APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1993, p. 837-8420099-2240/93/030837-06$02.00/0Copyright X 1993, American Society for Microbiology

Involvement of an Intracellular OligogalacturonateHydrolase in Metabolism of Pectin byClostridium thernosaccharolyticum

MARION VAN RIJSSEL, MARTEN P. SMIDT,t GISELLA VAN KOUWEN, AND THEO A. HANSEN*

Department of Microbiology, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands

Received 17 September 1992/Accepted 16 December 1992

The enzymes pectin methylesterase and polygalacturonate hydrolase, which are responsible for the initialsteps of pectin degradation by Clostridium thermosaccharolyticum, were shown to be induced on the polymericsubstrates pectin and pectate, as well as on oligogalacturonates, and to be repressed in the presence of glucose.The digalacturonate and trigalacturonate produced by the extraceliular pectin methylesterase-polygalactur-onate hydrolase complex were transported across the cytoplasmic membrane and hydrolyzed by an inducibleoligogalacturonate hydrolase to galacturonate. The oligogalacturonate hydrolase was separated from thepolygalacturonate hydrolase and characterized. Its temperature optimum was 65°C, and its pH optimum was

6. The native molecular size was 90 kDa, and the enzyme was stable for more than 1 h at 65°C. The maximumreaction rate on oligomers decreased with the increasing degree of polymerization. Galacturonate was releasedby hydrolysis from the nonreducing end of the oligomer. The amounts of pectinolytic enzymes produced were

all strictly correlated to the amount of biomass formed. Galacturonate was metabolized via a modifiedEntner-Doudoroff route.

Pectin is a heteropolysaccharide consisting mainly oflinear chains of a-1,4-linked D-galacturonan. Part of thegalacturonate residues is esterified with methanol. Neutralsugars are mainly present in the so-called hairy regions (23,25). Enzymes catalyzing the degradation of pectin have beenstudied extensively in the past decades because of theirinvolvement in food spoilage and plant diseases; in addition,some of them are used in food technology (6, 23). Pectinasescomprise pectin methylesterases, which liberate the metha-nol esterified to the C6 of galacturonate residues and depoly-merases. The depolymerases hydrolyze the glycosidic bondsor break them via n-elimination. The action of these hydro-lases and lyases can initiate from the end of the chains or

randomly. Some of these enzymes are specifically activetowards oligogalacturonates instead of the polymeric sub-strate and, therefore, are true oligomerases. The result ofdepolymerization is the formation of (oligo)galacturonate(s)or unsaturated (oligo)galacturonate(s). (Unsaturated) galac-turonate and/or small oligomers can be taken up by the cell,and the oligomers are further converted by oligogalactur-onases into (unsaturated) monomers (6, 23, 30).

In our laboratory, Clostridium thermosaccharolyticumHaren was isolated by enrichment on pectin; pectin degra-dation was catalyzed by an extracellular complex with bothpolygalacturonate (PGA) hydrolase and pectin methyl-esterase activity, resulting in the formation of digalactur-onate and some trigalacturonate (28, 29). The fate of theextracellularly produced oligogalacturonates in this organ-ism remained to be elucidated.Research on (galact)uronate metabolism has concentrated

on gram-negative bacteria of the genera Erwinia, Pseudo-monas, Escherichia, and Agrobacterium (6, 15, 21). Ur-onates are metabolized either via modified Entner-Doudoroffroutes with 2-keto-3-deoxygluconate (KDG) and phospho-

* Corresponding author.t Present address: Department of Biochemistry, University of

Groningen, NL-9747 AG Groningen, The Netherlands.

ketodeoxygluconate (KDPG) as intermediates or via a path-way yielding ketoglutaric acid which enters the tricarboxylicacid cycle (23). Until now, little information about uronatemetabolism in gram-positive bacteria has been available.The aim of this study was to describe how oligogalactur-

onates are metabolized by C. thermosaccharolyticum andhow the synthesis of the enzymes involved in the initial stepsof pectin degradation by this gram-positive organism isregulated.

