Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by...

Eur. J. Biochem. 268, 3907±3915 (2001) q FEBS 2001

Concurrent maltodextrin and cellodextrin synthesis by Fibrobactersuccinogenes S85 as identified by 2D NMR spectroscopy

Maria Matulova1,2, Anne-Marie Delort1, ReÂ gis Nouaille1,3, GenevieÁ ve Gaudet3 and Evelyne Forano3

1Laboratoire de SyntheÁse, ElectrosyntheÁse et Etude de SysteÁmes aÁ InteÂreÃt Biologique, UMR 6504, UniversiteÂ Blaise Pascal, CNRS,

AubieÁre, France; 2Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republik; 3UniteÂ de Microbiologie, INRA,

Centre de Recherches de Clermont-Ferrand-Theix, Saint-GeneÁs-Champanelle, France

1D and 2D NMR experiments were used to analyse

the synthesis of various metabolites by resting cells of

Fibrobacter succinogenes S85 when incubated with

[1-13C]glucose, in both extracellular and cellular media.

Besides the expected glycogen, succinate, acetate, glucose-

1-P and glucose-6-P, maltodextrins and cellodextrins were

detected. Maltodextrins were excreted into the external

medium. They were found to have linear structures with a

maximum degree of polymerization (DP) of about 6 or 7

units. Cellodextrins were located in the cells (cytoplasm

and/or periplasm), and their DP was # 4. Both labelled

(1-13C and 6-13C) and unlabelled maltodextrins and cello-

dextrins were detected, showing the contribution of carbo-

hydrate cycling in F. succinogenes, including the reversal

of glycolysis and the futile cycle of glycogen. The mech-

anisms of these oligosaccharide syntheses are discussed.

Keywords: cellodextrins; Fibrobacter succinogenes; malto-

dextrins; NMR; rumen.

Fibrobacter succinogenes, a predominant cellulolytic rumenbacterium, degrades cellulose by a very efficient cellulolyticsystem [1,2]. Cellulose is depolymerized at the bacterialsurface by various cellulases, and the released cellodextrinsare hydrolysed to give glucose (Glc) and cellobiose by acellodextrinase located in the periplasm [3]. The two sugarsare then taken up and metabolized by the cells to succinate,acetate and small amounts of formate [4±9]. When theextracellular sugar concentration is high, part of the sugar isstored as glycogen [5,8] or is polymerized as cellodextrins[6,10]. We previously used in vivo 13C-NMR spectroscopyto study the metabolism of [1-13C]Glc and its polymeriza-tion to cellodextrins in resting cells of F. succinogenes S85[5±9]. Accumulation of cellodextrins was particularlyimportant when resting cells were metabolizing Glc andcellobiose [6,9] simultaneously, but it was also found incells metabolizing Glc only. Wells et al. [10] showed that

cellodextrins were also produced by growing cells andexcreted into the culture medium. This cellodextrin releasecould have important ecological implications in the rumenecosystem because cellodextrins can be used as substrateby other rumen micro-organisms. Indeed, it was pre-viously suggested that cellodextrin release mediates anutritional interaction between cells adherent to celluloseand planktonic cells of F. succinogenes [10,11]. In addition,Wells et al. [10] showed that Streptococcus bovis, anoncellulolytic rumen bacterium, could be cocultured withF. succinogenes S85 on cellulose as sole carbon substrateand suggested that S. bovis was fed by the cellodextrinsexcreted by F. succinogenes.

13C and 1H NMR were also used to monitor in vivothe storage and degradation of glycogen in resting cellsof the strain S85 of F. succinogenes [5] and also in otherstrains from this genus [8]. We showed simultaneousstorage and degradation of glycogen when bacteria weresupplied with an exogenous carbon source, indicating thatglycogen was in a continuous flux. Also, glycogen wassynthesized during all growth phases [5,8], although bacteriausually accumulate glycogen during periods of slow or nogrowth, such as the stationary phase, in the presence ofexcess carbon. This finding, together with futile cycling ofglycogen in resting cells, suggested a deficient regulationof glycogen metabolism in F. succinogenes. In addition,intracellular glycogen can account for as much as 70% ofthe dry mass of the bacteria [12], and its concentration wasshown to be related to the cell viability [13]. Glycogen thusappears to have an important role in F. succinogenes whichhas not yet been clearly identified.

