, in 3.0 ml: cells(0.5 to 1.2 mg of protein) grown on methanol ormethylamine; 150 umoles of potassium phosphatebuffer, pH 7.0; 20 gmoles of methanol or methy-lamine; and 10 umoles of sodium acetate-1-"4C (2MCi). The center well contained 0.1 ml of 20% (w/v)KOH. A control flask lacking methanol or methyl-amine was included for each time sample. Flaskswere incubated at 30 C under air. At timed intervals,a control flask and a test flask were removed andput on ice, and 0.2-ml portions of the suspensionswere immediately filtered through membrane filters(Millipore Corp., Bedford, Mass.; pore size, 0.45Am), washed once with 10 ml of 0.01 M sodium ace-tate, and then twice with 10 ml of water. The filterswere mounted on aluminum planchets and counted ina gas flow planchet counter (Nuclear-Chicago Corp.,Des Plaines, Ill.).

For testing the effect of methanol concentration

on the extent of acetate incorporation, Warburgflasks contained, in 3.0 ml: cells (1.3 mg of protein)grown on methanol; 150 Amoles of potassium phos-phate buffer, pH 7.0; 2 to 20 Amoles of methanol;and 10 MLmoles of sodium acetate-1-14C (2 MCi). Thecenter well contained 0.1 ml of 20% (w/v) KOH. Acontrol flask lacking methanol was included for eachflask. After oxygen uptake ceased, the control andtest flasks were removed, and the amount of "4C-acetate incorporated was determined as describedabove.

Fractionation of cells grown in the presence ofacetate-1-"4C. Cells were grown in 50 ml of basalsalts medium containing 2 mm sodium acetate-1-14C(20 MiCi) and 0.1 M methanol or 20 mm methylamine.After 3 days, cells were harvested by centrifugation;washed twice with 0.05 M potassium phosphate buf-fer, pH 7.0; and fractionated by the method of Rob-erts et al. (27). The protein residues were hydrolyzedby refluxing in 10 ml of 6 N HCl for 18 hr. Samplesfrom each fraction were plated on aluminum plan-chets and counted in a gas flow planchet counter.

Electrophoresis, chromatography, and ra-dioautography. Amino acids were partially resolvedby high-voltage electrophoresis in pyridine-aceticacid buffer, pH 3.6 (28). Thq basic amino acids werefurther resolved by chromatography in n-butanol-acetone-water-dicyclohexylamine (10:10:5:2, byvolume) (7). The neutral amino acids were chromato-graphed in isobutanol-methyl ethyl ketone-water (7:5:3, v/v/v) or propanol-water (8:2, v/v) (12). Aminoacids were detected by spraying with a ninhydrinreagent.Preparation of cell extracts and enzyme as-

says. A paste (2 g, wet weight) of cells was sus-pended in 10 ml of 0.05 M potassium phosphate buf-fer, pH 7.0, with 3 g of washed, fine glass beads (Su-perbrite type 110, 3 M Co., St. Paul, Minn.). Sus-pensions were sonically treated for two periods of 1min with a sonic disintegrator (Measuring and Sci-entific Equipment, Ltd., London, England). The dis-integrated suspensions were centrifuged for 10 minat 20,000 x g at 4 C. The supernatant solutions wereagain centrifuged at 90,000 x g for 1 hr in a refriger-ated Spinco model L ultracentrifuge. Protein wasestimated by the procedure of Warburg and Chris-tian (31).

Standard methods were used to determine thespecific activities of the following enzymes in ex-tracts: methanol dehydrogenase (2), methylaminedehydrogenase (5), a-ketoglutarate dehydrogenase(20), pyruvate dehydrogenase (20), isocitrate dehy-drogenase (15), malate dehydrogenase (21), and suc-cinate dehydrogenase (9). The assay for "hexosephosphate synthetase" (16) contained, in 1.0 ml: 100mM potassium phosphate buffer, pH 7.0; 4 mM mag-nesium chloride; 0.25 mM nicotinamide adenine di-nucleotide phosphate (NADP+); 5 mM D-ribose-5-phosphate; 4 mM formaldehyde; 1.68 Mmolar units ofrabbit muscle glucosephosphate isomerase (EC5.3.1.9; crude, Sigma Chemical Co., St. Louis, Mo.);0.15 Mmolar units of yeast glucose-6-phosphate dehy-drogenase (EC 1.1.1.49; type VII, Sigma ChemicalCo., St. Louis, Mo.); and cell extract (0.05 to 0.15

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mg of protein). All spectrophotometric assays wereperformed at room temperature on a Beckman DU-2spectrophotometer with a multichannel chart re-corder (Gilford Instrument Laboratory, Inc., Oberlin,Ohio).