MATERIALS AND METHODS

Organisms and cultivation. C. thermosaccharolyticumHaren was originally isolated from pond sediment (29). Theorganism was cultivated at 58°C under an atmosphere of N2.The medium used was derived from low-phosphate-bufferedbasal medium as described previously (29); cysteine (1 mM)was added as a reducing agent. Alcaligenes sp. strain M250was kindly provided by K. Kersters (University of Ghent,Ghent, Belgium) and cultivated at 30°C in a rotary shaker(14). Growth was monitored by the measurement of theoptical density of the culture at 660 nm (OD6.) in a 1-cmcuvette with a Hitachi U-1100 spectrophotometer (an OD of1.0 was equivalent to 0.48 g of cell carbon per liter). Thisvalue was, if necessary, corrected for the OD of the pectinpresent.

Substrates. Citrus pectin, pectate from orange, galactur-onic acid, and 6-phosphogalacturonate were purchased fromSigma. A mixture of digalacturonate and trigalacturonateused for routine assays of oligogalacturonate hydrolaseactivity was obtained as follows. The supernatant of a

continuous culture of C. thermosaccharolyticum (5 g ofpectin per liter; dilution rate of 0.3 h-1) was concentrated35-fold by Millipore filtration (type PTGC, 10,000-molecular-weight cutoff) and brought to pH 4 with 0.1 N HCI. Theprecipitate formed was removed by centrifugation (39,000 xg for 30 min). The preparation was diluted 10-fold with 20 gof pectate (pH 4) per liter and incubated for 1 h at 60°C. The

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polymeric fraction was precipitated with cold ethanol (67%[vol/vol]) after conversion to the free acid by passage overDowex-1 (H+). The supernatant contained 23 mM reducingsugars after evaporation of the ethanol at 40°C. Thin-layerchromatography (16) showed that the preparation consistedmainly of digalacturonate and smaller amounts of trigalactu-ronate, whereas longer oligoniers were absent. Pure oligo-mers for kinetic studies were obtained as described previ-ously (28).

Reduction of a pentagalacturonate solution was achievedby adding an equal volume of NaBH4 (20 mg/ml) in 2 Nammonia and incubating the mixture for 15 min, after whichacetic acid was added until pH 4 was reached. The polymericfraction of pectate free from small oligomers was obtainedby precipitation of a 2% pectate solution with cold ethanol,followed by filtration (filter no. 860; Schleicher & Schuell).A mixture containing 2-keto-3-deoxygluconate and glu-

conate was obtained by the conversion of gluconate by a cellextract of Alcaligenes sp. strain M250 (13).

Substrates for growth were autoclaved for 20 min at 120°Cor, in the case of the oligomers, galacturonate, and galac-tose, filter sterilized.

Preparation of cell extracts. Cells were harvested in theexponential growth phase by centrifugation (15 min at 5,800x g) and washed three times with 50 mM sodium phosphatebuffer (pH 7.1). Cells were disrupted either by ultrasonicdisintegration or by passage through a French pressure celloperating at 106 MPa. Unbroken cells and debris wereremoved by centrifugation at 45,000 x g for 30 min. Thesupernatant thus obtained was stored under nitrogen at 0°C.Enzyme assays. Oligogalacturonate hydrolase (oligogalac-

turonase) and PGA hydrolase were measured discontinu-ously by monitoring the increase in reducing sugars (28).Both assays were carried out at 58°C unless stated other-wise. For the PGA hydrolase assay, a concentration of0.33% (wt/vol) pectate was used; during the oligogalactur-onase purification, PGA hydrolase activity was determinedwith oligomer-free pectate. The reaction mixture for theoligogalacturonase assay contained 10 ,u of enzyme sample,30 ,u of a dimer-trimer preparation (23 mM galacturonateequivalents) and 60 RlI of 150 mM potassium phosphatebuffer (pH 6.0). D-Galacturonic acid was used for calibrationof the reducing sugar assay. One unit of activity is defined asthe amount causing 1 ,umol of reducing sugar to be liberatedper min.