Surprisingly, while analysing the products of Glcmetabolism by F. succinogenes S85, we found that cellsalso synthesized and released oligosaccharides that wereidentified as maltodextrins. The present study was there-fore undertaken to quantify synthesis of maltodextrins inF. succinogenes S85 resting cells, in parallel with

Correspondence to M. Matulova, Laboratoire de SyntheÁse,

ElectrosyntheÁse et Etude de SysteÁmes aÁ InteÂreÃt Biologique,

UMR 6504 UniversiteÂ Blaise Pascal, CNRS, 63177 AubieÁre cedex,

France. Fax: 1 33 4 73 40 77 17, Tel.: 1 33 4 73 40 77 14,

E-mail: [email protected]

Abbreviations: MDt, CDt, terminal nonreducing unit of maltodextrin

and cellodextrin, respectively; MDnr, CDnr, nonreducing end; MDr,

CDr, reducing end unit; MDint, internal residues; DP, degree of

polymerization; HSQC, heteronuclear single-quantum coherence1H-13C correlated; XHCORR, 13C-1H correlated; TSP,

3-(trimethyl-silyl)-1-propanesulfonic acid.

Enzymes: aldolase (EC 2.2.1.2); triose phosphate isomerase

(EC 5.3.1.1); PPi±fructose-6-P phosphotransferase (EC 2.7.1.90);

amyloglucosidase (EC 3.2.1.3); cellodextrinase (EC 3.2.1.91);

cellobiase (EC 3.2.1.21); cellobiose phosphorylase (2.4.1.20);

cellodextrin phosphorylase (EC 2.4.1.49); maltose phosphorylase

(EC 2.4.1.8); glutamate dehydrogenase (EC 1.5.1.9).

(Received 16 March 2001, accepted 17 May 2001)

cellodextrins, and investigate their origin. We also analysedthe structure of the malto-oligosaccharides and cello-oligosaccharides produced and the contribution of endogen-ous glycogen to their synthesis, using 13C and 1H NMR.Because the concentration of these oligosaccharides is verylow, the advantage of the 13C enrichment in the 13C-NMRspectra of complex mixtures is obvious. In addition, the 13Cenrichment of the metabolites can be calculated from1H-NMR spectra and to quantify the extent to whichunlabelled molecules (released from prestored unlabelledglycogen for instance) are involved in the metabolism[8,14].

As 1H-NMR signals of saccharides are very close andoften overlap, 2D NMR techniques have been developed toassess structure determination of purified polymers. Mostof these structural studies were performed at naturalabundance [15]; a few of them were carried out with13C-labelled polymers [16,17].

The originality of the work presented here is that weused 2D NMR techniques [DQF COSY, heteronuclearsingle-quantum coherence 1H-13C correlated (HSQC),13C-1H-correlated (XHCORR)] to analyse oligosaccharidestructure (maltodextrins and cellodextrins) directly in super-natants and pellets of bacteria incubated with [1-13C]Glcwithout purification. Working on complex bacterial mediapresents major advantages: (a) it avoids time-consumingpurification; (b) global information is available on thedifferent metabolites, including their 13C enrichments(Glc, Glc-6-P, Glc-1-P, succinate, acetate¼). These dataallowed us to propose synthesis pathways for theseoligosaccharides.

M A T E R I A L S A N D M E T H O D S

Bacterial strain and culture conditions

F. succinogenes S85 (ATCC 19169) was grown for 15 h in achemically defined medium [5] with 3 g´L21 Glc.

Preparation of cells, extracellular and cellular media

Cells were prepared as described by Matheron et al. [6].Cells harvested in the late exponential phase werecentrifuged (6000 g, 10 min, 4 8C) and resuspended undera 100% CO2 atmosphere in a reduced 50 mm potassiumphosphate buffer containing 0.4% Na2CO3, 0.05% cysteineand 13 mm (NH4)2SO4 at pH 7.1. Cell suspensions (5 mgprotein´mL21) were incubated with labelled or unlabelledGlc at 37 8C either in the spectrometer (in vivo NMR) or ina water bath. At given times of incubation, the cell suspen-sion was centrifuged (13 000 g, 10 min, 4 8C), and thesupernatant was kept as extracellular medium. The pelletwas resuspended in water, and cells were broken bysuccessive freeze±thawing. Cell debris was eliminatedby centrifugation (14 000 g, 15 min) and the supernatantwas assimilated to cellular medium (cytoplasm andperiplasm).

