Oligosaccharide synthesis in fibrobacter succinogenes S85 and its modulation by the substrate

S85 and its modulation by the substrate

Re´gis Nouaille1,2, Maria Matulova1,3, Anne-Marie Delort1and Evelyne Forano2

1 Laboratoire de Synthe`se et Etude de Syste`mes a` Inte´reˆt Biologique, UMR 6504 Universite´ Blaise Pascal – CNRS, 63177 Aubie`re cedex, France

2 Unite´ de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Gene`s-Champanelle, France 3 Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 842 38 Bratislava, Slovak Republik

Cellulolytic bacteria play an important role in nature, borne out by the fact that microbial cellulose utiliza-tion is responsible for one of the largest material flows in the biosphere. Cellulolytic bacteria have thus been studied in detail, but, because of methodological diffi-culties in using solid cellulosic substrates, the majority of the studies were carried out on bacteria utilizing sol-uble substrates [1]. When data are available, it appears that most of the statements and concepts applicable to cellulolytic bacteria metabolizing soluble carbohydrates are not applicable to bacteria using cellulosic substrate [1]. In previous work, we showed that Fibrobacter suc-cinogenesS85, a cellulolytic rumen bacterium, was able to synthesize and release oligosaccharides, identified by 2D-NMR techniques as maltodextrins (MD) and

maltodextrin-1-phosphate (MD1P), upon incubation with glucose [2,3]. On the contrary, no cellodextrins were released by the bacteria. The synthesis of MD and MD1P was unexpected in a strain specialized in cellulose degradation and known to be unable to use maltose and starch [4]. These results prompted us to investigate whether such MD and MD1P were pro-duced in bacteria metabolizing their natural substrate, cellulose. More precisely, the objectives were to answer the following questions (i) is the synthesis of MD and MD1P a specific feature of bacteria metabolizing glu-cose, or does it occur also on other carbohydrates, (ii) is F. succinogenes able to utilize these MDs as a carbon source, (iii) are cellodextrins produced when bacteria degrade cellulose, and (iv) are such observations

physio-Keywords

cellodextrin; Fibrobacter succinogenes S85; maltodextrin; NMR; rumen

Correspondence

A.-M. Delort, Laboratoire de Synthe`se et Etude de Syste`mes a` Inte´reˆt Biologique, UMR 6504 Universite´ Blaise Pascal – CNRS, 63177 Aubie`re cedex, France

Fax: +33 473 407717 Tel: +33 473 407717

E-mail: amdelort@chimtp.univ-bpclermont.fr (Received 15 December 2004, revised 1 March 2005, accepted 14 March 2005) doi:10.1111/j.1742-4658.2005.04662.x

In this article we compared the metabolism of phosphorylated and unphosphorylated oligosaccharides (cellodextrins and maltodextrins) in Fibrobacter succinogenes S85 resting cells incubated with the following substrates: glucose; cellobiose; a mixture of glucose and cellobiose; and cellulose. Intracellular and extracellular media were analysed by 1H-NMR and by TLC. The first important finding is that no cellodextrins were found to accumulate in the extracellular media of cells, regardless of the substrate; this contrasts to what is generally reported in the literature. The second finding of this work is that maltodextrins of degree of poly-merization > 2 are synthesized regardless of the substrate, and can be used by the bacteria. Maltotriose plays a key role in this metabolism of maltodextrin. Maltodextrin-1-phosphate was detected in all the incuba-tions, and a new metabolite, corresponding to a phosphorylated glucose derivative, was produced in the extracellular medium when cells were incubated with cellulose. The accumulation of these phosphorylated sugars increased with the degree of polymerization of the substrate.

Abbreviations

cellobiose, 4-O-b-D-glucopyranosyl-D-glucose; DP, degree of polymerization; ge-DQF COSY, gradient-enhanced double quantum filtered1_H-1_H correlated COSY; ge-HSQC, gradient-enhanced single quantum coherence1H-13C; Glc1P, glucose-1-phosphate; Glc6P, glucose-6-phosphate; MD1P, maltodextrin-1-phosphate; MD, (malto-oligosaccharides) linear maltodextrins; MDt, the terminal Glc unit of MD; MDint, internal Glc units of MD and MD1P; TSP-d4, sodium 3-(trimethylsilyl)propionate-d4; X, unknown glucose-1-phosphate derivative.

logically relevant for life in the rumen where cellulose is the main carbon source?

To answer these questions, F. succinogenes resting cells were incubated with cellulose and with cellobiose (the main product of cellulolysis), and with a mixture of glucose and cellobiose. Cell extracts and extracellu-lar media were analysed by 2D NMR spectroscopy and by TLC to identify and quantify the oligosaccha-rides produced.