Pectin methylesterase activity was detected by the pro-duction of methanol in a reaction mixture containing culturesupernatant, 5 g of pectin per liter, and 150 mM potassiumphosphate (pH 6.0 at 60°C). The polymeric fraction wasprecipitated with 1 volume of cold isopropanol, and thesupernatant was analyzed for methanol (9).

All assays described below, which are based on theconsumption of NADH, were performed anaerobically toprevent substrate-independent oxidation of NADH cata-lyzed by the cell extract. The incubation temperature was42°C for technical reasons. One unit of activity is defined asthe amount of enzyme catalyzing the consumption of 1 ,umolof NADH per min (6340 = 6.22 mM-1 cm-1).The isomerization of galacturonate to tagaturonate by

D-galacturonate isomerase (EC 5.3.1.12) could only be mea-sured by monitoring the reduction of tagaturonate withNADH, which was catalyzed by D-tagaturonate reductase(EC 1.1.1.58); this was possible because of the higheractivity of the reductase (7). The reaction mixture contained33 mM sodium phosphate buffer (pH 7.0), 150 ,M NADH,330 ,uM D-galacturonic acid, and cell extract. D-Tagatur-

onate reductase was assayed in a similar way by starting thereaction with NADH after a preincubation time of 15 min toallow sufficient production of tagaturonate from galactur-onate.The reaction mixture was also used to detect the formation

of 2-keto-3-deoxyaldonic acids with the thiobarbiturate as-say (5) after 15 h of incubation.The overall activity of KDG kinase and KDPG aldolase

(EC 4.1.2.14) was assayed in a coupled reaction with lactatedehydrogenase. The reaction mixture contained 5 mM ATP,40 mM sodium phosphate buffer (pH 7.0), 0.8 mM KDG, 14mM gluconate, 2 U of lactate dehydrogenase per ml, 150 ,uMNADH, and cell extract.Transport studies. The presence of a phosphotransferase

system for digalacturonate was investigated by the methodof Robillard and Blaauw (22). To determine the action of aproton-motive-force-driven system, membrane vesicleswere energized by artificial gradients or by fusion withliposomes containing cytochrome c oxidase (26). After incu-bation of the energized vesicles with the substrate, they wererapidly applied to Sephadex G-50 fine columns (Pharmacia).The collected fractions with vesicles were washed and usedin uronate determinations after concentration by evapora-tion under vacuum.

Gel filtration chromatography. For the purification ofoligogalacturonate hydrolase activity, crude cell extract (300mg of protein in 5 ml) was applied to a Sephacryl S-300 HRcolumn (90 by 2.6 cm; Pharmacia) and eluted with 50 mMpotassium phosphate buffer (pH 6.0) at a flow rate of 0.5ml/min. Fractions of 5 ml were collected.

Molecular weight estimation. The native molecular weightof the oligogalacturonase was estimated by gel filtrationchromatography. Cell extract (0.2 mg of protein in 100 ,ul)was loaded on a Superose 12 HR 10/30 column (Pharmacia)and eluted with 50 mM potassium phosphate buffer (pH 6.5)at a flow rate of 1 ml/min. Fractions of 0.2 ml were collectedand analyzed for activity. Calibration was performed with aprotein mixture containing thyroglobulin (Mr, 670,000),gamma globulin (Mr, 158,000), ovalbumin (Mr, 44,000), myo-globin (Mr, 17,000) and cyanocobalamine (Mr 1,350).

Other methods. Pectin or pectate oligomers were detectedby thin-layer chromatography on Merck aluminum platescoated with silica (16) or by high-performance liquid chro-matography (17). The quantification of galacturonic equiva-lents was performed with the para-hydroxybiphenyl colori-metric assay for uronic acids (3).

Protein concentrations were determined by the method ofBradford (4), with the Bio-Rad protein assay kit and bovineserum albumin as a standard.

RESULTS

Growth on oligogalacturonates. The specific growth rate ofC. thermosaccharolyticum on the dimer, trimer, tetramer,and pentamer was the same as that for pectin (0.43 h-1). Forthe oligomers, growth ended abruptly when the substratewas depleted, while on pectin the growth rate graduallydecreased. During growth on the tetramer, a dimer appearedin the supernatant, and on the pentamer, a dimer and atrimer were formed. In a batch culture with initial concen-trations of 10mM galacturonate and 5 g of pectin per liter, noconsumption of the monomer was observed by thin-layerchromatography and the growth rate was unaffected.