We checked that no cell lysis occurred during the incu-bations by measuring the activity of l-glutamate dehydro-genase (as described in [18]), a cytoplasmic marker, in theextracellular medium and comparing it with that ofcorresponding cell extracts.

NMR spectroscopy

Measurement of NMR spectra at 25 8C were performedon 300-MHz Avance DSX and DPX spectrometersequipped with 5-mm TXI 1H,13C,15N and multinuclearprobes with inverse detection both with z gradients up to5 � 1023 T´cm21. Carbon spectra were measured in5-mm 1H,13C dual and 1H,13C,15N,31P quaternary nucleiprobes. 1H-NMR chemical shifts are given relative to internal3-(trimethyl-silyl)-1-propanesulfonic acid-d4 (TSP-d4 d0.0).

In the 5-mm sample tube, 400 mL cell-free supernatantwas mixed with 100 mL 25 mm TSP-d4 in D2O. Aftermeasurement of 13C enrichment of succinate and acetate[8], pH was corrected to 7.4, and the samples werefreeze-dried and dissolved in 99.98% D2O for furthermeasurements.

NMR signals of metabolites were assigned by comparingthem with NMR spectra of standard samples recordedunder the same conditions.

For identification of metabolite signals in the complexmixture of supernatants, 1H,13C-NMR spectra, the DQFCOSY sequence of Davies [19] with pulse field gradient,and 2D 1H-13C HSQC [20] with GARP decoupling andXHCORR spectra [21] were used.

13C-NMR spectra were acquired with 45 8 pulses,acquisition time 1.08 s, and relaxation time 1 s. Thechemical shifts of the a and b forms of Glc (d 92.93 and96.75, respectively), estimated with acetone as an internalstandard (d 31.07), was used to calibrate the chemical shiftsof the metabolites present in the mixture.

Metabolite assays

Protein concentration was determined by the Bradfordmethod [22], with BSA as standard.

Succinate, acetate and Glc were assayed using Boehrin-ger kits. Glycogen was assayed using amyloglucosidase aspreviously described [9].

TLC

TLC was carried out as described in [6], with a mixture ofGlc, cellobiose and cellodextrins (n � 3, n � 5 and n � 6;from Sigma; 4 g´L21) as standard.

Chemicals

[1-13C]Glc (99% labelled) and TSP-d4 were purchasedfrom Eurisotop (St Aubin, France). All enzymes andchemicals were purchased from Sigma or Boehringer.

R E S U L T S

Resting cells of F. succinogenes were incubated with[1-13C]Glc or unlabelled Glc, and the metabolites producedin the extracellular medium and in the cells were analysedin parallel by 1D and 2D 13C-NMR and 1H-NMR. For thispurpose, samples of the cell suspension were taken atdifferent time intervals, and extracellular and cellular mediawere separated by centrifugation before analysis asdescribed in Materials and methods.

3908 M. Matulova et al. (Eur. J. Biochem. 268) q FEBS 2001

Extracellular medium analysis

1D 13C-NMR experiments. Kinetics of [1-13C]Glc utiliza-tion were first monitored by 13C-NMR spectroscopy.Figure 1 shows 13C-NMR spectra of the extracellularmedium of samples collected after 9 and 18 min ofincubation. Signals corresponding to [2-13C]succinate and[2-13C]acetate (not shown) were observed at d 35.06 and24.10 p.p.m., respectively, as previously described forin vivo experiments [6]. Besides signals indicating thepresence of a,b-Glc, Glc-1-P and a,b-Glc-6-P (Table 1),additional ones were present between d 100.59 and d100.23 p.p.m. Their chemical shifts, together with theirH1 signals observed at d < 5.41, are characteristic ofC1 and H1 of nonreducing a-Glc units of aGlc(1!4)[aGlc(1!4)aGlc]n(1!4)Glc maltodextrin oligosaccharides(MD) [21,23]. The signal at d 100.59 was attributed toterminal nonreducing Glc (MDt), and those at d 100.42±100.37 to internal residues (MDint). Signals of reducing enda and b Glc (MDr) were present at d 92.74 and 96.60,respectively, while those next to them were observedbetween d 100.32 and d 100.23. In the region of C6resonances, a broad signal at d 63.65±63.50 was due to botha and b Glc-6-P, that at d 63.54 was due to fructose-6-P,

and that at d 65.24 was due to fructose-1,6-diP. In addition,signals of a and b MDr and MDint could be identified at61.56 and 61.31 p.p.m., respectively. Natural abundance C6signals of residual [1-13C]Glc were observed at d 61.60 and61.40.

