JOURNAL OF BAC-ERIOLoGY, Apr. 1975, p. 47-53Copyright 0 1975 American Society for Microbiology

Vol. 122, No. 1Printed in U.S.A.

Growth of Pseudomonas C on C1 Compounds: EnzymeActivities in Extracts of Pseudomonas C Cells Grown on

Methanol, Formaldehyde, and Formate as Sole Carbon SourcesI. GOLDBERG AND R. I. MATELES*

Laboratory of Applied Microbiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel

Received for publication 6 January 1975

Pseudomonas C can grow on methanol, formaldehyde, or formate as solecarbon source. It is proposed that the assimilation of carbon by Pseudomonas Cgrown on different Cl growth substrates proceeds via one of two metabolicpathways, the serine pathway or the allulose pathway (the ribose phosphate cycleof formaldehyde fixation). This contention is based on the distribution of two keyenzymes, each of which appears to be specifically involved in one of theassimilation pathways, glycerate dehydrogenase (serine pathway) and hexosephosphate synthetase (allulose pathway). The assimilation of methanol inPseudomonas C cells appears to occur via the allulose pathway, whereas theutilization of formaldehyde or formate in cells grown on formaldehyde or formateas sole carbon sources appears to proceed by the serine pathway. When methanolis present together with formaldehyde or formate in the growth medium, theformaldehyde or formate is utilized by the allulose pathway.

Many organisms can utilize reduced Cl com-pounds as their sole carbon and energy sources(4, 15). Bacteria that can utilize methane,methanol, formate, or methylamine use one oftwo carbon assimilation pathways: the serinepathway or the allulose pathway (15). An essen-tial reaction in the serine pathway is the hy-droxymethylation of glycine by N5' 10-tetrahy-drofolic acid, which is derived from formalde-hyde in a tetrahydrofolic acid-dependent reac-tion to give serine. This reaction is catalyzed bythe enzyme serine hydroxymethyl transferase(10, 15). The serine is converted to hydroxypyr-uvate, which is reduced to glycerate by dihydro-nicotinamide adenine dinucleotide (NADH)-dependent glycerate dehydrogenase (10, 15).The first reaction in the allulose pathway is theacyloin addition of formaldehyde to ribose-5-phosphate to give allulose-6-phosphate. Thisreaction is catalyzed by the enzyme hexosephosphate synthetase (7, 11, 15).

Stieglitz and Mateles (17) have shown thepresence of hexose phosphate synthetase activ-ity in extracts of Pseudomonas C grown onmethanol as a sole carbon source. Recently (1),we described the conditions required for growthof Pseudomonas C on methanol, formaldehyde,or formate as sole carbon source. To determinewhich assimilation pathway functions inPseudomonas C grown on different C1 com-pounds, key enzyme reactions of the allulose

(hexose phosphate synthetase) and serine (glyc-erate dehydrogenase) pathways were tested incell extracts.

MATERIALS AND METHODSChemicals. Analytical-grade methanol and form-

aldehyde (38 to 40%, containing about 10% methanolas a stabilizer) were purchased from Palestine Fruta-rom Ltd., Haifa, Israel. Paraformaldehyde and formicacid of analytical grade were obtained from BritishDrug Houses Ltd., Poole, England. Hydroxypyruvicacid (lithium salt, grade III), dithiothreitol, NADH,dihydronicotinamide adenine dinucleotide phosphate(NADPH), and D-ribose-5-phosphate (barium salt)were obtained from Sigma Chemical Co., St. Louis,Mo. [ 4CJformaldehyde (10.5 mCi/mmol) and sodium[4C Iformate (59.9 mCi/mmol) were obtained fromthe Radiochemical Centre, Amersham, England. Allother chemicals were of reagent grade.Growth of organisms. The organism used in this

study, Pseudomonas C, has been described (2). Cellswere grown in batch cultures in 250-ml baffled flaskscontaining 50 ml of M-3 medium, pH 7.0 (1), supple-mented with Na2HPO4 (2 g/liter) and NaH2PO4 (0.9g/liter). Methanol (final concentration of 10 g/liter) orglucose (final concentration of 2 to 4 g/liter) wasadded to cell suspensions, and incubations werecarried out at 32 C in a New Brunswick gyratoryshaker, model G-25, at a shaking rate of 210 rpm.Culture densities were measured at 650 nm with aGilford model 240 spectrophotometer (Gilford Instru-ment Laboratories Inc., Oberlin, Ohio). Cells from thelate exponential phase of growth were harvested forenzyme assays, about 5 to 6 h after inoculation.