Transport system for digalacturonate. In the supernatant ofcultures of C. thermosaccharolyticum, unless significant celllysis had occurred, a monomer was never found, and since

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EM

0co

7

6

5

4

3

2

1

0

100

800>t._

0._

c)._

40

20

0150 190 230 270 310 350

elution volume (ml)FIG. 1. Elution profile of oligogalacturonate hydrolase (0) and

PGA hydrolase (A) from a Sephacryl S-300 gel filtration columnloaded with cell extract of C. thermosaccharolyticum.

the products of pectin hydrolysis were digalacturonate andtrigalacturonate (28), it seemed logical that both of theseoligomers were taken up by the cell. The existence of aphosphotransferase system translocating digalacturonatewas investigated. We could not find phosphorylation of thedimer but did find phosphorylation of glucose. Membranevesicles made from pectin-grown C. thermosaccharolyticumcells could be energized either via artificial gradients or afterfusion with liposomes containing cytochrome c oxidase (A*4was 120 or 160 mV, respectively). Although an accumulationratio above 50 would have resulted in detectable amounts ofdigalacturonate, a dimer was not found in the vesicles.

Oligogalacturonate hydrolase. Cell extracts of C. thermo-saccharolyticum incubated with a di-, tri- and tetramerproduced a monomer, as determined by thin-layer chroma-tography. This oligogalacturonase activity was present in-side the cell because activity was observed only after dis-ruption of the cells by sonification. In the oligomerase assay,the initial reaction rate was proportional to the amount ofcell extract when the protein concentration in the reactionmixture was below 0.2 mg/ml.

Purification of the protein was troublesome because com-plete inactivation occurred during hydrophobic interactionchromatography or hydroxylapatite chromatography, and afivefold decrease in activity was found with a Mono-Qanion-exchange column by fast protein liquid chromatogra-phy (Pharmacia). The enzyme was also partially inactivatedduring ammonium sulfate precipitation and ultrafiltration.Gel filtration chromatography inactivated the enzyme by30%. However, although the purification factor of the col-lected enzyme pool (fraction 270 to 330 ml) was only 2.0,PGA hydrolase was separated effectively from part of theoligogalacturonase activity (Fig. 1). The characterization ofthe oligogalacturonase was done with this partially purifiedenzyme (specific activity, 3.5 U/mg on a mixture of di- andtrigalacturonate and 0.15 U/mg on oligomer-free pectate). Inthis preparation, the reaction rate was proportional to theamount of sample added and the production of reducingsugars was linear in time for each assay. Analysis of theproducts formed during degradation of NaBH4-reducedpentagalacturonate by the oligogalacturonase showed thatthe only monomer formed in the initial stage of the reactionwas galacturonate. This result indicated that the action of theoligomerase was from the nonreducing end of the oligomer.

0

.4

4-

100

80

60

40

20

0 ' l . . .

2 3 4 5 6 7 8 9

pH

FIG. 2. (A) Effect of pH on the activity of the partially purifiedoligogalacturonate hydrolase; (B) effect of incubation at different pHvalues on the activity of the oligogalacturonase (60°C). The bufferwas 100 mM potassium phosphate.

The native enzyme was eluted from a Superose 12 gelfiltration column at a molecular size of 90 kDa.The pH optimum of the enzyme was between 5.6 and 6.2

(Fig. 2A). The enzyme was stable for at least 1 h at 60°C inthe pH range of 5.5 to 7 (Fig. 2B). The activity graduallyincreased with increasing temperature up to 65°C. At highertemperatures, the activity rapidly decreased to zero at 70°C(Fig. 3; assay time, 30 min). The activity was stable for 1 h

0

0

I-

L._

100

80

60

40

20

030 40 50 60 70 80

temperature ( °C)FIG. 3. Effect of temperature on the activity of oligogalactur-

onate hydrolase.