No signal indicating the presence of (1!6)-linked Glcunits due to branched maltodextrin oligosaccharides atd 99.4 [23] was found in the spectra. Therefore, themaltodextrins produced by F. succinogenes in the extra-cellular medium were linear.

The maltodextrin spectral pattern (relative intensityMDr /MDnr) recorded after 9 and 18 min of incubationsuggests the presence of a mixture of maltodextrins with alow degree of polymerization (DP < 5±6). TLC analysis ofthese samples confirmed that the oligosaccharides had amaximal DP of 6±7 (not shown). The intensity of the[1-13C] signal of internal residues (d < 100.40) increasedwith time, whereas that of terminal nonreducing Glc (d100.59) remained constant, in parallel with Glc, and MDr

signals decreased. These results indicate an increase inmaltodextrin chain length with time.

Finally, no signal corresponding to nonreducing Glc unitsof [bGlc(1!4)bGlc]n(1!4)Glc cellodextrins at d 103.35[24] was observed in the spectra.

Fig. 1. C1 and C6 signal regions of 13C-NMR spectra of extracellular medium obtained after 9 and 18 min of F. succinogenes incubation

with [1-13C]Glc. MD*, maltodextrin Glc units neighbouring MDr Glca,b; **, unknown derivative of Glc-1-P.

q FEBS 2001 Maltodextrin synthesis by F. succinogenes (Eur. J. Biochem. 268) 3909

DQF COSY and HSQC 2D NMR experiments. The iden-tification of labelled maltodextrins in the extracellularmedium was unexpected and consequently led us to analysein more detail these molecules and their labelling, usinghomo-correlated DQF COSY and hetero-correlated HSQCspectra. The complexity of the mixture of metabolitesand their low concentrations promted us to concentrate onthe anomeric H1 and C1 signals and also, in the case ofmaltodextrins, H2 signals. The chemical shifts of thesesignals are a signature of the type of linkage of Glc units inmaltodextrins [23].

In our experiments, COSY DQF spectra of supernatantsof incubations of F. succinogenes with [1-13C]Glc wererather complex (Fig. 2B) because of 1H/13C coupling.Therefore, to facilitate assignment of the signals, cells werealso incubated with unlabelled Glc (Fig. 2A).

In the series designated A, three principal groups ofH1/H2 cross-peaks could be recognized. The first, due toGlc1-P, was present at d 5.46/3.52. The second was com-posed of two overlapping cross-peaks resonating at d 5.23/3.58 and d 5.23/3.54, the intensity of which was timedependent. The cross-peak at d 5.23/3.58 was more intenseat t � 18 min and was assigned to aGlc-6-P and MDr aGlcunits, while the other one at d 5.23/3.54 was more intense att � 9 min and was due to aGlc. The third group of H1/H2cross-peaks due to MDnr Glc units corresponded to threeoverlapping cross-peaks resonating at d 5.43/3.57, d 5.41/3.59 and d 5.41/3.63. The detailed assignment of MDnr

signals was made by comparison of their H1 and H2chemical shifts with those found for maltose and malto-triose by Morris & Hall [21]. The H1/H2 cross-peak at d5.41/3.63 was assigned to Glc residues next to MDr a,bGlcunits (MD*, Fig. 2) and that at d 5.41/3.59 to terminal MDt

Glc units. The last assignment of MDt was confirmed by1H/13C correlation in the 2D XHCORR spectrum (notshown) performed on the extracellular medium collectedafter 18 min of incubation with [1-13C]Glc. Finally the

remaining cross-peak at d 5.43/3.57 was assigned to theinternal maltodextrin Glc units (MDint).

Two other conclusions could be drawn from these DQFCOSY spectra (Fig. 2A): first, no cross-peak correspondingto cellodextrin nonreducing Glc units (CDnr) was detected(characteristic H1 chemical shift d 4.52); secondly, no H1signal due to branched (1!6)-linked Glc units, expected toappear at d< 4.97, was present. The latter is consistentwith that observed in 13C-NMR spectra (Fig. 1), confirmingthe presence of linear maltodextrin chains in the extra-cellular medium.