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When formate was used as a sole carbon source andgrowth-limiting nutrient, cells were grown in continu-ous culture in a New Brunswick BioFlo model C-30chemostat, at 35 C. The pH in the growth vessel was

maintained at 7.0 4 0.1 by an automatic titrationsystem that included a Radiometer pH meter model28, titrator model 11, and an Ingold combined elec-trode, type 401-MH. Cells were grown in M-3 medium(1) containing 3 g of methanol per liter with a dilutionrate of 0.29/h, 600 rpm, and 0.5 volume of air pervolume of growth medium per min. Formic acid was

added to the medium reservoir stepwise (0.2, 0.4, 0.8.... g/liter per 24 h) until the final concentration offormic acid was 5 g/liter. Then the medium was

changed to fresh M-3 medium containing formic acid(5 g/liter) and 0.1 to 0.15 mM p-aminobenzoic acid(PABA) without methanol. The dilution rate waschanged to 0.05/h, and after a steady state was

established the dilution rate was increased slowly to0.18/h. Samples were taken after a new steady statewas established, and the absorbance of the culture,measured at 650 nm, was about 1.0 (equivalent to 0.75mg [dry weightyml).When formaldehyde was used as a sole carbon

source and growth-limiting nutrient, Pseudomonas Ccells were grown in continuous culture on normal M-3medium (1), in which case an automatic titrationsystem was used, or on M-3 medium in whichammonium sulfate was replaced by urea (0.35 g/liter),in which case the pH remained at 7.0 without externalcontrol. Methanol (final concentration of 2 g/liter)was added to the mediunm and growth was at 35 Cwith a dilution rate of 0.3/h, 600 rpm, and aeration of0.5 volume of air per volume of growth medium permin. Heated paraformaldehyde was added stepwise tothe medium reservoir until the concentration of para-formaldehyde was 3 g/liter. After a steady state was

achieved, the medium was changed to methanol-freeM-3 medium containing heated paraformaldehyde(final concentration of 3 g/liter), PABA (0.15 mM),olic acid (0.1 mM), Tween-80 (0.1 ml/liter), andDL-serine (10 mg/liter). The dilution rate was changedto 0.05/h, and after a new steady state was establishedit was increased to 0.065/h. After a new steady statewas reached, samples were taken and the absorbanceof the culture was about 0.5.

For growth of Pseudomonas C on methanol pluseither formaldehyde or formate as growth-limitingnutrients, cells were grown in continuous culture onM-3 medium (1) supplemented with Na2HPO4 (0.9g/liter) and NaH2PO4 (0.45 g/liter). Methanol (1g/liter) was added to the medium and growth was at35 C with a dilution rate of 0.3/h, 600 rpm, and 0.5volume of air per volume of growth medium per min.After a steady state was achieved, heated paraformal-dehyde or formic acid was added stepwise to themedium reservoir until the concentration of formalde-hyde was 2 g/liter and that of formic acid was 3 g/liter.After a new steady state was achieved, cells were

harvested and processed as described below.For replica plating, M-3 medium was solidified

with 1.5% agar (Difco). The appropriate carbon source

was added before pouring plates. Colonies were repli-

cated by use of a velvet pad. From 100 to 200 colonieswere replicated on different agars.