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0

CO)

0

100

80

60

40

20

0

0 10 20 30 40 50 60

time (min)FIG. 4. Effect of temperature on the stability of the oligogalac-

turonate hydrolase. 0, 65°C; A, 70°C; V, 75'C.

at 65°C, but at 700 and 75°C, half of the activity was lost after45 and 4 min, respectively (Fig. 4).The kinetic parameters were calculated from Lineweaver-

Burk plots. The Vm. decreased with increasing chainlength: 15.1, 10.5, and 6.8 U/mg of protein for di-, tri- andtetragalacturonate, respectively. The Km decreased from 4.7mM for the dimer to 2.0 and 0.8 for the trimer and tetramer,respectively.

Uronate metabolism. Galacturonate was rapidly reducedby NADH upon the addition of cell extract, which indicatedthe presence of a reductase. Preincubation of galacturonatewith extract resulted in an even higher NADH consumptionrate. The specific reaction rates were 0.8 and 15.7 U/mg,respectively. 6-Phosphogalacturonate was not a substratefor this reaction. After prolonged incubation of the same

reaction mixture, a product was found with an absorptionpeak at 548 nm in the thiobarbiturate test, indicative for2-keto-3-deoxyaldonic acids (5). A mixture containing KDG,gluconate, ATP, and lactate dehydrogenase, after addition ofcell extract, showed NADH consumption as a result ofpyruvate production; the NADH consumption rate was 0.1U/mg. No activity was found without ATP. Gluconate was

not a substrate for this coupled reaction.Regulation of pectinolytic enzyme production. The activi-

ties of pectin methylesterase and PGA hydrolase were thesame for growth on pectin, pectate, and oligogalacturonates(di- and trimers). The amounts of both activities in batchcultures on pectin were proportional to the amount ofbiomass formed (OD660). For the PGA hydrolase, this wasalso found for steady states at different growth rates inpectin-limited continuous cultures (Fig. 5). The activity ofthe PGA hydrolase (in units per milliliter) is 0.13 x OD660value, and the activity of esterase (in units per milliliter) is0.52 x OD660 value (r2, 0.978 and 0.990, respectively). Thespecific activities of the oligogalacturonase on pectate andpectin were equal and constant during growth in batchculture on pectin (2.0 U/mg of protein).

Pectinolytic activities were not observed during growth on

glucose, galactose, xylose, glucose and pectin, or xylose andgalacturonate. An addition of glucose (10 mM) to a batchculture growing on pectin stopped the production of thethree pectinolytic enzymes. The addition of xylose did nothave an effect on the enzyme production.

0.28

0.24

0.20

0.16

> 0.12 X

0.08

0.04 A

0.00 j ~ l

0.0 0.2 0.4 0.6 0.8 1.0

optical densityFIG. 5. Activity of PGA hydrolase and pectin methylesterase as

a function of OD660 0, PGA hydrolase in batch culture; *, PGAhydrolase in continuous culture; A, pectin methylesterase in batchculture.

DISCUSSION

Earlier studies have shown that the initial degradation ofpectin by C. thennosaccharolyticum is catalyzed by theextracellular pectin methylesterase-PGA hydrolase com-plex. The products formed by the action of this complex onpectin were methanol, digalacturonate, traces of trigalactu-ronate, and limit pectins (28). In this study, we found that thedepolymerization of pectin was not the rate-limiting step forgrowth because the organism grew as fast on the polymer ason oligomeric products. The growth on oligogalacturonatesstopped abruptly when the substrate was depleted, while thegrowth rate on pectin slowed down and eventually stopped,leaving still 11% of the galacturonate equivalents uncon-sumed (29). This indicated that at the end of growth onpectin, apparently, the depolymerization did become ratelimiting.