In the series designated B, after incubation with[1-13C]Glc, the same types of metabolite were present.However, in addition to 12C-linked H1 signals, 13C-linkedH1 signals were split into 13C satellites. One bond couplingconstant 1JC1-H1 values were about 170 Hz for a and160 Hz for b forms. Thus at a given time of incubation thepresence of unlabelled and labelled Glc-1-P, Glc-6-P, andGlc, MDnr and MDr were detected. This indicates thatmaltodextrin originated from [1-13C]Glc but also fromunlabelled Glc units. The presence of cross-peak correla-tions in the HSQC spectrum shown in Fig. 3A confirmedthe assignments of various metabolite signals as well astheir 13C labelling, including Glc-1-P, Glc-6-P, Glc, MDnr

and MDr.A comparison of DQF COSY spectra of the extracellular

media of cells incubated with [1-13C]Glc (Fig. 2B) andthose of cells incubated with unlabelled Glc (Fig. 2A)allowed a more detail analysis of the evolution of malto-dextrin amount and the 13C enrichments with time. Theintensity of cross-peaks corresponding to MDnr (Fig. 2A)increased with time, showing the accumulation of thisoligosaccharide in the extracellular medium. Also note thatthe intensity of MDint signals was increased to a greaterextent than that of MDt and MD* signals. Interestingly,the intensity of 13C satellites of MDint signals (Fig. 2B)increased to a greater extent than 13C satellites of MDt andMD*. This last result is consistent with that observedpreviously in 13C-NMR spectra (Fig. 1), indicating thatmaltodextrin polymers lengthen with time.

Quantification of the metabolites. At the end of theincubation of F. succinogenes S85 resting cells (2.9 mgprotein´mL21) with 32 mm [1-13C]Glc, when Glc wasexhausted, the amount of succinate, acetate, maltodextrins,Glc-6-P and Glc-1-P produced in the extracellular mediumwere quantified either enzymatically or from 1H-NMRsignal integrals. In parallel, intracellular glycogen wasassayed in the corresponding cells (Table 2). These resultsshow that maltodextrins represent about 8% of equivalentGlc units when compared with the amount of Glc used andabout 40% of the newly synthesized glycogen. Succinate,the major product of F. succinogenes metabolism, andacetate were, respectively, 10 times and twice as concen-trated as maltodextrins, while the maltodextrin concentrationwas actually in the range of that of Glc-1-P and Glc-6-P(Table 2).

The 13C enrichment of CH2 succinate and CH3 acetatewas measured from 1D 1H-NMR spectra by the 13C-filteredspin echo difference pulse sequence as previously described[8]. The maximum labelling of C2 succinate and C2 acetateshould be 25% and 50%, respectively, if 100% labelled[1-13C]Glc was consumed. One [1-13C]Glc unit is cleaved

Table 1. Chemical shifts of metabolites identified in supernatants

and cell extracts. F. succinogenes S85 was incubated with [1-13C]Glc.

Spectra were recorded at 25 8C and pH 7.40 in D2O. MD*,

maltodextrin Glc unit neighbouring aMDr and bMDr; ±, not assigned.

Chemical shift (d, p.p.m.)

Metabolite H1 C1 H2 C6

aGlc 5�.23 92�.93 3�.54 61�.40

bGlc 4�.65 96�.75 3�.24 61�.60

aGlc-6-P 5�.23 93�.07 3�.58 63�.50±63.65

bGlc-6-P 4�.65 96�.92 3�.28 63�.61±63.50

Glc-1-P 5�.46 94�.26 3�.52 61�.25

aMDr 5�.23 92�.74 3�.58 61�.55

bMDr 4�.66 96�.60 3�.28 61�.55

MD* 5�.41 100�.32±100.23 3�.63 ±

MDint 5�.44 100�.42±100.37 3�.57 61�.31

MDt 5�.41 100�.59 3�.59 ±

Fru-6-P ± ± ± 63�.54

Fru-1,6-diP ± ± ± 65�.24

aCDr <5�.23 92�.66 ± ±

bCDr <4�.66 96�.59 ± ±

CDnr 4�.52 103�.41 103.40 103.12 ± 61�.42


into two triose phosphates and thus two molecules ofacetate and succinate are produced, half of them beinglabelled (50%). In addition, succinate is a symmetricalmolecule with two CH2 groups, thus only 25% of C2-succinate atoms are 13C labelled. The percentage labellingwas constant with time and was found to be 21% forsuccinate and 29% for acetate. These values are consistent

