Biochem. J. (1966) 98, 804

Sucrose Utilization by Zymomonas mobilis:Formation of a Levan

BY E. A. DAWES* AND D. W. RIBBONStDepartment of Biochemistry, University of Glasgow, and

Department of Biochemistry, University of Hull

AND D. A. REESDepartment of Chemistry, University of Edinburgh

(Received 10 August 1965)

1. Molar growth-yield coefficients of Zymomonas mobilis for glucose, fructose,glucose plus fructose, and sucrose are reported. Yield coefficients for sucrose areappreciably lower than those for the equivalent concentrations of glucose plusfructose. 2. Only 2.6% of [U-14C]glucose supplied in the growth medium is incor-porated into cell substance by Z. mobilis utilizing glucose as the energy source.3. During growth on sucrose a levan is formed. It has been characterized andshown to resemble other bacterial levans. 4. Levan formation from sucrose couldbe demonstrated with both washed cell suspensions and cell extracts of Z. mobilis.5. Sucrose phosphorylase could not be demonstrated in extracts of the organism.

Tracer studies indicated that Zymomonas mobilisferments glucose via an anaerobic Entner-Doudoroffpathway (Gibbs & DeMoss, 1954) and we havereported that the key enzymes are present inextracts of these cells (Ribbons & Dawes, 1961;Dawes, Ribbons & Large, 1966). As Bauchop &Elsden (1960) pointed out, this is an inefficientpathway when compared with the glycolytic routeof ethanol formation in yeast. They demonstratedthat the growth yield of Z. mobilis per mole ofglucose was 8-3, a value which is approximatelyone-half of that obtained for Saccharomyces cere-visiae, but which is accounted for by the provisionof only 1 mole of ATP/mole of glucose fermented byan Entner-Doudoroff pathway. Our studies in-cluded determination of molar growth yields forZ. mobilis when glucose, fructose and sucrose wereprovided as sources of energy in peptone-yeastextract media, so that we might see how efficientlysucrose is utilized and perhaps distinguish whetherhydrolysis or phosphorolysis occurred. Molargrowth yields on sucrose were, however, erratic andlow and this we have shown to be due to the forma-tion ofa levan. The isolation, analysis and synthesisof the levan are described together with the molar-growth-yield experiments that led to its discovery.A preliminary account of this work has been pre-sented to The Biochemical Society (Ribbons,Dawes & Rees, 1962).

* Present address: Department of Biochemistry, Univer-sity of Hull.

t Present address: Milstead Laboratory of ChemicalEnzymology, Shell' Research Ltd., Sittingbourne, Kent.

EXPERIMENTAL

Organism, cultural conditions and preparation of cellextracts. Z. mobilis (N.C.I.B. 8938) was maintained, cul-tured and extracts were prepared from it as described inthe preceding paper (Dawes et al. 1966) except that, whererequired, sucrose was added (2%, w/v) to the growthmedium as an alternative energy source.

Chemicals and buffer solutions. These were as describedin the preceding paper (Dawes et al. 1966). [U-14C]Glucosewas obtained from The Radiochemical Centre, Amersham,Bucks.

Analytical methods

Chemical estimations. Fructose was determined with theresorcinol reagent (Umbreit, Burris & Stauffer, 1957).Total carbohydrate was estimated by the anthrone methodof Trevelyan & Harrison (1952). For other estimations seeDawes et al. (1966). Elemental analyses were performed byMr J. M. L. Cameron, Chemistry Department, Universityof Glasgow.Levan determination. (a) Levan was estimated turbi-

dimetrically in 1cm. cells with a Spekker absorptiometer byusing filters H503 and 603; linearity of levan concentrationagainst drum reading was observed up to 2-5mg. (1543,u-moles of fructose equivalent) of levan/ml., when the valuewas 0-615. Thereafter increments of drum readings de-creased as the levan concentration was further raised, e.g.4-25mg. of levan/ml. gives a reading of 0-91. (b) Levanwas estimated by measurement of the amount of totalfructose that is precipitated by 75% ethanol, after washingthe precipitate in 75% ethanol. Results have been expressedas iLmoles of fructose equivalent, assuming a mean residueweight of 162.