RESULTSGeneral morphological characteristics.

On methanol agar plates, colonies of Wl were2 to 3 mm in diameter, mucoid, raised, andcircular with a smooth edge. Microscopic ex-amination revealed that the cells were motile,gram-negative rods. No endospores or cystswere observed. As judged by an India inkstain, cells were encapsulated. The Leifson (18)staining procedure showed that the cells had asingle polar flagellum. After centrifuging, therewas always a white gelatinous layer over thecell pellet. The cell pellet was buff-orange.The organism did not show an elaborate in-

ternal membrane system (Fig. 1). It containedonly membranous bodies of the mesosometype.The mean guanine-plus-cytosine content of

the deoxyribonucleic acid was 56.1 moles percent.Physiological characteristics. Although

the organism was isolated from enrichmentcultures yielding predominantly facultativemethylotrophs, WI was an obligate methylo-troph. Only methanol and methylamine sup-ported its growth. The following substratesfailed to support growth of the organism in thebasal salts medium: methane, formate, formal-dehyde, ethanol, n-propanol, n-butanol, n-pentanol, isopropanol, glycine, serine, gluta-mate, alanine, glucose, fructose, acetate, cit-rate, malate, succinate, lactate, oxalate, tar-trate, and nutrient broth. The organism grew

over a pH range of 5 to 9 with an optimum atpH 7.0. The optimal temperature for growthwas 30 C, although the organism did growslowly at 37 and 24 C.

Suspensions of methanol-grown Wl oxidizedmethanol, formaldehyde, formate, and severalprimary alcohols, but failed to oxidize methyl-amine and acetate (Table 1). Although ethanoland propanol were apparently oxidized to theircorresponding acids, acetaldehyde and pro-pionaldehyde were not oxidized by cell suspen-sions. Methanol-grown cells of Methylococcuscapsulatus, another obligate methylotroph,oxidized primary alcohols to their corre-

TABLE 1. Oxidation of various substrates by cellsuspensions of WI

Methanol-grown Methylamine-WI grown WI

Substrate(20 umoles) 0, con- 02 con-

sumed Q (0w)a sumed Q (O)(gumoles) (umoles)

Methanol 26.4 400 21.0 284Methylamine 0 0 15.0 815Formaldehyde 14.0 400 12.0 490Formate 8.8 75 9.5 55Ethanol 18.8 265 n.t.bn-Propanol 16.0 270 n.t.n-Butanol 18.4 265 n.t.n-Pentanolc 7.6 248 n.t.Acetate 0 0 n.t.Acetaldehyde 0 0 n.t.Propionaldehyde 0 0 n.t.

aMicroliters of oxygen consumed per hour permilligram of protein.

b Not tested.cReaction mixture contained 10 !Lmoles of pen-

tanol.*' t'-'t. 4i8. - *# ~-i5etes e.-

d ,,!M., X.-s4'

i i./

0_FIG. 1. Electron micrograph of Wl. Bar equals 0.1 gm.

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sponding aldehydes (22). Suspensions of meth-ylamine-grown Wl oxidized methylamine,formaldehyde, formate, and methanol (Table1).

Extracts of methanol-grown cells containeda soluble, phenazine methosulfate (PMS)-linked methanol dehydrogenase. It requiredammonium ions for activity and had a pH op-timum of 8.5 to 9.0. 2,6-Dichlorophenol indo-phenol also served as an electron acceptorwhen PMS was present. Ethanol, n-propanol,n-butanol, and n-pentanol, as well as metha-nol, were substrates for the enzyme. In thesecharacteristics the mqthanol dehydrogenase ofWl resembled that described in PseudomonasM27 by Anthony and Zatman (2).

Extracts of methylamine-grown cells con-

tained a soluble, PMS-linked methylaminedehydrogenase. Such an enzyme was describedpreviously by Eady and Large (5) in Pseudo-monas AML.Metabolic characteristics. Obligate methy-

lotrophs resemble obligate lithotrophs in theirstrict dependence on a specific energy source(23). Although Wl did not grow on acetate or

oxidize it, the cells assimilated acetate whenan appropriate energy source was present. Cellsuspensions incorporated acetate-i- 14C at ratesproportional to the rates of methanol or meth-ylamine oxidation (Fig. 2a and b). The extentof acetate-1-l4C incorporation was strictlydependent on the amount of methanol oxi-dized (Fig. 3). Similar results have been foundwith cell suspensions of M. capsulatus incu-bated with methanol and "4C-acetate (D. S.Hoare and M. Hensley, unpublished data).When Wl was grown on methanol or methyl-