Results

Synthesis of MDs and phosphorylated sugars Incubation in medium containing soluble sugars Resting cells (6 mgÆmL)1 of protein) of F. succinogenes S85 were incubated anaerobically with 32 mm glucose or 16 mm cellobiose, or with a mixture of 16 mm glu-cose and 8 mm cellobiose, in order to maintain the same glucose unit concentration. Samples of the cell suspension were taken at regular time-points, and extracellular and intracellular media (see the Experi-mental procedures) were separated by centrifugation and analysed, in parallel, by 1H NMR spectroscopy and by TLC. Consumption of the substrates and pro-duction of the final metabolites (succinate and acetate) were quantified from integrals in 1D1H NMR spectra (Fig. 1). Under these three different substrate condi-tions, the same concentrations of succinate and acetate were produced in the extracellular medium (Fig. 1B). Figure 1A shows that glucose was metabolized faster than cellobiose: when glucose or cellobiose were used as the only substrate, the rates of their utilization were 0.10 lmolÆmg of protein)1Æmin)1 and 0.06 lmolÆmg of protein)1Æmin)1, respectively, while the rates were 0.05 lmolÆmg of protein)1Æmin)1 and 0.03 lmolÆmg of protein)1Æmin)1 when these two sugars were used sim-ultaneously. It should also be noted that the rate of utilization of these sugars is two times slower when they are used as part of a mixture.

Detailed investigation of supernatant and cell extract composition was carried out using1H NMR gradient-enhanced double quantum filtered 1H-1H correlated COSY (ge-DQF COSY) and1H-13C gradient-enhanced single quantum coherence 1H-13C (ge-HSQC) spectra; identification of the various sugars was as described in detail previously [3]. Figure 2 shows the region of ge-DQF COSY NMR spectra that allowed a quantita-tive H1⁄ H2 cross-peak analysis of MDs, MD1P and glucose-1-phosphate (Glc1P) [3]. The same products were found under all three conditions of incubation (Fig. 2A–C). MDs and MD1P, which were first des-cribed when the cells metabolized glucose, were also

found in both the intracellular and extracellular media of cells metabolizing cellobiose or a mixture of the two sugars. Figure 3 shows the concentration of sugars obtained after cross-peak integration of the identified species in 2D ge-DQF COSY NMR spectra. It should be noted that the concentrations of all the intracellular sugars are much higher ( 30-fold) than those of the extracellular sugars. Furthermore, the ratio of phos-phorylated sugars (including Glc1P and MD1P) vs. MD is much higher in the supernatants than in the cells. This ratio increases when the substrates are chan-ged from glucose (Fig. 3A) to cellobiose (Fig. 3B); an

0 5 10 15 20 25 30 A B 0 10 20 30 40 time (min) 0 10 20 30 40 time (min) concentration (mM) 0 5 10 15 20 25 30 concentration (mM)

Fig. 1. Time dependence of sugar consumption (A) and metabolite production (B). Fibrobacter succinogenes cells were incubated with 32 mMglucose (white symbols), 16 mMcellobiose (black symbols),

or a mixture of glucose (16 mM) and cellobiose (8 mM) (grey sym-bols). (A) Consumption of glucose (squares) and cellobiose (tri-angles). (B) Production of succinate (squares) and acetate (circles). Glucose was assayed by using an enzymatic kit (Roche Diagnos-tics, Meylan, France); the concentrations of cellobiose, acetate and succinate were measured from 1D1H NMR spectra.

3.45 S S S S MD-1Pa +Glc1P MD-1Pa +Glc1P MD-1Pb MD-1Pb MD_t X MDint MD_t X MDint E E E E

A

B

C

D

intermediary situation is observed with a mixture of the two sugars (Fig. 3C). Note that, for clarity, ‘MD’ is used here for both maltose and MDs of larger size, and that, similarly, MD1P is used for both maltose-1P and MD1P of degree of polymerization (DP) > 2. The evolution of the various metabolite concentrations with time were similar regardless of the substrates used: they increased with time in the extracellular media, while in the cells they decreased at the end of the incubations.

Figure 4 shows TLC analyses of supernatants and cell extracts obtained following the incubation of rest-ing F. succinogenes with glucose (Fig. 4A), cellobiose (Fig. 4B), or glucose and cellobiose (Fig. 4C). Spots corresponding to MDs were detected and, in all cases, they had the same lengths, ranging from maltotriose to maltoheptaose. No maltose was detected, either in the supernatant or in the cell extracts, following incuba-tion with glucose. Maltose was not detected in cell extracts following incubation with cellobiose, or glu-cose and cellobiose. In the corresponding supernatants, spot T2 was cellobiose as its intensity decreased with time. Only maltotriose was present in the cell extracts of all incubations. In the supernatant obtained after incubation with cellobiose, the intensities of MD spots seemed to be lower (Fig. 4B), in agreement with the decrease of the terminal Glc unit of MD (MDt) and of the internal Glc units of MD and MD1P (MDint) cross-peaks in NMR spectra (Fig. 2B, plot S).