Incorporation of [14C]formaldehyde and ["C]-formate. Pseudomonas C cells were grown in con-

tinuous culture with methanol plus either formalde-hyde or formate as described above. Samples were

taken at steady-state conditions and centrifuged, andthe cells were resuspended in M-3 medium containing80 mM methanol. Cell suspensions were incubated inbaffled flasks at 35 C with shaking at 210 rpm. At zerotime, [4C ]formaldehyde (final concentration of 5mM, 0.23 gCi/ml) or ["4C]formate (final concentra-tion of 10 mM, 0.2 gCi/ml) was added to cellsuspensions. At different times samples were takenand cells were centrifuged and washed with twoportions of 10 ml of M-3 medium. The cells wereextracted with 80% acetone, and the pellet obtainedafter centrifugation was heated with 5% trichloroace-tic acid at 90 C for 20 min. After cooling, thesuspension was centrifuged and the insoluble materialobtained was dissolved in a solution of 0.1 N NaOHcontaining 0.2% sodium lauryl sulfate. Samples weretaken and counted in Bray solution in a liquidscintillation counter.

Preparation of cell extracts. Cells were harvestedby centrifugation for 5 min at 10,000 x g (allmanipulations were carried out at 4 C). Cells were

then washed and suspended in about 10 ml of 50 mMsodium phosphate buffer, pH 7.0, containing 5 mMMgCl2. The washed cells were ruptured in a Frenchpressure cell (American Instrument Co. Inc., SilverSpring, Md.) operating at 20,000 lb/in2. The crudeextract was centrifuged for 20 min at 25,000 x g. Thesupernatant fluid (referred to as "extract") was as-sayed immediately. The cell sediment fraction was

suspended in 2 ml of the above-mentioned buffer andwas assayed for hexose phosphate synthetase activity.

Fractionations of the 25,000 x g supernatant solu-tions with (NH4)2SO4 were carried out by the slowaddition of solid ammonium sulfate at 4 C. Afterstirring for about 15 min, the suspension was cen-

trifuged and the precipitates were dissolved in 5 ml of50 mM sodium phosphate buffer, pH 7.0, containing 1mM dithiothreitol and 5 mM MgC92.Enzyme assays. (i) Hexose phosphate synthe-

tase. This activity was assayed according to Law-rence et al. (11), using the modification of Stieglitzand Mateles (17).

It was established that in several isolates of meth-ane-utilizing bacteria (9) as well as in PseudomonasC (17), considerable hexose phosphate synthetaseactivity was found in the cell sediment fractionsobtained from centrifugation of the crude cell ex-tracts. Thus, the activity of the enzyme was assayedwith the supernatant fluids and the cell sedimentfractions obtained after centrifugation of the crudeextracts at 25,000 x g for 20 min. The reaction was

linear for 3 min at 30 C with up to 100 ,g of protein inthe assay mixtures, using 4 gmol of ribose-5-phos-phate and 2 umol of ['4CJformaldehyde (0.04 MCi) inthe reaction volume. Zero-time reactions and assays

minus ribose-5-phosphate gave very small backgroundvalues as compared with the complete assays. One

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unit of enzyme activity is defmed as the amount ofenzyme reqired for the incorporation of 1 jmol offormaldehyde/h. Specific activity is expressed asenzyme units per milIigram of protein.

(li) Glyewate dehydrogease (hydroxypyruvatereductase; EC 1.1.1.29). The enzyme activity wasassayed as desribed by Large and Quayle (10). Theasay mixtures contaned, in a final volume of 3 ml,100 pmol of sodium phosphate buffer (pH 7.0), 0.4ismol of NADH or NADPH, and enzyme preparationcontaining up to 1.7 mg of protein. Lithium hydroxy-pyruvate (2 Mmol) was added, and the decrease inextinction at 340 nm was measured. For each concen-tration of protein, a control was run without lithiumhydroxypyruvate. The difference between the activi-ties in the presence and absence of hydroxypyruvateconstitutes the glycerate dehydrogenase activity. Par-allel samples in which pyruvate was used in place ofhydroxypyruvate showed no reaction and thus ruleout the possibility that hydroxypyruvate reductionwas due to lactate dehydrogenase (16). One unit ofenzyme activity is defined as the amount of enzymerequired for the oxidation of 1 gmol of NADH orNADPH/h. Specific activity is expressed as enzymeunits per milligram of protein.