C. thermosaccharolyticum was unable to produce or con-sume galacturonate, a phenomenon which was observedearlier with other pectin-degrading bacteria, such as C.thermosulfurigenes, C. rubrum, C. thermocellum, andBacteroides pectinophilus (12, 20, 24, 27). The dimer andtrimer, therefore, seem to be taken up by the cell. Produc-tion of small oligomers by extracellular enzymes and spe-cialized oligomer uptake by the cells could have an advan-tage over the production of monomers which might bemetabolized by a larger number of organisms, resulting incompetition for the substrate. Another advantage could belower energy costs for the translocation of oligomers than formonomers.The involvement of a phosphotransferase transport system

for the uptake of digalacturonate in C. thermosaccharolyti-cum is not likely because digalacturonate, in contrast toglucose, was not phosphorylated and phosphogalacturonatewas not a substrate for the first enzyme of the modifiedEntner-Doudoroff route (see below). Our attempts to finduptake of the dimer by a proton-motive-force-driven systemin energized membrane vesicles were hampered by the highdetection limit of the substrate, which was not available in aradioactive form. However, in case of a A/*-driven uptakesystem, a detectable amount of digalacturonate was expectedon the basis of the A/I measured and the sensitivity of theuronate assay. The nature of the system for the uptake of

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these charged molecules, therefore, needs further investiga-tion.The oligomers were intracellularly hydrolyzed to yield

galacturonate by an oligogalacturonate hydrolase in C. ther-mosaccharolyticum. Until now, only a few bacterial oligoga-lacturonases have been described. Hydrolases and lyaseswith, in certain cases, preference for a galacturonic residueadjacent to an unsaturated residue have been found inBacillus, Erwinia, and Pseudomonas species (30). Anotheroligogalacturonate hydrolase has been reported to be presentin Selenomonas ruminantium, but this enzyme has not beenfurther characterized (10). The reaction rate of the partiallypurified clostridial enzyme on oligomers decreased with theincreasing degree of polymerization. There was hardly anyactivity towards the polymer pectate: a phenomenon char-acteristic for oligomerases (the low activity on pectate wasprobably due to traces of PGA hydrolase present). Theexo-acting oligomerase liberated galacturonate from thenonreducing end of the oligomer. The enzyme resembled theoligogalacturonate hydrolase of a Bacillus species describedby Hasegawa and Nagel (8). The pH optimum of the C.thermosaccharolyticum enzyme was similar, but the temper-ature optimum was higher. The enzyme was also more stableat high temperatures.Gluconate is metabolized in some clostridia (2) by a

modified Entner-Doudoroff route, and we assumed thatgalacturonate would also be metabolized via a modifiedKDPG pathway, as has been described for Erwinia species(15). The route involves a galacturonate isomerase, tagatur-onate reductase, altronate dehydratase, KDG kinase, andKDPG aldolase (Fig. 6). The cell extract-dependent reduc-tion of galacturonate with NADH in a two-step processindicated the presence of both galacturonate isomerase andtagaturonate reductase in this organism. The formation of a2-keto-3-deoxyaldonic acid could be explained by the actionof an altronate dehydratase forming KDG. KDG was thesubstrate for the coupled reaction of KDG kinase, KDPGaldolase, and lactate dehydrogenase. Most of the enzymeactivities had to be measured indirectly because of theunavailability of the intermediates; however, we can con-clude that the modified Entner-Doudoroff route is operativein this organism.

All three pectinolytic enzymes PGA hydrolase, pectinmethylesterase, and oligogalacturonase are induced on pec-tin and pectate. PGA hydrolase and pectin methylesterasewere also found during growth on oligogalacturonates. Oneshould note that the pectin methylesterase is not necessaryfor growth on pectate or oligomers. Expression of the threeenzymes seems to be controlled in a coordinated way. Thegenes may be organized in an operon or perhaps may beregulated by the same inducer-repressor system. From ourresults, it is clear that the inducer of the pectinolytic en-zymes must be produced at or below the degradation level ofoligogalacturonates. Galacturonate, 5-keto-4-deoxyuronate,2,5-diketo-3-deoxygluconate, and KDG have been shown toinduce the production of pectinolytic enzymes in Erwiniastrains (19). Both galacturonic acid and KDG could also bethe inducer in C. thermosaccharolyticum.The amounts of PGA hydrolase, pectin methylesterase,

and oligogalacturonase were strictly coupled to the amountof biomass formed. The ratio of PGA hydrolase to celldensity was independent of the dilution rate in a continuousculture similar to the situation of PGA lyase production inBacillus subtilis (18). In Aeromonas liquefaciens and Er-winia carotovora, pectin limitation resulted in higheramounts of PGA lyase and pectin lyase, respectively (1, 11);