Cell fractionation. The procedures of Roberts, Abelson,Cowie, Bolton & Britten (1957) were used.

804

LEVAN FORMATION BY ZYMOMONAS MOBILISIsotopic procedures. The radioactivity ofthe fermentation

C02 trapped in alkali was measured by precipitation ofreplicate portions as BaCO3 by the methods described bySakami (1955). The radioactivity of cells was determinedby spreading portions (0.1 and 0-2ml.) of suitably dilutedsuspensions on stainless-steel planchets having an effectiveraised area of 1-5 cm.2 (Dawes & Holms,1958). Cell fractionswere plated in a similar manner. Ethanol was steam-distilled from supernatants at neutral pH and collected indichromate (Neish, 1952). The acetate formed was distilled,titrated with 0-05N-NaOH and portions (0.1 and 0-2ml.)of the resulting sodium acetate solution were spread onplanchets.

soo(a) "1

400 -

300 -

- 200

-, 100

, 0 10 20 30 40 50

us Conen. of glucose (mM)

c5 500

C) (C) I

c 400 ,

ann 7,

0 0 20 30 40 50

Concn. oftotal hexose (mM)

Conen. offructose (mM)

500(d)

400-

300

200 -

100 ~ /;

0 10 20 30 40 50

Concn. of hexoseequivalent (mM)

Fig. 1. Relationship between concentration of energysource and cell yield of Z. mobilis. Growth was measuredturbidimetrically and the experimental protocol was asdescribed by Bauchop & Elsden (1960). (a) Glucose; (b)fructose; (c) equimolar mixture of glucose and fructose;(d) sucrose. The points are the average values of six indi-vidual determinations for each concentration.

All estimations were made at or corrected to infinitethinness by using an end-window Geiger-Miller tube andan Ekco automatic scaler type N530 G. Counts were col-lected for a period sufficient to ensure that the countingerror was less than +2%. Corrections were applied forbackground and coincidence.Paper chromatography. Descending paper chromato-

grams were developed in the solvent systems: (a) water-satd. phenol+ 1% of aq. NH3 (sp.gr. 0.88); (b) butan-l-ol-acetic acid-water (4:1:5, by vol.); (c) propan-l-ol-ethylacetate-water (7:1:2, by vol.) (Feingold, Avigad & Hestrin,1956); (d) butan-l-ol-ethanol-water (5:1:4, by vol.; upperphase). Carbohydrates were detected with ammoniacalAgNO3 (Partridge, 1948; Trevelyan, Procter & Harrison,1950), aniline oxalate (Horrocks & Manning, 1949), urea-phosphate (Wise, Dimler, Davis & Rist, 1955) and triphenyl.tetrazolium chloride (Wallenfels, 1950).

Gas-phase chromatography. This was carried out with aPye Argon Gas Chromatograph with a 90Sr detector.Molar growth yields. The yields of Z. mobilis on glucose,

fructose and sucrose were generally determined turbidi-metrically in a manner identical with that employed byBauchop & Elsden (1960). Occasionally direct weightmeasurements were made on centrifuged and washed cul-tures as described by the same authors.

Spectrophotometry. Ultraviolet and visible spectra wereestablished as before (Dawes et al. 1966). Infrared spectrawere determined with a Perkin-Elmer Infracord withliquid paraffin (Nujol) mulls.

Ultracentrifuge. A Spinco model E ultracentrifuge wasused. The polysaccharide concentration was 2-7mg./ml.(water, solvent) and the rotor speed 11573 rev./min. Aschlieren angle of 550 and exposures of 15see. at 4min.intervals were employed. The temperature was 17.40.

