View metadata, citation and similar papers at core.ac.ukBiochimica et Biophysica Acta 1355Ž. 1997 50±60 brought to you by CORE

provided by Elsevier - Publisher Connector

Re-investigation of glucose in Fibrobacter succinogenes, using NMR spectroscopy and enzymatic assays. Evidence for pentose phosphates phosphoketolase and pyruvate formate activities Christelle Matheron a, Anne-Marie Delort a,), GenevieveÁ Gaudet b,c, Evelyne Forano b a Laboratoire de SyntheseÁÁÁÂà et Etudes de Systemes a Interet Biologique, URA 485 du CNRS, UniÕersite  Blaise Pascal, 63177 Aubiere, Á France b Laboratoire de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genes-Champanelle,Á France c Centre UniÕersitaire des Sciences et Techniques, UniÕersiteÂÁ Blaise Pascal, 63177 Aubiere, France Received 21 May 1996; revised 16 July 1996; accepted 12 August 1996

Abstract

The glucose metabolism of Fibrobacter succinogenes S85 was studied in detail; key intermediates and alternative pathways were evidenced by NMR andror enzymatic assays. A high phosphoketolase activity was detected in four strains of Fibrobacter under strictly anaerobic conditions, with ribose-5-phosphate as substrate, no activity was evidenced with fructose-6-phosphate. This is the first report of a pentose phosphates phosphoketolase in bacteria unable to use pentoses. In contrast, the Entner-Doudoroff pathway and the oxidative branch of the pentose phosphate pathway could not be evidenced. 13 13 Incubation of living cells of F. succinogenes with Na 23CO confirmed the incorporation of CO 2 in the carboxylic group of succinate. The presence of fumarase was evidenced by in vivo 13C-NMR using 2-heptyl-4-hydroxyquinoline-N-oxide Ž.HQNO ; the showed a high reversibility under physiological conditions. The production of formate from glucose catabolism was evidenced by enzymatic assay and by NMR and a pyruvate formate lyase activity was detected using strictly anaerobic conditions.

Keywords: Anaerobic bacterium; Rumen; Glucose metabolism; Phosphoketolase; Pyruvate formate lyase; NMR,13 C; ŽFibrobacter succinogenes.

1. Introduction acetate and little formate from hexoseswx 1,2 . Glucose is metabolized through the Embden-Meyerhof-Parnas Fibrobacter succinogenes is a strictly anaerobic pathway 3±5 . It is proposed that oxaloacetate, cellulolytic bacterium living in the rumen. This bac- wx formed from phosphoenolpyruvate, was reduced to terium uses cellulose, glucose and cellobiose as car- succinate via malate and fumarate, and that pyruvate bon and energy sources, and produces succinate, led to the synthesis of acetate via acetyl-CoA and acetyl-phosphatewx 4Ž. Fig. 1 . The pathway of formate ) Corresponding author. Fax: 33 73407717; e-mail: production is not known. We studied glucose q 13 1 [email protected] metabolism in F. succinogenes by C and Hin

0167-4889r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S0167-4889Ž. 96 00118-8 C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 51 vivo NMR spectroscopywx 5 . This study revealed imum value 20%.Ž and CH3 of acetate maximum simultaneous storage and degradation of glycogen, value 25%. , measured by1 H-NMR, were different which was degraded even in the presence of exoge- from those expectedwx 5 . Indeed, if labelled exoge- nous glucose, and consequently led to the production nous glucose was degraded only via the pathway of unlabelled metabolites when the cells were incu- previously describedwx 4 , the theoretical percentages 13 bated with C-enriched glucose. In addition, the of enrichment of CH23 of succinate and CH of percentages of enrichment of CH2 of succinateŽ max- acetate would be 25% and 50%, respectively, or

13 Fig. 1. Pathway of formation of succinate and acetate fromw 1- Cx glucose via EMP pathway in F. succinogenes. Ž.™ Enzymatic 13 activities evidenced previouslywx 3,4 , Ž.´ Stages deduced from our work, Ž.) C-enrichment. 52 C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 slightly less due to utilization of endogenous unla- would thus be the presence of the pentose phosphate belled glycogen. This discrepancy suggested that an pathway connected with either a phosphoketolase alternative pathway lead to the production of unla- activity or the Entner-Doudoroff pathway:Ž. i In the belled acetate. pentose phosphate pathwayŽ. Fig. 2 , glucose-6-phos- In addition, F. succinogenes possesses the essen- phate undergoes oxidation to 6-phospho-gluconic tial of the non-oxidative branch of the pen- acid, followed by an oxidative decarboxylation to tose phosphate pathwaywx 6 , though known to be form ribulose-5-phosphate. This C1 decarboxylation 13 13 unable to metabolize pentoses. One possible explaina- would release CO2 duringw 1- Cx glucose tion of the low percentage of enrichment of acetate metabolism by F. succinogenes cells, and would led