The two large spots (spot 1 and spot 2), previously observed following incubation with glucose [3], were also detected in cells metabolizing cellobiose (Fig. 4B) or a mixture of glucose and cellobiose (Fig. 4C). Spot 1 was present in both supernatants and cell extracts, while spot 2 was found only in the cells. The spots were previously shown to contain phosphorylated sugars. In the case of incubation with glucose, spot 1 was shown to include Glc1P, Glc6P and MD1P [3]. As these phos-phorylated sugars were detected in 2D NMR spectra of cellobiose incubation media, we can assume that they are also present in spot 1 of the corresponding TLC. Unfortunately, under these TLC conditions it was not possible to separate MD1P according to their length.

The kinetics of production of the different metabo-lites was not changed significantly by the nature of the substrate.

Incubation in medium containing cellulose

Resting cells of F. succinogenes (10 mgÆmL)1 of pro-tein) were preincubated with 0.2 g of cellulose (Sigma-cell 20), for 1 h in order to allow a maximal adhesion of the bacteria to cellulose [5,6]. Then, centrifugation was carried out to separate adherent and nonadherent cells. As shown previously, under these conditions only one-third of the bacteria were adherent to cellulose [5]. Adherent cells (final concentration 3.5 mgÆmL)1 of protein) were then further incubated with their sub-strate for 3 h. Samples of the incubation were taken at 1, 2 and 3 h, and extracellular and intracellular media were separated by centrifugation and analysed in par-allel by 1H NMR spectroscopy and by TLC. Under these conditions, 12.5 mm succinate was produced in the extracellular medium after 3 h of incubation. With respect to the cell concentration (3.5 mgÆmL)1 of pro-tein) which was 1.7 times lower than that of the cells incubated with soluble sugars (6 mgÆmL)1 of protein), the production of succinate after 3 h of incubation with cellulose (21 mm) was comparable to that obtained after 40 min of incubation with soluble sugars (19 mm, Fig. 1B). Consequently, we decided to compare the production of other metabolites, including Glc1P, MDs and MD1P, when soluble sugars and cel-lulose were metabolized by F. succinogenes S85.

In the 2D1H NMR spectra of samples collected after 3 h of incubation with cellulose (Fig. 2D), the same metabolites were identified as occurring after incu-bation with glucose or cellobiose (Fig. 2A,B) or their mixture (Fig. 2C). However, in the spectrum of the supernatant (Fig. 2D, panel S) a new, high-intensity H1⁄ H2 cross-peak appeared at 5.46⁄ 3.48 p.p.m. Although the structure of this new metabolite (named X) was not completely determined, both the H1⁄ H2 chemical shift values and the same cross-peak pattern as that of Glc1P indicated that Glc1P was a part of the X molecule. X was thus a Glc1P derivative. A precise quantification of signals was carried out by 2D 1_{H NMR. As already noted for incubation with} soluble sugars, the intracellular concentrations were much higher than the extracellular concentrations (Fig. 3D). In the extracellular medium (Fig. 3D, panel S), metabolite X was the major product, while it could not be detected in the cells (Fig. 3D, panel E). The ratio

Fig. 2. a-Anomeric region of gradient-enhanced double quantum filtered1_H-1_{H correlated COSY (ge-DQF COSY) NMR spectra of supernatant} (S) and cell extract (E). Incubation of Fibrobacter succinogenes resting cells for 40 min with 32 mMglucose (A), 16 mMcellobiose (B), or a mix-ture of 16 mMglucose and 8 mMcellobiose (C), and for 3 h with 0.2 g of cellulose Sigmacell 20 (Sigma-Aldrich, Saint Quentin Fallavier, France) (D). Glc1P, glucose-1-phosphate; MD1P, maltodextrin-1-phosphate; MD1Pa, Glc1P unit of MD1P; MD1Pb, Glc unit neighbouring the Glc1P of MD1P; MD, linear maltodextrins; MDt, the terminal Glc unit of MD; MDint, internal Glc units of MD and MD1P. X, unknown Glc1P derivative.