Chemical deterninations. Protein was deter-mined by the method of Lowry et al. (13), with bovineserum albumin as a standard. Methanol was deter-mined by direct injection of the growth medium into acolumn (50/80 mesh, Porapak Q) of a Varian Aero-graph 1200 gas chromatograph with a hydrogen flamedetector. For injections of 3 to 5 ;d, a direct relationwas observed between peak height and methanolconcentrations between 0 and 20 g/liter (2). Formatewas determined in the growth medium with potas-sium permanganate (1). Formaldehyde was deter-mined either by the acetylacetone method of Nash (6)with formaldehyde as the standard or by the chromo-tropic method (14) with formaldehyde-sulfite as thestandard.

RESULTSGlycerate dehydrogenase activity in ex-

tracts of cells grown on different carbonsources. Extracts of Pseudomonas C cellsgrown on formate as a sole carbon source had anNADH-dependent glycerate dehydrogenase ac-tivity that was almost completely dependent onthe presence of hydroxypyruvate (Fig. 1A).Similar results were obtained in extracts offormaldehyde-grown cells (results not shown).An NADH-oxidizing activity independent of

hydroxypyruvate was found in extracts of meth-anol-grown cells (Fig. 10). The specific activ-ity of the hydroxypyruvate-independent NADHoxidase in these cells was 13.8 (Fig. 10). Sincethe demonstration of NADH-dependent glycer-ate dehydrogenase in methanol-grown cells iscomplicated by the presence of relatively highactivity of NADH oxidase, an attempt wasmade to separate these activities.

About 84% of the total NADH-dependentglycerate dehydrogenase activity in extracts offormate-grown cells was found in the 50 to 70%(NH.)SO4 fraction of the 25,000 x g superna-tant fluid (Table 1). This activity was purifiedby about 2.9-fold by (NH4)2SO fractionation.In the 50 to 70% (NH4)2SO fraction, as well asin other fractions, there was no detectableNADH-oxidizing activity in the absence ofhydroxypyruvate. When the same procedure of(NH4)2SO4 fractionation was applied to the25,000 x g supernatant fluid of methanol-growncells, no detectable NADH-oxidizing activity,with or without hydroxypyruvate, was found inthe 50 to 70%o (NH4)2SO4 fraction (Table 1).

1.3 A

0.9

Eca

-4>%

U

c

w

.2

.4,06

0.5

1 3

0.9

0 .5

I 1 62 4 6

-c

2 6 10

1.1

071

_B

0

H

0 3h_

I I

2 6 10

-D I

Time of incubation ( min.)FIG. 1. NADH- and NADPH-oxidizing activities

in extracts of Pseudomonas C grown on methanol orformate as sole carbon source. Cells were grown withmethanol in a batch culture and with formate in acontinuous culture as described in the text. Assayswere carried out as described for glycerate dehydro-genase. Symbols: (0) incubation mixture withouthydroxypyruvate; (0) incubation mixture includinghydroxypyruvate. (A) NADH-oxidizing activity informate-grown cells. Assay mixture contained 62.5 ,gofprotein. (B) NADPH-oxidizing activity in formate-grown cells. Assay mixture contained 250 ug ofprotein. (C) NADH-oxidizing activity in methanol-grown cells. Assay mixture contained 330 Ag ofprotein. (D) NADPH-oxidizing activity in extracts ofmethanol-grown cells. Assay mixture contained 1,650Mg of protein.

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Activity of NADH oxidation independent ofhydroxypyruvate was found in the 0 to 25% andin the 25 to 50% (NH4)2SO4 fractions of the25,000 x g supernatant fluid obtained frommethanol-grown cells. These results indicatethe absence of NADH-dependent glycerate de-hydrogenase activity in methanol-grown cells ofPseudomonas C (Tables 1 and 2).When Pseudomonas C cells were grown with

formaldehyde or formate as sole carbon source,the specific activities of NADH-dependent glyc-erate dehydrogenase was 55.3 and 74.4, respec-tively (Table 2). The specific activity of thisenzyme found in extracts of glucose-grown cellswas 3.69 (Table 2).