PECTIN

H20

methyl esterasepolygalacturonate hydrolase

limit pectins

DI (TRI )GALACTURONATE

OUT

I NDI (TRI ) GALACTURONATE

H20oligogalacturonatehydrolase

GALACTURONATE

1 galacturonate isomerase

TAGATURONATE

NADH | tagaturonate reductase

NAD+ALTRONATE

H20 4 altronate dehydratase

KETO-DEOXYGLUCONATE

ATP |KDG kinase

ADP 4

KETO-DEOXYPHOSPHOGLUCONATE

g KDPG aldolase

PYRUVATE + GLYCERALDEHYDE-3-PHOSPHATEFIG. 6. Proposed pathway for pectin metabolism by C. thermo-

saccharolyticum.

this might be due to a relief of a catabolite repression causedby degradation products of pectin.

In this study, the synthesis of pectinolytic enzymes by C.thermosaccharolyticum was found to be controlled by (atleast) two mechanisms: (i) induction by (di)galacturonate orits metabolites and (ii) catabolite repression in the presenceof glucose.

ACKNOWLEDGMENTS

We thank Wim Harder and Lubbert Dijkhuizen for valuablesuggestions, Henk Schols and Margien Mutter (Department of FoodScience, Wageningen Agricultural University) for the characteriza-tion of pectinic substrates and products, Gea Speelmans and KlaasDoesburg for contribution to transport studies, Irma van der Veenfor technical assistance, and Jaap Visser (Department of Genetics,Wageningen Agricultural University) for useful comments.

This work was supported by the Program Committee for Indus-trial Biotechnology of the Netherlands Ministry of Economic Af-fairs.

REFERENCES1. Almenchor-Hecht, M. L., and A. T. Bull. 1978. Continuous-flow

enrichment of a strain of Erwinia carotovora having specificityfor highly methylated pectin. Arch. Microbiol. 119:163-166.

2. Bender, R., J. R. Andreesen, and G. Gottschalk. 1971. 2-Keto-3-deoxygluconate, an intermediate in the fermentation of glu-conate by clostridia. J. Bacteriol. 107:570-573.

3. Blumenkrantz, N., and G. Asboe-Hansen. 1973. New method for

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quantitative determination of uronic acids. Anal. Biochem.54:484-489.

4. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

5. Chaby, R., D. Charon, R. S. Sarfati, L. Szab6, and F. Trigalo.1975. Estimation of 3-deoxy-2-ketoaldonic acids. Methods En-zymol. 41B:32-34.

6. Fogarty, W. M., and C. T. Kelly. 1983. Pectic enzymes, p.131-182. In W. M. Fogarty (ed.), Microbial enzymes andbiotechnology. Elsevier Applied Science Publishers, London.

7. Gierschner, K., and H. Endress. 1984. D-Galacturonate andD-tagaturonate p. 313-320. In H. U. Bergmeyer (ed.), Methodsof enzymatic analysis, vol. 6. Metabolites I-Carbohydrates,3rd ed. VCH Publishers, New York.

8. Hasegawa, S., and C. W. Nagel. 1968. Isolation of an oligoga-lacturonate hydrolase from a Bacillus specie. Arch. Biochem.Biophys. 124:513-520.

9. Heijthuijsen, J. H. F. G., and T. A. Hansen. 1989. Betainefermentation and oxidation by marine Desufuromonas strains.Appl. Environ. Microbiol. 55:965-969.

10. Heinrichowa, K., M. Wojciechowicz, and A. Ziolecki. 1989. Thepectinolytic enzyme of Selenomonas ruminantium. J. Appl.Bacteriol. 66:169-174.

11. Hsu, E. J., and R. H. Vaughn. 1969. Production and cataboliterepression of the constitutive polygalacturonic acid trans-elim-inase of Aeromonas liquefaciens. J. Bacteriol. 98:172-181.