amine in the presence of acetate-1-'4C therewas limited incorporation of acetate into cellconstituents. Acetate was incorporated mainlyinto the lipid and protein fractions of the cell(Table 2). Very little radioactivity was foundin the polysaccharide and nucleic acid frac-tion. This distribution pattern also occurs inM. capsulatus grown on "4C-acetate andmethane (Patel et al., Bacteriol. Proc., p. 128,1969). For comparison, the pink facultativemethylotroph was grown on methylamine andacetate-1-"4C under the same conditions. Inthis organism, acetate was incorporated intoall major cell components, lipids, polysaccha-rides, nucleic acids, and proteins (Table 2).The hydrolyzed protein residues from Wl

and the pink organism were analyzed for la-beled amino acids by high-voltage paper elec-trophoresis (28), paper chromatography (7, 12),and radioautography. In Wl only glutamate,proline, arginine, and leucine were labeled. No

zc--4

OXYGE,N CONSUMED (Cmoles)

al l> col l

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_\o-~~~~~~~~~~~~

_ j o M olC

_ \ % 0

I I I I I

C-ACETATE INCORPORATED ( nmoles)

OXYGEN CONSUMED C(Amoles)

z

m

FIG. 2. Time course of acetate-1-'4C incorporationby cell suspensions of Wl. (a) Warburg flasks con-

tained, in 3.0 ml: 125 umoles of potassium phosphatebuffer (pH 7.0); 10 ,umoles of sodium acetate-1- 4C (2IiCi); 20 gmoles of methanol; and cells (1.2 mg ofprotein). (b) Warburg flasks contained, in 3.0 ml: 125,gmoles of potassium phosphate buffer (pH 7.0); 10;imoles of sodium acetate-1-"4C (2 1Ci); 20 umoles ofmethylamine; and cells (0.5 mg of protein). Centerwells contained 0.1 ml of 20%o potassium hydroxide.Flasks were incubated in air at 30 C. Symbols: (0)oxygen consumed; (0) "4C-acetate incorporated.

radioactivity was found in asparate or theother neutral and basic amino acids. In thefacultative methylotroph virtually all of theamino acids were labeled.

Inability to incorporate acetate into aspar-tate or polysaccharides may result from a

block in the tricarboxylic acid cycle at a-keto-glutarate dehydrogenase (29). The facultativemethylotroph contained all of the tricarboxylic

I I I I I.

al ol a A.S

C'1 0 0 C' 0

I a I I v"-ACETATE INCORPORATEr" %.nmoles)

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OXYGEN CONSUMED (smoles)

14C ACETATE INCORPORATED ( n moles)

FIG. 3. Acetate-1-"4C incorporation by cell sus-pensions of Wl. Warburg flasks contained, in 3.0 ml:125 Mmoles of potassium phosphate buffer (pH 7.0);10 ,qmoles of sodium acetate-1-'4C acetate (2 MCi); 2to 20 ,moles of methanol; and cells (1.3 mg of pro-tein). Center wells contained 0.1 ml of 20% potas-sium hydroxide. Flasks were incubated at 30 Cunder air until oxygen consumption stopped. Sym-bols: (0) oxygen consumed; (0) "4C-acetate incorpo-rated.

TABLE 2. Distribution of '4C in Wl and a facultativemethylotroph on "4C-acetate and methylamine

Per cent distribu-tion of 14C

Fraction Faculta-

wi tiveWi methyl-otroph

Cold 5% trichloroacetic acid ... 8.1 3.3Ethyl alcohol ................ 46.0 20.0Ethyl alcohol/ether ........... 9.6 1.8Hot 5% trichloroacetic acid .... 6.0 19.6Acid/ethyl alcohol/ether ....... 9.5 5.2Protein hydrolysate ........... 21.0 49.0

acid cycle dehydrogenases, whereas Wlshowed only isocitrate dehydrogenase, suc-cinate dehydrogenase, and malate dehydro-genase activities (Table 3). The dehydrogen-ases found in Wl had much lower specific ac-tivities than those found in the pink organism.Although a-ketoglutarate dehydrogenase ac-

tivity could not be detected in Wi, pyruvatedehydrogenase was present.

Extracts of methanol- and methylamine-grown Wl were assayed for hexose phosphatesynthetase. The specific activities were thesame under both growth conditions (296Mmoles of NADP+ reduced per min per mg ofprotein).