Activity of glycerate dehydrogenase, withNADPH as the reductant, could be measured in

extracts of cells grown on different carbonsources (Fig. 1B and 1D; Table 2). This activityin extracts of formaldehyde- or formate-growncells was about 10% of the activity found withNADH as the coenzyme. An NADPH-depend-ent glycerate dehydrogenase activity was foundin methanol-grown cells, but this activity wasonly 4 to 7% of the activity found in formalde-hyde- or formate-grown cells (Table 2, Fig. 1D).Low activity of NADPH-dependent glyceratedehydrogenase was found in extracts of glucose-grown cells.Hexose phosphate synthetase activity in

extracts of cells grown on different carbonsources. There was hexose phosphate synthe-tase activity in the supernatant fluid from25,000 x g centrifugation of the crude extract of

TABLE 1. Ammonium sulfate fractionation of NADH-oxidizing activities in extracts ofPseudomonas C grownon methanol or formate as sole carbon sourcea

Methanol' Formateb

Fraction -Li HPc +Li HP- -Li HP +Li HP

EUd SP act, EU Sp act EU Sp act EU Sp act

Supernatant fluid, 25,000 x g 33 14 33 14 0.25 0.03 535 74Precipitate, 0-25% 14 35 14 35 ND' ND 10 7.5(NH4)2SO4

Precipitate, 25-50% 18 17 18 17 ND ND 60 9(NH4)2S04

Precipitate, 50-70% ND ND ND 450 214(NH4)2S04

Supernatant fluid, 70% ND ND ND 6.7 7.5(NH4)2S04

a For experimental conditions see text.Cells were grown on methanol (10 g/liter) in batch culture and on formate in continuous culture.

cLi HP, Lithium hydroxypyruvate. Minus indicates the absence and plus the presence of lithiumhydroxypyruvate in the assay mixtures.

d Enzyme units; micromoles of NADH oxidized per hour.e'Expressed as enzyme units per milligram of protein.I ND, No detectable activity was observed.

TABLE 2. Enzyme activities in extracts of Pseudomonas C cells grown on different carbon sourcesa

Carbon source (U/mg of protein)Enzyme

Methanol" Formaldehyde Formate Glucoseb

Hexose phosphate synthetase-superna-tant fluidc................ 125 < 1 < 1 < 1

Hexose phosphate synthetase-cell sedi-ment fractionc ................... .. 25 0.1 <0.1 <0.1

Glycerate dehydrogenase-NADH ........ < 0. ld 55.3 74.4 3.69Glycerate dehydrogenase-NADPH 0.33d 4.5 7.9 0.8

a Numbers represent values for specific activities defined as enzyme units per milligram of protein. Eachvalue represents the average of two or three determinations of separate extracts.

b Cells were grown with methanol or glucose in batch cultures.c The supernatant fluids and the cell sediment fractions were obtained by centrifugation of the crude cell

extracts at 25,000 x g for 20 min.dActivity was measured as described in Table 1.

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Pseudomonas C cells grown in M-3 mediumwith methanol (10 g/liter) (Table 2). Aboutone-sixth of the total hexose phosphate synthe-tase activity was found in the cell sedimentfraction obtained from centrifugation of thecrude extract.Hexose phosphate synthetase activity could

not be detected (Table 2) in the extracts ofPseudomonas C cells grown on formaldehyde,formate, or glucose as sole growth substrate.Mixing extracts of methanol-grown cells withextracts of formate- or glucose-grown cells didnot decrease the activity found in extracts ofmethanol-grown cells.The question arose whether the same cells

that can utilize methanol as a sole carbonsource can utilize other C, compounds in thepresence or absence of methanol. To answer thisquestion, formate-grown cells were diluted andplated on agar plates containing 2.5 g of sodiumformate per liter. After 48 h, replica platingswere performed to agar plates containing 2.5 g

of sodium formate, 10 g of methanol, or 4 g ofglucose per liter, or no added carbon source.Identical numbers and positions of colonieswere obtained on the three plates containingadded carbon sources, whereas no colonies werefound on the plate containing only salts andagar.Enzyme activities in extracts of cells

grown on methanol plus either formaldehydeor formate. The conditions required for thegrowth of Pseudomonas C in medium contain-ing methanol plus either formaldehyde or for-mate have been recently described (1). It was