with those measured previously when resting cells wereincubated with ammonia and Glc. The labelling deficit (4%of one CH2 of succinate, and thus 8% of the succinatemolecules) corresponds to the degradation of endogenousglycogen due to the futile cycling phenomenon [14](Fig. 4). It shows that, during these experiments, futilecycling of glycogen is occurring and that about 16% of Glc-6-P molecules entering glycolysis come from prestoredunlabelled glycogen, contributing to the synthesis ofsuccinate and acetate. In the case of acetate, in additionto the glycogen cycling, another phenomenon is contribut-ing to the isotopic dilution of C2, namely the reversion ofsuccinate synthesis pathway [8].

Intracellular medium analysis

We previously found, by in vivo 13C-NMR spectrscopy,small amounts of cellodextrins during incubation ofF. succinogenes resting cells with [1-13C]Glc [6]. However,in the 13C-NMR spectra of the extracellular fluid obtainedafter incubation of cells with [1-13C]Glc, no signals due tocellodextrins were observed (Fig. 1). Their absence in theextracellular medium was also confirmed by the 1H-NMR,DQF COSY and HSQC spectra (Fig. 2 and Fig. 3A).

Fig. 2. Region of a anomeric signals of the DQF COSY spectra of extracellular medium obtained after incubation of F. succinogenes with

nonlabelled Glc (Series A) and [1-13C]Glc (Series B) after 9 and 18 min. Top traces represent the 1H-NMR spectra. MD*, maltodextrin Glc unit

neighbouring reducing end aGlc; 12C, protons linked to 12C atom; 13C, protons linked to 13C atom.

Table 2. Metabolite content. Metabolite content was measured in

the extracellular medium (succinate, acetate, Glc-6-P, maltodextrin,

Glc-1-P) or in the cells (proteins, glycogen) after incubation of

F. succinogenes S85 with 32 mm [1-13C]Glc. Glycogen, succinate,

acetate and Glc-6-P were measured enzymatically, and maltodextrin

and Glc-1-P were estimated from 1H-NMR spectra after freeze-drying

of the sample. The value is relative to TSP-d4 content, which was

added to the supernatant before freeze-drying. Glycogen was newly

synthesized. MD, Maltodextrin.

Proteins

(mg´mL21)

Glycogen

(mm)

Succinate

(mm)

Acetate

(mm)

Glc-6-P

(mm)

MD

(mm)

Glc-1-P

(mm)

2�.9 6�.40 28�.8 5�.8 4�.7 2�.6 0�.7


Therefore we analysed the metabolites of the cellularmedium (cytoplasm 1 periplasm) by 13C NMR (notshown); besides C1 signals of MDnr Glc units (d100.59±100.32), those of CDnr were present at d 103.41, 103.40 and103.12 p.p.m. together with that of CDr a,bGlc at d92.66 p.p.m. and 96.59 p.p.m. C6 signals of CDnr Glc unitswere detected at d 61.42 p.p.m.; all the other skeletalcarbon atom signals (C2-C5) were missing. The fact thatin the 13C-NMR spectrum only C1 and C6 signals of

cellodextrin were present suggests that [1-13C]Glc was usedfor their synthesis. However, in the 1H-NMR spectrum ofthe cell extract (Fig. 3B, top trace) the 12C-linked H1 signalof nonreducing Glc units was also present at d 4.52 p.p.m.(13C satellites were not identified as they were hidden byother signals.) This means that, for the synthesis ofcellodextrins, nonlabelled and labelled Glc units wereused, as observed for maltodextrin. The HSQC spectrumrecorded on cell extract (Fig. 3B) also confirmed thepresence of the cross-peak at d 4.52/103.40 due to CDnr

units.Finally, the intensities of CDr, CDnr of cellodextrins in

the 13C-NMR spectra suggest that cellodextrin chains haveat least a DP of 4. The intracellular medium was alsoanalysed by TLC, and the DP of the oligosaccharides wasfound up to 4, confirming NMR data.

D I S C U S S I O N

In this work, by using 1D and 2D NMR experiments, wewere able to analyse the synthesis of various metabolitesby resting cells of F. succinogenes when incubated with[1-13C]Glc in both extracellular and cellular media.Besides the expected glycogen, succinate, acetate, Glc-1-P and Glc-6-P, maltodextrins and cellodextrins werealso detected.