45 37.5 30 22.5 15 7.5 0 45 37.5 30 22.5 15 7.5 0 45 37.5 30 22.5 15 7.5 0 60 52.5 45 37.5 30 22.5 15 7.5 0 1.5 1.25 1 0.75 0.5 0.25 concentration (mM) concentration (mM) concentration (mM) concentration (mM) _{concentration (mM)} concentration (mM) concentration (mM) concentration (mM) 0 0 10 20 time (min) S A B C D S S S E E E E time (min)

time (min) time (min)

30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 1 2 3 1 2 time (h) time (h) 3 1.5 1.25 1 0.75 0.5 0.25 0 1.5 1.25 1 0.75 0.5 0.25 0 2 1.75 1.5 1.25 1 0.75 0.5 0.25 0

Fig. 3. Time courses of the concentrations of glucose-1-phosphate (Glc1P) (black squares), maltodextrin-1-phosphate (MD1P) (stripped), maltodextrin (MD) (empty squares) and X (light grey). Supernatants (S) and cell extracts (E) were obtained after the incubation of Fibrobacter succinogenes resting cells for 40 min with 32 mMglucose (A), 16 mMcellobiose (B), or a mixture of 16 mMglucose and 8 mMcellobiose (C), and for 3 h with 0.2 g of cellulose Sigmacell 20 (D). Quantification of metabolite concentrations was obtained by integrating 2D NMR signal volumes. For the supernatants obtained, values were directly reported, while for cell extracts, the intracellular concentrations were cal-culated relative to an intracellular volume of 5.5 lLÆmg)1of protein [21]. In the case of Sigmacell 20 cellulose, obtained and calculated values were multiplied by 1.7 to take into account the lower cell concentration. X, unknown Glc1P derivative.

of phosphorylated sugars (Glc1P, md1P and X) vs. MD was also much higher in supernatants than in cell extracts. This ratio was dramatically increased following incubation with cellulose compared to incubation with glucose or cellobiose.

All the metabolite concentrations increased with time in the supernatant; the concentration of metabo-lite X increased by sevenfold between 1 and 2 h of incubation and reached its maximum after 3 h of incu-bation.

TLC experiments confirmed the presence of spot 1 and spot 2 in these incubations with cellulose (data not shown).

Synthesis of cellodextrins

The synthesis of cellodextrins was monitored by 1H NMR spectra after incubation with various substrates: glucose, cellobiose, a mixture of glucose and cellobiose, and cellulose; the spectra of supernatants and cell extracts after 40 min of incubation are shown in Fig. 5. The H1 signal of nonreducing end glucose units of cello-biose or cellodextrins resonating at d 4.52 p.p.m. was used to monitor the metabolism of these sugars. In the spectra of supernatants after incubation with

cello-biose, the 1H signal at 4.52 p.p.m. was detected at the start of the incubation; it decreased with time and was barely detectable at 40 min (Fig. 5B, plots S). The kinetics of disappearance of this signal was consistent with the consumption of cellobiose without simulta-neous accumulation of cellodextrins in the supernatant (Fig. 1A). Consequently, we assumed that no cello-dextrins were produced in the supernatants of cells metabolizing cellobiose. Similarly, no cellodextrins were produced in the supernatants of cells metabolizing glu-cose and a mixture of gluglu-cose and cellobiose (Fig. 5A,C, plot S). When cells metabolized cellulose, the signal at 4.52 p.p.m. was not detected at any point in the kinetics (Fig. 5D, plot S). This result showed that no cello-dextrins accumulated in the extracellular medium when cellulose was used as substrate. We recently showed that a low concentration of cellodextrins was present in cell extracts when cells metabolized glucose [2]. The pres-ence of cellodextrins was also confirmed in the spectra of cell extracts obtained after incubation with the four different substrates tested (Fig. 5A–D, plots E), in agreement with this previous finding.

Following culture with the four different substrates (Fig. 5A–D), MD and MD1P H1 signals were clearly seen in1_{H NMR spectra.} Glc T2 M3 M4 M5 M6 Spot 1 Spot 2

A

B

S

C

time (min)

S

E

S

E

S

E

Fig. 4. TLC of saccharide derivatives present in supernatants (S) or cell extracts (E). Incubation of Fibrobacter succinogenes resting cells with 32 mMglucose (A), 16 mMcellobiose (B), or a mixture of 16 mMglucose and 8 mMcellobiose (C). Glc, glucose; T2, disaccharide; M3, malto-triose; M4, maltotetraose; M5, maltopentaose; M6, maltohexaose; Spot 1, Spot 2, spots containing phosphorylated sugar species. The same amount of sample was spotted on TLC and the same treatment (intensity and contrast) was applied to the different images.