found that both formaldehyde and formate are

incorporated and utilized by the cells togetherwith methanol. The steady-state concentrationsfor residual methanol in the growth vessel wereless than 50 mg/liter at all times, either in theabsence or presence of formaldehyde or formate.The steady-state concentrations of residualformaldehyde or formate were less than 20 and 5mg/liter, respectively, at all times. Cells grownon methanol plus formaldehyde or methanolplus formate could incorporate formaldehyde or

formate into trichloroacetic acid-insoluble ma-

terial (Table 3).There was hexose phosphate synthetase ac-

tivity in extracts of Pseudomonas C cells grownon methanol plus formaldehyde or on methanolplus formate as the only carbon sources (Table3). The activity of the enzyme in extracts ofcells grown on methanol alone was substantiallythe same as that found in extracts of cells grownon methanol plus either formate or formalde-hyde. About 30% of the total hexose phosphatesynthetase activity of extracts prepared from

TABLE 3. Incorporation of [14C]formaldehyde and[14Cjformate into Pseudomonas C cells grown incontinuous culture with methanol plus either

formaldehyde or formatea

Growth substrates

Incubation Methanol + formalde- Methanol + formatetime (min) hyde (incorporation of (incorporation of

[14C]formaldehyde)" [14C formate)b

10 4,000 4,26020 8,650 6,81030 15,625 10,38040 21,250 13,14050 26,786 17,66060 33,125 22,800

aFor experimental conditions see the text.b Numbers represent counts per minute per milli-

liter of cell suspension. Radioactivity was measured inthe hot trichloroacetic acid-insoluble fraction of thecells.

these cells was present in the cell sedimentfractions. On the other hand, there was nodetectable activity of NADH-dependent glycer-ate dehydrogenase in extracts of cells grown onmethanol, methanol plus formaldehyde, ormethanol plus formate (Table 4). The activitiesof NADPH-dependent glycerate dehydrogenasefound in extracts of cells grown on methanolplus either formaldehyde or formate (Table 4)were only 0.4 to 2% of the activities of thisenzyme found in extracts of formaldehyde- orformate-grown cells (Table 2).

DISCUSSIONFrom the results presented here it seems that

Pseudomonas C can utilize different Cl com-pounds by two alternative assimilation path-ways. The presence of hexose phosphate synthe-tase and the finding of little, if any, glyceratedehydrogenase activity in extracts of Pseudo-monas C cells grown on methanol (Tables 1 and2; 17) indicated the presence of the allulosepathway of carbon assimilation in these cells.On the other hand, the presence of glyceratedehydrogenase activity and the absence of hex-ose phosphate synthetase activity in extractsof Pseudomonas C grown on formaldehyde orformate as sole carbon sources (Tables 1 and 2)indicated the assimilation of these C1 com-pounds via the serine pathway.An additional indication for this contention,

that methanol is utilized by one metabolicpathway and formaldehyde or formate by an-other, is indicated by the effect of PABA on thegrowth of Pseudomonas C on different C1 com-pounds (1). PABA is a precursor of tetrahy-drofolic acid that is involved in the assimilation

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TABLE 4. Enzyme activities in extracts of Pseudomonas C cells grown on different C, growth substratesa

Carbon source" (U/mg of protein)Enzyme Methanol + Methanol

Methanol formaldehyde + formate

Hexose phosphate synthetase-supernatant fluidc ..... ..... 102 85 108Hexose phosphate synthetase-cell sediment fractionc .... 41 36.8 56Glycerate dehydrogenase-NADH ........................ < 0.01 < 0.01 < 0.01Glycerate dehydrogenase-NADPH ........ ............... 0.08 0.02 0.16

a Numbers represent values for specific activities defined as enzyme units per milligram of protein.b Cells were grown with methanol, methanol plus formaldehyde, and methanol plus formate in continuous

culture as described in the text.c The supernatant fluids and the cell sediment fractions were obtained by centrifugation of the crude cell

extracts at 25,000 x g for 20 min.