RESULTS

Molar growth-yield coefficients

The relationship between total bacterial growthand the concentration of energy source provided isshown in Fig. 1. Glucose and fructose and anequimolar mixture of glucose and fructose allowgrowth yields (g. dry wt. of bacterial cells/mole ofhexose equivalent) of 9-32, 9-21 and 9-16, whereas

Table 1. Growth-yield coefficients of Zymomonas mobilis with glucose and sucrose assources of energy

Cultures were grown in 1% Bacto-Peptone-1% yeast-extract media supplemented with the energy sourcein 250ml. conical flasks. The culture volume was 155ml., and was inoculated with a loop of an exponential-phase culture developing on media of similar energy source which was limiting total growth. Cell densities weredetermined turbidimetrically and by direct weighing.

Total cell yield (mg.)

Conen. Turbidi- Direct(mM) metric weighing29-0 37-2 39-214-5 28-1 31-014-5 28-5 34-5

Yield coefficient

Turbidi-metric8-306-226-32

Directweighing

8-716-907-45

Vol. 98 805

Energysource

GlucoseSucroseSucrose

E. A. DAWES, D. W. RIBBONS AND D. A. REES

sucrose gives a yield of only 7-36, which variesfrom experiment to experiment between 5-35 and8-0. A direct determination of the weight of bac-terial cells is illustrated in Table 1, where a consis-tently lower yield of cells is obtained on sucrosethan on glucose. The yield of ethanol from thesesubstrates was 1-68 (glucose) and 1-35 (sucrose)moles/mole of hexose equivalent.We had previously demonstrated that the energy

source (with glucose) is not incorporated appreciablyinto cellular materials (Table 2); hence it was con-sidered likely that other metabolic products wereformed from sucrose as no sucrose could be detectedafter fermentation. The values which trace the fateof the carbon from [U-14C]glucose after fermenta-tion agree favourably with those obtained recentlyby Belaich & Senez (1965) and demonstrate that,even under conditions of excess of energy source,there is little incorporation into cellular material.Assuming a carbon content of cells of 50%, thenthe ratio of the specific activity of cellular carbonto the substrate glucose carbon is 0-83:1 0, i.e.

considerable dilution of the glucose into cellularmaterial has not occurred. The distribution of the14C in the cells was also fairly uniform, indicatingthat it is not accounted for by a specific storagematerial or by structural carbohydrate as inZymosarcina ventriculi (E. A. Dawes & D. W.Ribbons, unpublished work) (Table 3). The carbo-hydrate content of the organism is, in fact, verylow, representing only some 3% of the dry weight.

Isolation and analysi8 of the levan

Isolation. Centrifugation of sucrose cultures ofZ. mobilis for 1-2min. at 15000g yielded a cellpellet and an opalescent supernatant fluid whichcould be clarified by centrifugation at 25 000-30000g for 30min. The pellets obtained weretranslucent gels, and were easily resuspended andprecipitated by further centrifugation. Isolation ofsufficient material for its chemical characterizationwas achieved as follows.Four cultures (81. each) of Z. mobilis on 2%

Table 2. Fate of [U-14C]glucose during growth of Zymomonas mobilis in glucose-limiting cultures

A culture medium (53ml., 2% glucose, 1% Bacto-Peptone and 1% yeast extract) containing [U-14C]gluCose(3 x 106 counts/min.) was inoculated with a stationary-phase culture of Z. mobilis (5 ml.). A sample (5 ml.) wasimmediately withdrawn for chemical and radiochemical analysis and the rest of the culture harvested after16-5hr., washed twice with the same volume of water and then with 25ml. of water. The cells were suspended inwater and made up to 50ml., giving a cell density 581 ,ug. dry wt./ml. The culture supernatant (Iml.) was steam-distilled and excess of potassium dichromate oxidizing mixture (Neish, 1952) added to the distillate (4ml.).After standing for lhr. at 220 the acetic acid formed was colleeted by steam-distillation in a volume of 25ml. andportions were titrated with C02-free 0-05N-NaOH and plated and counted as sodium acetate. The radiochemicalyield of CO2 was calculated from the yield of ethanol in the fermentation, i.e. 1.6moles/mole of hexose.