Incubation with maltose and MDs

It has previously been shown that F. succinogenes S85 was not able to grow on maltose [4], but as MD syn-thesis was clearly shown under various conditions, we

decided to test the ability of the strain to grow on dif-ferent MDs, ranging from maltotriose to maltohexaose alone, or in a mixture. The largest MD tested was maltohexaose, as it was considered in Escherichia coli as the maximal length able to cross the maltoporin,

Fig. 5. 1D1_{H NMR spectra of supernatant (S) and cell extract (E). Incubation of Fibrobacter succinogenes resting cells for 40 min with 32 m} M

glucose (A), 16 mMcellobiose (B), or a mixture of 16 mMglucose and 8 mMcellobiose (C), and for 3 h with 0.2 g of cellulose Sigmacell 20 (D). CD, cellodextrins; Glc1P, glucose-1-phosphate; MD, linear maltodextrins; MD1P, maltodextrin-1-phosphate. *The presence of CD signals.

LamB [7]. The results are reported in Table 1. We also verified that F. succinogenes S85 was not able to grow on starch (data not shown). Growth and metabolism were monitored by measuring the attenuance (D) at 600 nm and by succinate and acetate assays from 1H NMR spectra, respectively. Maltodextrins of DP > 2 were clearly metabolized by the cells, as shown by the following that (i) their presence allowed the mainten-ance of cells in a metabolically active state as acetate and succinate were produced, (ii) an increase in D600 (maximal increase of 0.2) was observed, suggesting slight growth (data not shown) and (iii) bacteria cul-tured in the presence of MDs of DP > 2 could grow rapidly when subcultured on cellobiose (D600of > 1.5) and produced large amounts of succinate and acetate (Table 1). On the contrary, no growth was observed in a culture medium containing maltose, and no acetate and succinate were produced. In addition, when cells were first incubated with maltose and then subcultured on cellobiose, no growth was observed, indicating that the cells were no longer viable. Also, under these con-ditions, the amount of acetate and succinate produced was much lower when compared to that produced when cells were first cultured with maltotriose, malto-tetraose or maltohexaose. Moreover, on MDs of DP > 2, the acetate : succinate ratio (> 1) was inverted compared to that measured after culture on glucose or cellobiose (< 1). This shift to acetate pro-duction could be explained by a need for ATP produc-tion to maintain cell survival.

From the results obtained above, there are two stri-king points concerning MD chain lengths, namely (i) maltose was not used for growth and was not present in either the supernatant or in the cell extracts of rest-ing cells incubated with or without glucose, and (ii) maltotriose was the minimum MD ‘building block’ allowing bacterial metabolism and observed in cell extracts, and only MDs of DP > 3 were detected in the extracellular medium (Fig. 4). These observations prompted us to design experiments to test the minimum MD length necessary to promote their synthesis. There-fore, resting cells were incubated with 16 mm maltose (Fig. 6A), 11 mm maltotriose (Fig. 6B) and 8 mm maltotetraose (Fig. 6C); these concentrations are all equivalent to 32 mm glucose. In all the samples, the metabolites previously observed were present, including MDs, glucose, spot 1 and spot 2. When incubated with maltose (Fig. 6A), the spots were similar to those formed in the absence of exogenous substrate (Fig. 4B). This fact suggests their origin in endogenous glycogen degradation. TLC clearly showed that, although malt-ose was able to enter the cell, it was not used as the smallest substrate for the synthesis of longer MDs in either the extracellular or the intracellular compart-ment. These MDs probably came from endogenous glycogen. On the contrary, in the case of incubation with maltotriose (Fig. 6B) and maltotetraose (Fig. 6C), the spots were larger than those in Fig. 6A,B. This showed that maltotriose and maltotetraose, which were also transported into the bacteria, were used to synthes-ize longer MDs: these MDs (mainly maltotetraose, maltopentaose and maltohexaose, but also longer chains in lower concentrations) were present both in supernatants and in the cell extracts (Fig. 6B,C). Again, no maltose was detected while glucose was present.

Discussion

It is usually assumed that the degradation of cellulose by F. succinogenes produces cellodextrins that are released into the medium [8]. In a previous work, we were unable to detect cellodextrins, but found MDs and MD1P, in culture media from F. succinogenes cells incubated with glucose. In the present study we pro-vide further epro-vidence that cellodextrins are released on neither cellobiose nor cellulose, i.e. the natural sub-strate in the rumen. A small amount of cellodextrins was detected in cell extracts, but the absence of detect-able cellodextrins in the medium, regardless of the type of carbon source, suggests that these compounds are used internally rather than released into the medium.

The present work provides further details regarding the metabolism of MDs in F. succinogenes. These

Table 1. Production of succinate and acetate by Fibrobacter succin-ogenes grown on various carbon sources. CB, cellobiose; Glc, glu-cose; M2, maltose; M3, maltotriose; M4, maltotetraose; M6, maltohexaose or a mixture of maltodextrins (M2M3, M2M4, M3M4 and M3M6). M to CB means that bacteria were first grown on the carbon source M and then subcultured on cellobiose. Succinate and acetate concentrations were determined from1_{H NMR spectra} of the culture media after 48 h. Average standard deviations were ± 0.7 and 0.2 for acetate and succinate, respectively.