Carbohydrate cycling in F. succinogenes S85

The use of [1-13C]Glc combined with 1H-NMR and13C-NMR techniques provided clear evidence of carbo-hydrate cycling in F. succinogenes.

First, various metabolites detected by 13C-NMR analysisof supernatants were labelled on the C1 but also C6positions; they include Glc-1-P, Glc-6-P, maltodextrins andcellodextrins. The isotopic transfer from C1 to C6 resultsfrom the reversibility of the aldolase/triose phosphateisomerase triangle. It also implies the reversibility of theglycolytic pathway through the PPi±fructose-6-phosphatephosphotransferase (step 1 in Fig. 4). In our previousstudies we could detect this istotopic transfer in in vivo13C-NMR spectra of glycogen and Glc-6-P [8,9]. It is alsoshown here with other metabolites: Glc-1-P, maltodextrinsand cellodextrins. Excretion of sugar phosphates into theextracellular medium is not very common in bacteria. Wepreviously showed that Glc-6-P was excreted by variousstrains of Fibrobacter genus [9]. The mechanism of thisexcretion is not known, but it could involve a specificpremease. Wild-type Escherichia coli cells were shownto secrete Glc-6-P when an uncoupler of oxidative phos-phorylation was added [25].

Secondly, futile cycling of glycogen was also clearlyshown and quantified by 1H-NMR spectrscopy. Thistechnique, by measuring the 12C/13C ratio of CH2 ofsuccinate, allowed us to assess the isotopic dilution of thefinal metabolites resulting from the simultaneous degra-dation of prestored unlabelled glycogen and [1-13C]Glc.It was shown that, under our experimental conditions,about 16% of unlabelled Glc-6-P molecules wereentering glycolysis. This is consistent with resultsobtained previously [14].

Fig. 3. HSQC spectra of extracellular medium (A) and broken cell

extract (B) obtained after incubation of F. succinogenes with

[1-13C]Glc in the magnet. 12C, Protons linked to 12C atom; 13C,

protons linked to 13C atom.


Maltodextrin synthesis

In this work, we have demonstrated for the first time thesynthesis of maltodextrins by F. succinogenes S85. Thesemaltodextrins showed a maximum degree of polymeriza-tion of about 6±7 units and no branching by (1!6) linkageof Glc units; their chain length increased with time. Theseresults indicated that maltodextrins could not be the directproduct of glycogen hydrolysis, as we previously checkedthat intracellular glycogen of F. succinogenes was com-posed, as expected, of Glc moieties linked both by (1!4)and (1!6) bonds (unpublished results). In 2D DQF-COSYNMR spectra, the presence of both 13C-linked and12C-linked H1 signals of maltodextrins clearly showed thatthese oligosaccharides originated from [1-13C]Glc but alsofrom a nonlabelled carbon source (Fig. 2). [12C]Glc unitsactually resulted from the carbohydrate cycling reportedabove: the reversal of glycolysis (step 1 in Fig. 4; the triosephosphate isomerase generates [12C]dihydroxyacetonephosphate and [12C]glyceraldehyde 3-phosphate) and thefutile cycle of glycogen (generating [12C]Glc-1-P, step 2 ofFig. 4).

Because some strains of F. succinogenes have beenreported to use starch or maltose, we checked that strainS85 was unable to use maltose as growth substrate (notshown). It was therefore surprising to note the synthesis ofmaltodextrins in the presence of Glc by this strain, whichdoes not have a functional maltose system. However,Decker et al. [26] clearly showed that maltose andmaltotriose can be formed endogenously in E. coli fromGlc and Glc-1-P independently of enzymes of the maltose

system. It was shown by using a range of mutants thatmaltodextrins can be formed from glycogen. Two differentmechanisms were proposed [26,27]: (a) glycogen isdegraded to Glc-1-P, and then maltose and maltotriose aresynthesized from Glc-1-P and Glc (or maltose) through thereverse action of a putative maltose/maltotriose phos-phorylase; (b) maltotriose units are directly generatedfrom glycogen by a specific enzyme and can be furtherextended to longer maltodextrins. In E. coli, there was noexperimental evidence to discriminate between these twohypothesis. Our results are consistent with the synthesis ofmaltodextrins from glycogen in F. succinogenes but theyare not discriminatory either. Generally it is admitted thatglycogen is degraded to Glc-1-P in all cell systems [28],and the detection of unlabelled Glc-1-P in 1H-NMR spectraproves it is also the case in F. succinogenes. Thus malto-dextrins can be formed by condensation of this basic unitwith Glc (step 3 in Fig. 4), but direct cleavage of glycogenin short maltodextrins (maltotriose) (step 4 in Fig. 4) cannotbe excluded.