Fermentation yields-- I Radiochemical distribution (counts/min.)

Glucosefermented(m-moles)

4-67

Ethanolformed

(m-moles)7-45

Cellyield(mg.)20-91

Glucoseutilized Ethanol2-6 x 106 1-32 x 106(100%) (50.8%)

CO27-6 x 105(26-7%)

Cells6-7 x 104(2.6%)

Table 3. Distribution of 14C in cellularfractions after growth on [U-14C]glucose

Cells from the experiment of Table 2 were used. Duplicate portions (5ml., 5-8mg. dry wt.)of a cell suspensionin water were centrifuged and subjected to the fractionation procedure of Roberts et al. (1957).

FractionWhole cells

Cold trichloroacetic acid (5%)Ethanol (75%, v/v)Hot trichloroacetic acid (5%)ResidueRecovery

Radioactivity(counts/min.)

27000

1920 16994280 52105600 62409788 10080

Percentageof total

radioactivity100

7-1 6-2515-8 19-220-8 23-036-0 37-279.7 85-65

806 1966

LEVAN FORMATION BY ZYMOMONAS MOBILIS

sucrose-Bacto-Peptone-yeast-extractmediumweregrown into the stationary phase. Centrifugation at50000g with a fast flow rate (500ml./min.) on theSharples Super-Centrifuge yielded a cloudy super-natant which was almost devoid of bacterial cells.The supernatants were cooled to 0-2° and centri-fuged in batches at 27000g for 30min. on an MSEangle 13 centrifuge. Supernatants were discardedand the sedimented gel was taken up into water(11.) and resedimented at 27 OOOg for 90min.; theprocess was repeated. Suspension of the pellets in600ml. of water yielded a grey-brown opalescentsolution which was treated with 1800ml. of indus-trial spirit. A brown gum immediately separatedto the bottom of the beaker, the cloudy whitesupernatant was clarified at 20000g for 5min. andthe pellets were combined with the gum. Solutionof the solid in 300ml. of water and addition ofethanol to 75% (v/v) reprecipitated the levan,which was taken up in 250-300ml. of water,filtered and dialysed for 24hr. against distilledwater at room temperature. Filtration of thedialysed preparation and freeze-drying produced afluffy white material which was dried over phos-phorus pentoxide. The yield was 4-5g.

Characterization of the polysaccharide as a fructo-san. Fructose was the only sugar that could bedetected in this hydrolysate by paper chromato-graphy with solvents (a) and (b). Polysaccharide(300mg.) was heated in 0-02 N-sulphuric acid (20ml.)at 1000 for 45min. The solution was neutralizedwith barium carbonate and concentrated to drynessunder diminished pressure. The resulting syrupwas thoroughly dried in a vacuum desiccator overphosphorus pentoxide and treated with acetone inthe presence of conc. sulphuric acid as described byBell (1947) to give 2,3: 4,5-di-isopropylidene-D-fructose: 250mg.; m.p. and mixed m.p. withauthentic material, 93.5-95°, [a]D - 34.50 (c 0.8 inwater). Analysis of a sample of levan which hadbeen pre-dried at 600 in vacuo for 24hr. by themethod of Roe (Umbreit et al. 1957) showed 94.5%of anhydrofructose residues. Nitrogen analysis gave< 0-01 %.Periodate oxidation. Fructosan (15-3mg.) was

dissolved in sodium metaperiodate (0-07M; 3ml.)and the solution left at room temperature, theperiodate uptake being determined from time totime by Aspinall & Ferrier's (1957) method. Therewas an initial rapid consumption of 0-96mole ofoxidant (within 5hr.), rising finally to a value of1-OOmole/mole of anhydrofructose (24hr.). Theseresults are consistent with a formula for the fructo-san consisting entirely of fructofuranose residueslinked 2,1 or 2,6 or both.