Concentration⁄ mM Concentration⁄ mM

Sugar Succinate Acetate Sugar Succinate Acetate

No 0.0 0.0 Glc 12.6 6.4 CB 13.6 6.5 M2 0.0 0.0 M2 to CB 0.4 1.1 M3 0.9 1.5 M3 to CB 8.5 5.7 M4 0.5 1.4 M4 to CB 8.9 5.5 M6 0.5 2.1 M6 to CB 9.0 6.0 M2M3 1.4 2.9 M2M3 to CB 9.4 6.2 M2M4 1.5 3.0 M2M4 to CB 9.2 5.9 M3M4 1.0 2.1 M3M4 to CB 8.3 4.2 M3M6 1.3 2.0 M3M6 to CB 8.7 6.0

compounds were synthesized from either glucose or cellobiose, or cellulose, with DP ranging from 3 to 7, regardless of the sugar. They were detected both in cell extracts and in media but, interestingly, maltotri-ose (DP 3) was found only in cell extracts. Maltmaltotri-ose was detected in neither the cell extracts nor media. F. succinogenes did not grow when MDs were provi-ded as carbon sources, but MDs of DP‡ 3 did main-tain cells in an active metabolic state. In addition, the elongation of MD chains was observed when the MD primers had a DP of ‡ 3. Taken together, these obser-vations suggest that maltotriose is a key oligomer for MD metabolism in F. succingenes, having the mini-mum size required for chain elongation or to support bacterial metabolism, whereas maltose is not used. The situation in this bacterium may share some anal-ogy with the amylomaltase (MalQ) (EC 2.4.1.25) sys-tem of E. coli, where maltotriose is the shortest oligomer substrate for chain elongation [7]. An ortho-log of malQ is found in the genome of F. succino-genes, sharing 30% homology, but the inability of maltose and longer MDs to sustain growth, as well as the apparent lack of any role for maltose, makes the MD metabolism in this bacterium different from that depicted in E. coli.

In addition to MDs, F. succinogenes synthesizes and releases a number of phosphorylated sugars, including Glc1P, MD1P, and some compounds that still remain to be identified. All of these metabolites are found both in cell extracts and in extracellular media, sug-gesting that they are generated inside the cells before being released into the medium, but it cannot be exclu-ded that they might be – at least partly – also gener-ated extracellularly. The accumulation of Glc1P is likely to result from the action of cellobiose phos-phorylase (EC 2.4.1.20) when the carbon source is cellobiose. Indeed, intracellular accumulation and excretion of Glc1P and Glc6P were previously des-cribed in F. succinogenes metabolizing cellobiose [9,10]. Cellobiose phosphorylase may be responsible for the Glc1P accumulation on cellulose, provided that cello-biose is produced, but because the main products of cellulolysis are cellodextrins, a cellodextrin phosphory-lase (EC 2.4.1.49) could be additionally involved. The two enzymes are often found in anaerobic cellulolytic bacteria [1,11,12]. The export of Glc1P may be caused by a specific permease, such as that reported to occur for Glc6P in E. coli [13].

The synthesis and release of MD1P is more puzzling. We previously hypothesized that these compounds 0 10 20 40 0 10 20 40 standards 0 10 20 40 0 10 20 40 0 10 20 30 40 0 10 20 30 40

time (min)

time (min)

Glc M2 M3 M4 M5 M6 Spot 1 Spot 2 A B C

S

E

S

E

S

E

Fig. 6. TLC of saccharide derivatives present in supernatants (S) or cell extracts (E). Incubation of Fibrobacter succinogenes resting cells with 16 mMmaltose (A), 11 mMmaltotriose (B) or 8 mMmaltotetraose (C). Samples taken at the same time-points were applied to the TLC. Glc, glucose; M2, maltose; M3, maltotriose; M4, maltotetraose; M5, maltopentaose; M6, maltohexaose; Spot 1, Spot 2, spots containing phos-phorylated sugar species. Standards: Glc, M2, M3. Note that the intensity and contrast of the image presented in panel A are higher than those shown in panels B and C, although the sample deposits were similar.

might be intermediates of MD synthesis [3], and the occurrence of both MDs and MD1P in all situations examined here is consistent with such a hypothesis. Because we previously reported such synthesis for glu-cose, a soluble sugar not relevant to the physiology of F. succinogenesin the rumen, the present work was ini-tially undertaken in order to assess whether the synthe-sis of such compounds was relevant to cells utilizing cellulose or cellulose-like compounds (i.e. cellobiose). The results showed not only that MD1P were synthes-ized on both cellobiose and cellulose, but also that the amounts of MD1P produced increased with the com-plexity of the substrate (cellulose>cellobiose>glucose). The latter observation also holds for the other phos-phorylated sugars detected. All these results are consis-tent with a physiological role of MD1P synthesis for F. succinogenescells living in the rumen.