The detection of maltodextrins in the extracellularmedium suggests that they are either exported through thecytoplasmic membrane or synthesized in the periplasm andthen cross the outer membrane.

Synthesis of cellodextrins

Wells et al. [10] previously showed that cells of F. succino-genes growing on Glc or cellobiose were synthesizingcellodextrins and excreting them into the culture medium.We also previously showed cellodextrin synthesis in resting

Fig. 4. Possible pathways of maltodextrin and cellodextrin synthesis.


cells, particularly when they are metabolizing Glc andcellobiose. In this study, we characterized the cellodextrins inmore detail and showed their intracellular location in restingcells metabolizing Glc.

The analysis of the pellets of resting cells incubatedwith [1-13C]Glc showed the presence of C1-labelled andC6-labelled cellodextrins as well as unlabelled ones. Thepresence of [12C]cellodextrins and [6-13C]cellodextrinsproves the contribution of [12C]Glc-1-P and [6-13C]Glc-1-P resulting from carbohydrate cycling (reaction 5 inFig. 4). This excludes completely their synthesis from[1-13C]Glc only (reaction 5 in Fig. 4). NMR and TLCanalysis suggested that the cellodextrin had a maximum DPof 4.

The enzymes responsible for the synthesis of cello-dextrins have not been identified. The activity of bothcellobiose phosphorylase and cellobiase has previouslybeen demonstrated in this bacterium [6,9]. Wells et al. [10]proposed that cellodextrins were produced by a reversiblephosphorylase reaction (cellobiose and/or cellodextrinphosphorylase), but it is also possible that they aresynthesized through transglucosylation reaction of theb-glucosidase (cellobiase). Indeed, many bacterial and otherb-glucosidase have been shown to catalyse, in parallel withcellobiose hydrolysis, such transglucosylation reactions[29,30]. However, in this last case, the first step would besynthesis, by the phosphorylase, of cellobiose necessary toinitiate the transglucosylation reaction. In either case, thecellodextrin synthesis would have to take place in thecytoplasm, as these enzymes are likely to be intracellularlylocated [10,31]. They would then have to be transportedthrough the cytoplasmic membrane.

Another possibility is the synthesis of cellodextrins bythe reverse action, or transglucosylation reaction, of theperiplasmic cellodextrinase of F. succinogenes. This enzymehas been characterized previously. It is produced constitu-tively [1], cleaves only cello-oligosaccharides, and appearsto be strictly located in the periplasmic space [1,3]. In thiscase, the cellodextrins would not have to cross the innermembrane to be excreted by the growing cells.

In conclusion, 1D and 2D NMR techniques performeddirectly on complex biological media allowed us to performstructural analysis of cellodextrins and maltodextrins with-out purification and also to analyse the other bacterialmetabolites. In addition, when [1-13C]Glc was used, theNMR analysis of various isotopomers provided valuableinformation about bacterial metabolism. In particular, weshowed for the first time the capacity of F. succinogenes tosynthesize and excrete maltodextrins. We will now monitorand quantify the maltodextrin synthesis by F. succinogenesunder different conditions, such as various growth substrates,to assess the physiological impact of this maltodextrinproduction. Indeed, a great variety of noncellulolytic rumenbacteria are able to use maltodextrins. Thus, a mechanismof cross-feeding could take place between F. succinogenesand these species, similarly to that previously suggested forthe cellodextrins. F. succinogenes was indeed coculturedwith Selenomonas ruminantium [32], Treponema bryantii[33] or S. bovis [10] with cellulose as sole substrate. Suchmaltodextrin cross-feeding may help to explain previousresults indicating that starch-degrading bacteria alwaysoutnumbered cellulolytic species in animals fed a cellulose-rich diet such as wheat straw [34].

A C K N O W L E D G E M E N T S

M. M. is invited professor of the University Blaise Pascal, AubieÁre,

France. R. N. is grateful to ReÂgion Auvergne, Centre National de la

Recherche Scientifique and Institut National de la Recherche

Agronomique for a PhD grant. We thank Professor P. Hoggan for his

careful reading of the manuscript.

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