Partial acidic hydrolysis. Fructosan (20mg.) waspartially hydrolysed in 0- 1N-sulphuric acid (2 ml.)at 600 for lhr. After neutralization on IR-4B resin

and concentration the hydrolysate was examinedby paper chromatography with solvent (c). Forcomparison, samples of dahlia inulin and levan ofperennial rye-grass tops were treated in exactly thesame way. Zymomonas fructosan gave a seriesof oligosaccharides (detected up to the penta-saccharide) which resembled in Rp value and inbehaviour towards triphenyltetrazolium chloridespray (cf. Feingold et al. 1956) the series fromperennial rye-grass levan. In contrast the oligo-saccharides from dahlia inulin had different RFvalues and could not be detected with triphenyl-tetrazolium chloride, though they were readilydetected with alkaline silver nitrate (Trevelyan etal. 1950).

Methylation of levan. Levan (10g.) was stirredwith water (25ml.) until it dissolved and the flaskwas then cooled in an ice bath. Sodium hydroxide(30%, w/v; 45ml.) and dimethyl sulphate (15ml.)were added simultaneously in portions with vigor-ous stirring over 5hr. The reaction mixture wasallowed to reach room temperature and stirredovernight. The same reagents were added similarlythe following day (at room temperature) and theprocedure was repeated a further three times.Acetone was added from time to time to maintaincomplete solution. Finally the mixture was stirredat 60° for lhr., cooled, neutralized with ION-sulphuric acid, and the methylated polysaccharideextracted with chloroform. The combined extractswere washed once with water, evaporated underdiminished pressure to small volume and pouredinto excess of light petroleum (b.p. 60 80°). Theprecipitate was removed on the centrifuge, washedthoroughly with light petroleum, and dried in vacuoat 600 to give a buff amorphous solid (0-9g.), whichshowed no hydroxyl group absorption in the infra-red spectrum [Found: OCH3, 45-6; fully methy-lated fructosan requires OCH3, 45-5%. [(X]D-59.4+10 (c 0-6 in chloroform)].Paper chrotnatography of hydrolysed methylated

levan. The material was hydrolysed by the methodof Arni & Percival (1951) and the resulting syrupcompared with authentic specimens of methylatedfructoses by chromatography in solvent (d) withurea-phosphate spray. The hydrolysate containedthree components. The first (R, relative to tetra-methylfructose, 0-65) moved at the same rate asauthentic 3,4-di-0-methylfructose. The secondcomponent (relative RpO-84) moved at the samerate as authentic 1,3,4-tri-0-methylfructose.Authentic 3,4,6-tri-0-methylfructose on the samechromatogram could be distinguished in that (i) ittravelled a little faster, and (ii) it gave a verystrong reaction with triphenyltetrazolium spray,whereas the tri-O-methylfructose in the hydroly-sate, like the authentic 1,3,4-tri-0-methylfructose,gave no reaction. The third component moved at

Vol. 98 807

E. A. DAWES, D. W. RIBBONS AND D. A. REES

the same rate as authentic 1,3,4,6-tetra-O-methyl-fructose and gave a similar inky blue-green colourwith urea-phosphate spray. Similar amounts of di-and tetra-O-methylfructoses were present, asjudged from spot intensities, with larger amounts(six to ten times as much) of the tri-O-methyl-fructose.