TABLE 3. Enzyme activities in extracts of methanol-grown Wl and a facultative methylotroph

Specific activity (nmoles/min/mg of protein)

Enzyme Facultative

Wi methylo-troph

Isocitrate dehydrogenase(NADP+) ........... 4.6 200.0

a-Ketoglutarate dehydro-genase ............. 0 3.5

Succinate dehydrogenase(particulate) ........ 0.8 68.5

Malate dehydrogenase(NAD+) ............ 69.0 1,020.0

Pyruvate dehydrogenase 17.4 1.6

DISCUSSIONBacteria which grow on methanol as the sole

source of carbon and energy fall into two phys-iological groups. "Facultative methylotrophs"grow on methanol as well as many other or-

ganic compounds. There are many organismsof this type (24, 26) including the pink isolateused in our studies. "Obligate methylotrophs"grow only on methanol and methane (26, 33).Our new obligate methylotroph is unique inthat it grows only on methanol and methylam-ine, and not on methane. It also differs fromall other obligate methylotrophs with respectto its ultrastructure. All methane-oxidizingbacteria have a complex cytoplasmic mem-brane system (3). The fact that our new isolatelacks a complex ultrastructure is probably re-lated to its inability to utilize methane.

In most physiological respects our new iso-late is analogous to the obligate lithotrophssuch as Thiobacillus thioparus (29) and Thio-bacillus neapolitanus (11). It assimilates acetateinto a limited number of cell constituents.Acetate assimilation by cell suspensions isstrictly dependent on the specific energysources, methanol or methylamine. Similarly,the obligate methylotroph M. capsulatus (D. S.Hoare and M. Hensley, unpublished data) as-similates acetate only in the presence of meth-anol or methane. Obligate methylotrophs andobligate lithotrophs also have in common ametabolic block in the tricarboxylic acid cycle.Whittenbury et al. (33) have proposed a new

classification scheme for the methane-oxi-dizing bacteria in which the names of all thegenera begin with the prefix "Methylo-" asfirst proposed by Foster and Davis (6). Sinceour organism is clearly an obligate methylo-troph it could be placed in a genus with theprefix "Methylo-." We have chosen the arbi-

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trary designation organism "Wl" pending a

more extensive survey for additional isolateswith similar physiological properties.

Obligate methylotrophs can be divided intotwo groups regarding their pathway of C1 as-

similation (17). The genera Methylomonas,Methylococcus, and Methylobacter use theribose phosphate cycle, whereas Methylosinusand Methylocystis use the serine pathway. Wlcontains hexose phosphate synthetase, a keyenzyme of the ribose phosphate cycle. Whetheror not this is the only or major pathway of C,metabolism in this organism is not known.

ACKNOWLEDGMENTSThe authors gratefully acknowledge the technical assist-

ance of Margaret Hensley. We are indebted to M. Mandel ofThe University of Texas M. D. Anderson Hospital andTumor Institute, Houston, Texas, for the DNA analyses andto L. Pope of The University of Texas at Austin Micro-biology Department for the Electron microscopy.

J.S.D. was a postdoctoral fellow supported by the RobertA. Welch Foundation.

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3. Davies, S. L., and R. Whittenbury. 1970. Fine structureof methane and other hydrocarbon-utilizing bacteria.J. Gen. Microbiol. 61:227-232.

4. Dixon, G. H., and H. L. Kornberg. 1959. Assay methodsfor key enzymes of the glyoxylate cycle. Biochem. J.72:3 P.

5. Eady, R. R., and P. J. Large. 1968. Purification andproperties of an amine dehydrogenase from Pseudo-monas AM1 and its role in growth on methylamine.Biochem. J. 106:245-255.

6. Foster, J. W., and R. H. Davis. 1966. A methane-depen-dent coccus, with notes on classification and nomen-

clature of obligate, methane-utilizing bacteria. J. Bac-teriol. 91:1924-1931.

7. Hardy, T. L., D. 0. Holland, and J. H. C. Nayler. 1955.One-phase solvent mixtures for the separation ofamino acids. Anal. Chem. 27:971-974.

8. Hemptinstall, J., and J. R. Quayle. 1970. Pathwaysleading to and from serine during growth of Pseudo-monas AM1 on C, compounds or succinate. Biochem.J. 117:563-572.

9. Jurtshuk, P., A. K. May, L. M. Pope, and P. R. Aston.1969. Comparative studies on succinate and terminaloxidase activity in microbial and mammalian elec-tron-transport systems. Can. J. Microbiol. 15:797-807.