of Cl compounds via the serine pathway, butnot in the assimilation of these compounds bythe allulose pathway (15). PABA significantlyraised the yield of formate-grown cells, had lesseffect on the yield of formaldehyde-grown cells,and had no effect on the yield of methanol-grown cells.The operation of two different metabolic

pathways for the utilization of different C,compounds in Pseudomonas C is in contrastwith the findings obtained with PseudomonasAM-1 cells (10). Pseudomonas AM-1 is capableof growth on methanol or formate as sole carbonsource (10). The activities of the enzymes in-volved in the serine pathway are similar inextracts of methanol- or formate-grown cells.The specific activity of NADH-dependent glyc-erate dehydrogenase was 94.2 and 41.6 in ex-tracts of Pseudomonas AM-1 grown on metha-nol or formate, respectively (10). In addition,the radioactivity from ["C]methanol or[I4C ]formate was initially incorporated into ser-ine when the labeled compounds were added tomethanol - or formate-grown PseudomonasAM-1 cells, respectively (9).The specific activity of hexose phosphate

synthetase in Pseudomonas C cells grown onmethanol as a sole carbon source is comparableto that found in those of the methane-utilizingbacteria, where they range from 18 to 132 (12).The activity of this enzyme in Pseudomonas Ccells grown on methanol was about two timeshigher than the activity found in bacterium 4B6grown on trimethylamine as a sole carbonsource (3). In all these bacteria no detectableactivity of NADH-dependent glycerate dehy-drogenase was found (3, 12).The specific activities of NADH-dependent

glycerate dehydrogenase detected in extracts ofPseudomonas C grown on formaldehyde or for-mate as sole carbon and energy sources (Table2) are comparable to the activities reported in

other bacteria, falling in the range of 35 to 94 (3,10, 12). In all these bacteria no detectablehexose phosphate synthetase activity was found(3, 10, 12).The activity of glycerate dehydrogenase in

Pseudomonas C was dependent on NADH.Activity on NADPH was about 10% of thatobserved on NADH (Fig. 1, Table 2).The utilization of formaldehyde and formate,

when utilized together with methanol byPseudomonas C cells, appears to occur via theallulose pathway. Evidence for this is that inextracts of Pseudomonas C cells grown in con-tinuous culture with methanol plus formalde-hyde or methanol plus formate as the onlycarbon substrate, hexose phosphate synthetaseactivity was present, whereas there was virtu-ally no activity of NADH-dependent glyceratedehydrogenase (Table 4). The label from["C ]formate or ['IC Iformaldehyde was incorpo-rated into trichloroacetic acid-insoluble mate-rial when cells were grown on methanol plusformate or methanol plus formaldehyde (Table3). These results are analogous to those ob-tained by Stieglitz and Mateles (17), who foundthat cells grown batchwise on methanol couldincorporate [1 'C ]formate or [4C ]formaldehydeinto trichloroacetic acid-insoluble material ifmethanol was present.The possible existence of two pathways for

assimilation of different C, in a single organismis suggested by the results of Kemp and Quayle(8) working with P. methanica. In that bacte-rium, which can grow on methane as a solecarbon source, [1 C]methane is rapidly assimi-lated mainly into sugar phosphates. When["IC ]methanol or [ l4C ]formaldehyde was addedto methane-grown cells, these labeled com-pounds were also incorporated mainly intophosphorylated sugars. However, when ["C ]-formate was added to methane-grown cells, itwas incorporated into serine and malate, sug-

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gesting two different metabolic pathways forthe assimilation of different Cl compounds inthe presence of methane.

It was shown that growing Pseudomonas C inthe presence of methanol will result in thepresence of hexose phosphate synthetase activ-ity and in the absence of glycerate dehydrogen-ase activity, even if formaldehyde or formateare utilized simultaneously with methanol. Thefact that in formaldehyde-, formate-, or glucose-grown cells hexose phosphate synthetase activ-ity was not detected whereas glycerate dehydro-genase was present seems to indicate thatmethanol will induce the synthesis of hexosephosphate synthetase and will cause a repres-sion of synthesis of glycerate dehydrogenase.