Gas-phase chromatography of methanolysed methy-lated levan. The material (10mg.) was treated in asealed Pyrex tube with methanolic hydrogenchloride (2.5%; 1.5ml.) at 60° for 3hr. Afterneutralization with silver carbonate the filtrate wasconcentrated to a syrup and compared by gas-phasechromatography with authentic di-, tri- and tetra-O-methylfructoses which had been treated in thesame way. The systems used were (i) 15% butane-diol succinate polyester on Celite at 1750 and (ii)10% polyphenol on Celite at 2000 (Aspinall, 1963).The two tri-O-methylfructoses could be readilydistinguished (Aspinall, 1963) and peaks cor-responding to those given by the authentic1,3,4-isomer were found to be present. No 3,4,6-tri-O-methylfructose was detected. Other peaks on

the chart indicated the presence of 3,4-di- and1,3,4,6-tetra-O-methylfructose derivatives.

Ultracentrifugal analysis. Concentrated solutionsofthe levan showed one major and one slower minorpeak. Results obtained gave S20 w as 390 xl1-13sec.

Formation of levan

Formation during growth. Yields of levan from2% sucrose cultures varied between 100 and200mg./l. Neither glucose nor fructose supportedlevan synthesis. Raffinose was not tested as agrowth substrate.Formation by washed cell suspension. Fig. 2

demonstrates the course of sucrose degradation andlevan formation by non-growing cells. This experi-ment clearly shows that glucose is utilized fasterthan the fructose portion, and that levan synthesisstops when, at the most, the sucrose concentrationis 500,ug. (1-46,umoles)/ml. (i.e. the differencebetween total carbohydrate and fructose present).After about 4hr. incubation only free fructose isleft and this continues to be metabolized by thecells without levan formation.Enzymic synthesis. Levan is readily synthesized

by crude extracts of Z. mobilis, without addedprimer (Fig. 3). Approximately 10% of the sucrose

w0

So

o -fZ a

0

ox0 0

4 o0o) v

04fo oE°<

30 60 90 120 150 180 210Time (min.)

Fig. 2. Synthesis of levan by washed cell suspensions. Awashed cell suspension (15ml., 11-7mg./ml.) was obtainedfrom a 350ml. sucrose culture and added to phosphate-citrate buffer, pH5.6 (15ml.), and 15% (w/v) sucrose solu-tion (15ml.), all previously incubated at 300, and samples(2ml.) were taken as indicated. Samples were immediatelycentrifuged for 1-2min. on a bench centrifuge (2000g) toremove cells and subjected to analysis of levan (turbidi-metrically) (o), total carbohydrate (including levan) (A)and total fructose (-). Note that the levan scale differs bya factor of 10.

'5r-4

0 _

"o00

0

:a.

-4

;.4

0 c0o

C)C x

v ^r-

0 2 3 4 5 6Time (hr.)

Fig. 3. Enzymic formation of levan. The reaction mixturecontained: citrate-phosphate buffer, pH5-0 (3ml.); 15%(w/v) sucrose (3 ml.); enzyme solution (crude extract, 3 ml.,containing 74mg. of protein). Temperature, 32°. Samples(1 ml.) were taken for analysis as indicated. *, Levanformation (turbidimetric); o, total non-levan carbohydrate(anthrone), i.e. carbohydrate soluble in 75% ethanol; A,reducing sugar formed. Controls containing 75% ofethanolwere included in the anthrone assays (Binnie, Dawes &Holms, 1960). Reducing sugar was determined beforeethanol treatment on the supernatant after deprotein-ization by the method of Somogyi (1930).

808 1966

LEVAN FORMATION BY ZYMOMONAS MOBILIS

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Vol. 98 809

E. A. DAWES, D. W. RIBBONS AND D. A. REES

utilized by extracts is converted into levan, theremainder of the sucrose being hydrolysed toglucose and fructose. As might be expected, levanformation does not occur at a linear rate, as judgedby the methods we have employed to assay itsformation, namely increased turbidity during incu-bation, analysis of total carbohydrate that is in-soluble in 75% ethanol (not shown in this experi-ment), and also by difference from the analysis oftotal carbohydrate that is soluble in 75% ethanol(Fig. 3). This latter value also shows a lag beforeit starts to decline. The molecular size which mustbe attained before these turbidimetric or precipita-tion methods of analysis are accurate is not known.The reaction mixtures were not investigated furtherfor other products such as fructosylsucrose. How-ever, there seems little reason to doubt that thislevan sucrase differs from those previously studiedin Acetobacter levanicum (Hestrin, Feingold &Avigad, 1956) and Bacillu8 MUbtili8 (Dedonder,1960). The pH optimum is about 5 and the speci-