10. Kellenberger, E., A. R. Ryter, and J. Sechaud. 1958.Electron microscope study of DNA-containingplasms. II. Vegetative and mature phage DNA as

compared with normal bacterial nucleoids in differentphysiological states. J. Biophys. Biochem. Cytol. 4:671-678.

11. Kelly, D. P. 1967. The incorporation of acetate by thechemo-autotroph Thiobacillus neapolitanus strain C.Arch. Mikrobiol. 58:99-116.

12. Kemble, A. R., and H. T. MacPherson. 1954. Determi-nation of monoamino monocarboxylic acids by quan-titative paper chromatography. Biochem. J. 56:548-

921

555.13. Kemp, M. B., and J. R. Quayle. 1966. Microbial growth

on C, compounds. Incorporation of C, units into allu-lose phosphate by extracts of Pseudomonas meth-anica. Biochem. J. 99:41-48.

14. Kemp, M. B., and J. R. Quayle. 1966. Microbial growthon C, compounds. Uptake of '4C-formate bymethane-grown Pseudomonws methanica and deter-mination of the hexose labeling pattern after briefincubations with '4C-methanol. Biochem. J. 102:94-102.

15. Kornberg, A. 1955. Isocitric dehydrogenase of yeast(TPN), p. 705-707. In S. P. Colowick and N. 0. Ka-plan (ed.), Methods in enzymology, vol. 1. AcademicPress Inc., New York.

16. Lawrence, A. J., M. B. Kemp, and J. R. Quayle. 1970.Synthesis of cell constituents by methane-grownMethylococcus capsulatus and Methanomorws meth-anooxidans. Biochem. J. 116:631-639.

17. Lawrence, A. J., and J. R. Quayle. 1970. Alternativecarbon assimilation pathways in methane-utilizingbacteria. J. Gen. Microbiol. 63:371-374.

18. Leifson, E. 1951. Staining, shape, and arrangement ofbacterial flagella. J. Bacteriol. 62:377-389.

19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

20. Mukherjee, B. B., J. Mathews, D. L. Horney, and L. J.Reed. 1965. Resolution and reconstitution of the Esch-erichia coli a-ketoglutarate dehydrogenase complex. J.Biol. Chem. 240:PC 2268.

21. Ochoa, S. 1955. Malic dehydrogenase from pig heart, p.735-739. In S. P. Colowick and N. 0. Kaplan (ed.),Methods in enzymology, vol. 1. Academic Press Inc.,New York.

22. Patel, R. N., and D. S. Hoare. 1971. Physiologicalstudies of methanol and methanol-oxidizing bacteria:oxidation of C-1 compounds by Methylococcus capsu-latus. J. Bacteriol. 107:187-192.

23. Peck, H. D., Jr. 1968. Energy-coupling mechanisms inchemolithotrophic bacteria. Annu. Rev. Microbiol. 22:489-518.

24. Quayle, J. R. 1961. Metabolism of C, compounds in au-

totrophic and heterotrophic microorganisms. Annu.Rev. Microbiol. 15:119-152.

25. Reynolds, E. S. 1963. The use of lead citrate at high pHas an electron-opaque stain in electron microscopy. J.Cell Biol. 17:208-212.

26. Ribbons, D. W., J. E. Harrison, and A. M. Wadzinski.1970. Metabolism of single carbon compounds. Annu.Rev. Microbiol. 24:135-158.

27. Roberts, R. B., D. B. Cowie, P. H. Abelson, E. T. Bol-ton, and R. J. Britten. 1955. Studies of biosynthesis inEscherichia coli. Carnegie Inst. Wash. Publ. 607:13-30.

28. Ryle, A. P., F. Sanger, L. F. Smith, and R. Kitai. 1955.The disulfide bonds of insulin. Biochem. J. 60:541-556.

29. Smith, A. J., J. London, and R. Y. Stanier. 1967. Bio-chemical basis of obligate autotrophy in blue-greenalgae and thiobacilli. J. Bacteriol. 94:972-983.

30. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966.The aerobic pseudomonads: a taxonomic survey. J.Gen. Microbiol. 43:159-271.

31. Warburg, O., and W. Christian. 1942. Isolierung undKristallisation des Garungsferments Enolase.Biochem. Z. 310:384-421.

32. Whittenbury, R., S. L. Davis, and J. F. Davey. 1970.Exospores and cysts formed by methane-utilizing bac-teria. J. Gen. Microbiol. 61:219-226.

33. Whittenbury, R., K. C. Phillips, and J. F. Wilkinson.1970. Enrichment, isolation and some properties ofmethane-utilizing bacteria. J. Gen. Microbiol. 61:205-218.

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