It is currently thought that there are twocategories of organisms able to grow on reducedC1 compounds as their sole source of carbon andenergy (5). These are: (i) the obligate methylo-trophs, whose growth is strictly dependent onthe presence of methane or methanol; and (ii)the facultative methylotrophs, which do notgrow on methane but can use methanol, meth-ylamine, formate, or other organic compoundsas their sole carbon and energy sources. Thefirst group can use one of two pathways toassimilate Cl compounds: the serine pathway orthe allulose pathway (5). The second group usesthe serine pathway (5).

Until recently, the allulose pathway had beenobserved only in those obligate methylotrophsable to grow on methane (5). The results pre-sented in this work indicating that Pseudomo-nas C uses different pathways of carbon assimi-lation when grown on different C1 compoundsprovide further support for this non-obligatoryassociation of hexose phosphate synthetasewith methane-utilizing bacteria.Since Pseudomonas C is able to grow on

methanol and on formaldehyde or formate, itsproposed oxidation intermediates (15), use ofthis organism facilitates the study of the mech-anisms involved in the oxidation and in theassimilation of these C1 compounds.

ACKNOWLEDGMENTSThis research was supported in part by grants from the

United States-Israel Binational Science Foundation and the

Bat-Sheva de Rothschild Fund for the Advancement ofScience and Technology.

LITERATURE CITED

1. Battat, E., I. Goldberg, and R. I. Mateles. 1974. Growthof Pseudomonas C on C, compounds: Continuousculture. Appl. Microbiol. 28:906-911.

2. Chalfan, Y., and R. I. Mateles. 1972. New pseudomonadutilizing methanol for growth. Appl. Microbiol.23:135-140.

3. Colby, J., and L. J. Zatman. 1972. Hexose phosphatesynthetase and tricarboxylic acid-cycle enzymes inbacterium 4B6, an obligate methylotroph. Biochem. J.128:1373-1376.

4. Cooney, C. L., and W. D. Levine. 1972. Microbialutilization of methanol. Adv. Appl. Microbiol.15:337-365.

5. Dahl, J. S., R. J. Mehta, and D. S. Hoare. 1972. Newobligate methylotroph. J. Bacteriol. 109:916-921.

6. Horwitz, W. (ed.) 1967. Methods of analysis. Assoc. Offic.Agric. Chem. 512.

7. Kemp, M. B., and J. R. Quayle. 1966. Microbial growthon C, compounds. Incorporation of C, units intoallulose phosphate by extracts of Pseudomonasmethanica. Biochem. J. 99:41-48.

8. Kemp, M. B., and J. R. Quayle. 1967. Microbial growthon C, compounds. Uptake of "4C-formaldehyde and"lC-formate by methane-grown Pseudomonasmethanica and determination of the hexose labellingpattern after brief incubation with "4C-methanol. Bio-chem. J. 102:94-102.

9. Large, P. J., D. Peel, and J. R. Quayle. 1961. Microbialgrowth on C, compounds. 2. Synthesis of cell constitu-ents by methanol- and formate-grown PseudomonasAM-1, and methanol-grown Hyphomicrobium vulgare.Biochem. J. 81:470-480.

10. Large, P. J., and J. R. Quayle. 1963. Microbial growth onC, compounds. 5. Enzyme activities in extracts ofPseudomonas AM-1. Biochem. J. 87:386-396.

11. Lawrence, A. J., M. B. Kemp, and J. R. Quayle. 1970.Synthesis of cell constituents by methane-grown Me-thylococcus capsulatus and Methylomonasmethanooxidans. Biochem. J. 116:631-639.

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

13. 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.

14. MacFadyen, D. A. 1945. Estimation of formaldehyde inbiological mixtures. J. Biol. Chem. 158:107-133.

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

16. Stafford, H. A., A. Magoldi, and B. J. Vennesland. 1954.The enzymatic reduction of hydroxypyruvic acid toD-glyceric acid in higher plants. J. Biol. Chem.207:621-629.

17. Stieglitz, B., and R. I. Mateles. 1973. Methanol metabo-lism in pseudomonad C. J. Bacteriol. 114:390-398.

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