ficity of reaction extends to other fructofuranosyl-oc-D-aldosides, e.g. raffinose (Table 4).In crude unsupplemented extracts the main

products of sucrose metabolism are glucose andfructose. Addition of inorganic phosphate (or itsuse in buffer systems) neither stimulates nor inhibitsthe rate of sucrose metabolism, nor do the productsalter appreciably. It seems therefore that phos-phorolysis plays little part in sucrose degradationby Z. mobili8. Only when ATP is added to extractsdoes further metabolism of the hexoses occur.Table 5 records the formation of pyruvate fromglucose, fructose and sucrose by extracts supple-mented with ATP and NAD. The rate of sucrosedegradation is relatively low, irrespective of theenergy source used in the growth media, a factnoted earlier when washed suspensions of Z.mobili were allowed to ferment glucose, fructoseor sucrose (Table 6). However, this is a propertywhich may easily vary with pH value. The ratesof oxygen consumption by whole cells metabolizing

Table 5. Formation of pyruvate from sucrose, glUco8e and fructto8e

Reaction mixtures contained: 0 2M-glycylglycine, pH7.3 (2.Oml.); 20mM-carbohydrate as indicated (1Oml.);20mM-NAD (0-1 ml.); 40mM-MgCl2 (0-3ml.); 50mM-ATP (1-Oml.); 0-56M-hydrazine (1-2ml.); cell-free extract(0.5 ml., 12-5mg. of protein), the last-named addition initiating the reactions. Temperature, 300. Samples (lml.)were taken at intervals for analysis of pyruvate and carbohydrate (anthrone method).

Time(min.)

1-02-55102030

Carbohydrate substrate

Glucose Fructose Sucrose

Pyruvate Carbohydrate Pyruvate Carbohydrate Pyruvate Carbohydrate(,umoles/ml.) (ttmoles/ml.) (,umoles/ml.) (,umoles/ml.) (,umoles/ml.) (,umoles/ml.)

0X02 3X73 0410 3-32 0*08 9*400.08 3-21 0-20 3-28 9.000-39 2-52 0*33 2-60 9.000-52 1-74 0-52 1*64 0.19 8-950-76 0*53 0-79 0 39 0-251-26 0-58 1-16 0 40 0-36 8-60

Table 6. Relative rate8 of carbohydrate utilization by wa8hed 8w9pen8ion8 of Zymomonas mobilisgrown with different carbohydrate8

Carbon dioxide formation was measured manometrically under an N2+ CO2 (95:5) atmosphere. Each flaskcontained: O-lM-citrate buffer, pH6.6 (l1Oml.); substrate (I Oml., 10/tmoles); cell suspension (1Oml.). Tem-perature, 30°. Dry wt. (mg.) of cells per flask: (a) 3-3; (b) 5-5; (c) 7-2; (d) 4-2; (e) 4-2; (f) 8-6. Rates are expressedas pl. of CO2 evolved/hr./mg. bacterial dry wt. (Qco,).

Carbohydrate in _ _ _growth medium ... Glucose

Fermentationsubstrate (a) (b)

GlucoseFructoseSucrose

31954-53-6

Fermentation rates (Qco,)

Fructose

(c)

317 34660-0 1543-6 14-7

(d)358178

6-6

Sucrose

(e) (f)293 206137 7712-3 24-4

810 1966

Vol. 98 LEVAN FORMATION BY ZYMOMONAS MOBILIS 811

sucrose were also much slower than for glucose orfructose.