12. Jensen, N. S., and E. Canale-Parola. 1986. Bacteroides pectino-philus sp. nov. and Bacteroides galacturonicus sp. nov.: twopectinolytic bacteria from the human intestinal tract. Appl.Environ. Microbiol. 52:880-887.

13. Kersters, K., and J. de Ley. 1975. 2-Keto-3-deoxy-D-gluconate.Methods Enzymol. 41B:99-101.

14. Kersters, K. and J. de Ley. 1975. D-Gluconate dehydratase fromAlcaligenes. Methods Enzymol. 42C:301-304.

15. Kilgore, W. W., and M. P. Starr. 1959. Catabolism of galactu-ronic and glucuronic acids by Erwinia carotovora. J. Biol.Chem. 234:2227-2235.

16. Koller, A., and H. Neukom. 1964. Detection of oligogalacturonicacids by thin-layer chromatography. Biochim. Biophys. Acta83:366-367.

17. Kravchenko, T. P., A. G. J. Voragen, and W. Pilnilk 1992.Analytical comparison of three industrial pectin preparations.Carbohydr. Polym. 18:17-25.

18. Kurowski, W. M., and J. A. Dunleavy. 1976. Cellular andenvironmental factors affecting the synthesis of polygalactur-

onate lyase by Bacillus subtilis. Eur. J. Appl. Microbiol. 2:103-112.

19. Nasser, W., S. Reverchon, and J. Robert-Baudouy. 1992. Purifi-cation and functional characterization of the KdgR protein, amajor repressor of pectinolysis genes of Erwinia chrysanthemi.Mol. Microbiol. 6:257-265.

20. Ng, H., and R. H. Vaughn. 1963. Clostridium rubrum sp. n. andother pectinolytic clostridia from soil. J. Bacteriol. 85:1104-1113.

21. Preiss, J., and G. Ashwell. 1963. Polygalacturonic acid metabo-lism in bacteria. II. Formation of 3-deoxy-D-glycero-2,5-hexo-diulosonic acid. J. Biol. Chem. 238:1577-1583.

22. Robillard, G. T., and M. Blaauw. 1987. Enzyme II of theEscherichia coli phosphoenolpyruvate-dependent phospho-transferase system: protein-protein and protein-phospholipidinteractions. Biochemistry 26:5796-5803.

23. Rombouts, F. M., and W. Pilnik. 1980. Pectic enzymes. Econ.Microbiol. 5:227-282.

24. Schink, B., and J. G. Zeikus. 1983. Clostidium thermosulfuro-genes sp. nov., a new thermophile that produces elementalsulphur from thiosulphate. J. Gen. Microbiol. 129:1149-1158.

25. Schols, H. A., M. A. Posthumus, and A. G. J. Voragen. 1990.Structural features of hairy regions of pectins isolated fromapple juice produced by the liquefaction process. Carbohydr.Res. 206:117-129.

26. Speelmans, G., W. de Vrij, and W. N. Konings. 1989. Charac-terization of amino acid transport in membrane vesicles fromthe thermophilic fermentative bacterium Clostidium fervidus.J. Bacteriol. 171:3788-3795.

27. Spinnler, H. E., B. Lavigne, and H. Blachere. 1986. Pectinolyticactivity of Clostridium thermocellum: its use for anaerobicfermentation of sugar beet pulp. Appl. Microbiol. Biotechnol.23:434-437.

28. van Rijssel, M., G. J. Gerwig, and T. A. Hansen. 1993. Isolationand characterization of an extracellular glycosylated proteincomplex from Clostridium thermosaccharolyticum with pectinmethylesterase and polygalacturonate hydrolase activity. Appl.Environ. Microbiol. 59:828-836.

29. van Rijssel, M., and T. A. Hansen. 1989. Fermentation of pectinby a newly isolated Clostridium thermosaccharolyticum strain.FEMS Microbiol. Lett. 61:41-46.

30. Whitaker, J. R. 1991. Microbial pectolytic enzymes, p. 133-176.In W. M. Fogarty, and C. T. Kelly (ed.), Microbial enzymes andbiotechnology, 2nd ed. Elsevier Applied Science Publishers,London.

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