In addition, the conditions of incubation with cellu-lose are different from those of soluble sugars, namely (i) adherent cells to a solid support can display a dif-ferent metabolism to planktonic cells [5], and (ii) dur-ing the whole incubation time the substrate is not limiting in the case of cellulose, while this is not the case for soluble sugars. This great difference of incuba-tion condiincuba-tions might also explain the increase of M_{D-1P concentration and the presence of compound} X when cells metabolized cellulose. It can be also noted that the MD⁄ MD1P ratio decreased with the sugar size, reaching a ratio of 1.0 in the case of cellu-lose. Two interpretations of this can be given, namely (i) the length of MD1P decreased, or (ii) the concen-tration of MD decreased, suggesting that MD might be phosphorylated.

F. succinogenes plays a key role in the rumen by degrading cellulose into metabolic products that are available to noncellulolytic species such as Streptococ-cus bovis, Selenomonas ruminantium or Treponema bry-antii, which have been shown to grow on cellulose in the presence of F. succinogenes [8,14,15]. It is usually accepted that cellodextrins are responsible for such cross-feeding effects. From the results presented here, where both MDs and MD1P, but not cellodextrins, were released by F. succinogenes metabolizing cellulose, we hypothesize that MDs and MD1P, rather than cel-lodextrins, may be responsible for such effects. Indeed, Strep. bovis is a major starch-degrading rumen bacter-ium and probably utilizes MDs efficiently. Several strains of Sel. ruminantium have been shown to be able to use MDs [16]. In addition, our hypothesis is consis-tent with the observation that starch-degrading bac-teria are always found in high numbers, together with cellulolytic bacteria, in animals fed a cellulose-rich diet [17]. Further investigations should be undertaken to

investigate the contribution of MD1P into the exchange of carbon between F. succinogenes and other rumen microorganisms.

In conclusion, we showed that, contrary to what was previously thought, cellodextrins do not accumu-late in the extracellular medium of cells degrading cel-lulose. In addition, we showed that maltotriose plays a key role in MD metabolism in F. succinogenes. Finally, the two main findings in this work are the detection of unusual phophorylated sugars (MD1P and X), and their differential accumulation, depending on the sub-strate. These results open new perspectives for the understanding of modulation of metabolism by the substrate, particularly the increase in production of phosphorylated species with the degree of polymeriza-tion of the b(1,4) saccharide. Also, the origin and the role of these phosphorylated sugars remains to be dis-covered; they might be involved in the regulation of specific gene expression, or in signalling mechanisms.

In current studies we are analysing sugar metabolism in growing cells to check whether the presence of phos-phorylated metabolites and the absence of cellodextrins in the culture medium still occur under conditions closer to those found physiologically.

Experimental procedures

Bacterial strain and culture conditions

F. succinogenes S85 (ATCC 19169) was cultured anaerobi-cally for 15 h in a chemianaerobi-cally defined medium [18] contain-ing 3 gÆL)1cellobiose, or in a rumen fluid-based medium [9] with 3 gÆL)1 of one (or a mixture) of the following sugars: glucose, cellobiose, maltose, maltotriose, maltotetraose, maltohexaose, or MD mixtures.

Preparation of cells, and extracellular and cellular media

Cells were prepared as described previously [10]: cells har-vested in the late exponential phase were centrifuged (6000 g, 10 min, 4
C) and resuspended under a 100% CO2 atmosphere in a reduced 50 mm potassium phosphate buf-fer containing 40 mm Na2CO3, 3 mm cysteine and 13 mm (NH4)2SO4, at pH 7.1.

Incubation with soluble sugars

Cell suspensions (6 mgÆmL)1 of protein) were incubated at 38
C, in a water bath, in medium containing 32 mm glu-cose, 16 mm cellobiose, a mixture of 16 mm glucose and 8 mm cellobiose, or MDs (maltose, maltotriose or malto-tetraose, each at 32 mm glucose unit equivalents). At

regu-lar time-points (10 min) 4 mL of the cell suspension was removed from the incubation medium, centrifuged (13 000 g, 10 min, 4
C), and the supernatant retained as extracellular medium. The pellet was resuspended in water and the cells were broken by successive freeze⁄ thawing procedures. Cell debris was pelleted by centrifugation (14 000 g, 15 min) and the supernatant was assimilated to cellular medium (cytoplasm and periplasm).