DISCUSSION

The observed low values for the molar growthyield of Z. mobili8 utilizing sucrose, which led tothe discovery of the levan, can be explained by thediversion of some of the energy source to fructanformation. Variations in the quantity of levanformed would account for the differences in yieldcoefficient recorded. Furthermore, the determina-tion of yield coefficient was subject to variationdue to (a) the light-scattering properties of thelevan and (b) the ease with which it is centrifugedwhen bacterial dry weights are measured.

Utilization of sucrose could conceivably occur byhydrolysis or by phosphorolysis. A sucrose phos-phorylase which catalyses reaction (1) is present in

Sucrose+ Pi= glucose 1-phosphate+ fructose (1)

P8eudomona8 8accharophila and Leuconostoc me8en-teroide8 (Doudoroff, 1955), but no evidence for theactivity of this enzyme in Z. mobili8 could beobtained. An organism which possesses sucrosephosphorylase is energetically more efficient in itsutilization of sucrose than one which does not, forphosphate is introduced into the glucose moleculewithout the expenditure of ATP. Consequently,where the Entner-Doudoroff pathway operates,the ATP yield by phosphorolysis would be 3, asopposed to 2moles/mole of sucrose by hydrolysis.The molar growth yield on sucrose should thus begreater than that on the equivalent concentrationof glucose plus fructose by a factor of 1-5 if phos-phorolysis occurs. The observed yields were, infact, substantially lower and, together with theenzymic evidence, it is concluded that Z. mobili8utilizes sucrose by a hydrolytic mechanism.Our results show clearly that levan formation is

accompanied by hydrolysis to glucose and fructose,but we have not established whether an invertase ispresent. Levan sucrase catalyses the reaction (2),

nC12H22011 + ROH >Sucrose acceptor

H(C6H1oO5).OH + n C6H1206 (2)levan glucose

where the acceptor is fructose and the donor eithersucrose or raffinose; if water is the acceptor, thenthe products will be fructose and glucose. Withcrude extracts the products of sucrose metabolismare glucose, fructose and levan; the hexoses are notfurther metabolized unless ATP is added. Thelevan sucrase alone could thus account for ourfindings.

Bacterial levans are a family of high-molecular-

weight fructans in which D-fructofuranosyl unitsare joined mainly by 2,6-linkages but with aboutevery ninth unit carrying a 2,1-branch-point(Hestrin, 1959), the glycosidic configuration prob-ably being , throughout. Z. mobil8 levan wouldappear to be a typical member of this family inevery respect. Periodate oxidation revealed that ifthe sugar units were in the furanose ring form, thenthe glycosidic linkages were attached exclusively toprimary hydroxyl groups. Partial hydrolysisshowed that the major linkage was 2,6, that largenumbers of such linkages were consecutive and thatmany (probably all) of the units were indeed in thefuranose ring form. Methylation analysis gaveresults that agreed with these conclusions andshowed further that 2,1-linkages were present inabout the usual proportions for bacterial levans,and that all these linkages were present at branch-points.The specific optical rotation of the methylation

polysaccharide confirmed the general similarity toother bacterial levans. The results of the partialhydrolysis study, together with the value for theoptical rotation of the methylated derivative,suggest that most, if not all, of the glycosidic link-ages have the f-configuration. The sedimentationcoefficient (390s) was similar to the values quoted(Feingold & Gehatia, 1957) for levans with mole-cular weights of the order of 107.

Dr G. 0. Aspinall very generously provided referencesamples of fructans and methylated sugars, and carriedout the analyses by gas chromatography. We thankProfessor SirEdmund Hirst, C.B.E., F.R.S., for his interest.Dr J. Pitts kindly provided the ultracentrifuge data. Weare grateful to Mrs I. Thomson and Mr J. Hastings fortechnical assistance.

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