We checked that no cell lysis occurred during the incuba-tions by measuring the activity of a cytoplasmic marker, l-glutamate dehydrogenase [19], in the extracellular med-ium and compared it with that of the corresponding cell extracts.

Incubation with cellulose

F. succinogenesS85 cells (10 mgÆmL)1of protein) were pre-incubated for 60 min at 38
C in the reduced buffer (50 mm potassium phosphate, 40 mm Na2CO3, 3 mm cysteine, pH 7.1) containing 13 mm NH4+ [6.5 mm (NH4)2SO4], in the presence of 0.2 g of cellulose (Sigmacell 20, Sigma). The cell suspension was centrifuged for 4 min at 2000 g. The pellet constituted the ‘adherent’ cells, which were suspended in the reduced buffer (3.5 mgÆmL)1 of protein). At regular intervals (1, 2 and 3 h), 3 mL of the cell suspension was removed from the incubation medium, centrifuged (10 000 g, 10 min, 4
C), and the supernatant retained as extracellular medium; the pellet was resuspended in water and the cells were broken by three successive freeze⁄ thaw-ing procedures. Cell debris and cellulose were pelleted by centrifugation (10 000 g, 10 min) and the supernatant was assimilated to cellular medium (cytoplasm and periplasm).

NMR spectroscopy

Measurements of NMR spectra were performed at 27
C on a 300 MHz or 500 MHz Avance Bruker spectrometer equipped with 5 mm TXI 1H, 13C, 15N with inverse detec-tion.1H NMR chemical shifts are given relative to internal sodium 3-(trimethylsilyl)propionate-d4 (TSP-d4; d 0.0). A sample volume of 3 mL was taken at regular intervals dur-ing incubation with different carbon sources (glucose, malt-ose, maltotriose or maltotetraose). After separation of pellets, the pH of cell-free supernatants was corrected to 7.40 and the supernatants were then freeze-dried twice with D2O. The same procedure was adopted for intracellular media obtained from pellets. Samples were further dissolved in a mixture of 470 lL of 99.98% D2O, 20 lL of 10 mm TSP-d4and 10 lL of 50 mm 1-O-methyl-b-d-xylopyranose.

Basic quantification of H1 signals at d 5.46 and 5.43–5.41 (each representing a mixture of metabolites, species) was per-formed in the1_{H NMR spectra taking TSP-d}

4as a reference. The values obtained served as the reference for calculation of metabolite concentrations based on H1⁄ H2 cross-peak integrals taken in ge-DQF COSY spectra (standard Bruker

program). Because of the identical H1⁄ H2 chemical shifts of free Glc1P and MD1Pa(the Glc1P part of the MD1P mole-cule), a very well separated cross-peak signal of MD1Pb(d 5.43⁄ 3.58, owing to the Glc unit next to Glc1P in MD1P) was used for calculating the free Glc1P concentration.

TLC

TLC was carried out as described previously [10] by using a mixture of glucose, cellobiose or maltose, maltotriose and maltotetraose, or phosphorylated sugars (3 gÆL)1; Sigma) as a standard. Samples were spotted onto thin-layer silica gel plates (Silica gel 60 F254; Merck, VWR International SAS, Strasbourg, France). Sugars were separated by the solvent mixture composed of propanol⁄ 1-ethylacetate ⁄ water ⁄ ethanol⁄ pyridine ⁄ acetic acid (7 : 3 : 5 : 3 : 2 : 2; v ⁄ v ⁄ v ⁄ v ⁄ v ⁄ v) and revealed by spraying with ethanol⁄ H2SO4⁄ thymol (95 : 5 : 0.5; v⁄ v ⁄ v) and heating at 110
C.

Metabolite assays

Protein concentration was determined by the Bradford method [20], using BSA as standard. Succinate, acetate and glucose were assayed by using enzymatic kits (Roche). In the case of bacterial cultures, acetate and succinate produc-tion was directly quantified from the 1_{H NMR spectra of} culture medium using internal sodium TSP-d4as standard. The same procedure was used to quantify MD,M_{D1P and} Glc1P in supernatant and cell extract samples issued from the culture of resting cells with glucose.

Chemicals

TSP-d4 was purchased from Eurisotop (Saint Aubin, France). Glucose, cellobiose, maltose, maltotriose, malto-tetraose, Sigmacell 20 cellulose and 1-O-methyl-b-d-xylopyranose were from Sigma. All enzymes and other chemicals were purchased from Sigma or Roche.

Acknowledgements

M. Matulova is a visiting professor at the University Blaise Pascal, Aubie`re, France. R. Nouaille is grateful to Re´gion Auvergne, Centre National de la Recherche Scientifique and Institut National de la Recherche Agronomique for a PhD grant.

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