Fig. 2. Catabolism ofw 1-13 Cx glucose via pentose phosphates or Entner-Doudoroff or phosphoketolase pathways. 1, Glucose-6-phosphate dehydrogenase; 2, 6-phosphogluconate dehydrogenase;, 3, transketolase; 4, transaldolase; 5, 6-phosphogluconate dehydrase, 6, KDPG aldolase, 7, phosphoketolase Ž.) 13C- enrichment. C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 53 to unlabelled metabolites. If this pathway was con- Na23 CO , 0.05% cysteine, pH 7.1. containing 5 mM nected to a phosphoketolase activity, unlabelled ac- dithiothreitolŽ. DTT and 3 mM MgCl2 for phospho- etate would be produced without concomitant synthe- ketolase assay, or 10 mM DTT and 0.3 mM sis of unlabelled succinate.Ž. ii In the Entner- FeŽ.Ž. NH42 SO 42 for pyruvate formate lyase assay Doudoroff pathway, the 6-phosphogluconic acid and were finally concentrated 100 times. Cells were would be converted into glyceraldehyde 3-phosphate disrupted anaerobically by sonication with a Branson and pyruvateŽ. Fig. 2 . Thusw 1-13 Cx glucose metabo- Sonifier Cell Disrupter B15Ž three treatments of 10 s lized by this pathway would give rise tow 1- each, 30 s apart. in an ice bath, that constituted crude 13Cx pyruvate that could decarboxylate to release extracts. Crude extracts were centrifugedŽ 15 000=g 13 CO2 and then give rise to unlabelled acetate. 5 min, 108C. in CO2 -filled centrifuge tubes, and The aim of this work was to revisit the glucose supernatants were immediately used as enzyme ex- metabolism of F. succinogenes S85, to identify any tract or stored at y208C. All steps were carried out alternative to the Embden-Meyerhof-Parnas pathway under a 100% CO2 athmosphereŽ. Table 1 . and evidence key intermediates of the pathway of formation of succinate and formate using 13C-NMR 2.3. Proteins and enzymatic assays. High pentose phosphates phos- phoketolase activity was evidenced in F. succino- Protein concentration was determined by the genes S85, but also in other Fibrobacter strains Ž F. method of Lowrywx 10 , using bovine serum albumin succinogenes HM2, 095 and F. intestinalis.. A pyru- as the standard. vate formate lyase activity, not previously identified, was shown in F. succinogenes extracts. The re- 2.4. Enzymatic assays versibility of fumarase in resting cells was shown to be very efficient under anaerobic conditions. Enzymatic assays were performed at 378Cin50 mM potassium phosphate buffer, pH 7.6Ž final vol- ume of 1 ml. unless otherwise stated. All assays were 2. Materials and methods at least done in triplicate with two or three enzyme extracts prepared from separate cultures. 2.1. Culture conditions Phosphoketolase Ž.EC 4.1.2.9 activity was deter- mined by spectrophotometric measurement of acetate Fibrobacter succinogenes S85Ž. ATCC 19169 was formationŽ. Boehringer kit after incubation of the grown on either a chemically defined mediumwx 5 , or enzyme extracts with either ribose-5-phosphate or a medium containing 40% rumen fluidwx 7 with 3 g fructose-6-phosphate. A reaction mixture composed ly1 cellobiose. F. succinogenes 095Ž obtained from of reduced bufferŽ 50 mM potassium phosphate, 0.4%

K-J Cheng, Lethbridge, Alberta, Canada. , HM2 Na23 CO , 0.05% cysteine, pH 7.1. , 0.6 mM TPP, 10 Ž.ATCC 43856 and F. intestinalis Ž.ATCC 43854 mM ADP, 10 mM DTT, 3.3 mM MgCl2 and 8 mM were grown on 40% rumen fluid mediumŽ 3 g ly1 of substrate was pre-incubated 90 s at 378C. The cellobiose. wx 7 . Lactobacillus plantarum ŽDSM reaction was initiated by addition of enzyme extract 20205. was grown in MRS mediumwx 8 with xylose Ž0.3 mg protein mly1. . The reaction was stopped at as substrate. Pseudomonas aeruginosa Ž.ATCC 9027 2, 5, 8, 11 and 15 min by heating for 15 min in a and Escherichia coli Ž.JM 109 were grown in Luria- water bath at 1008C,Ž controls on a standard solution Bertani mediumwx 9 . of acetate showed identical concentrations of acetate before and after 15 min at 1008C. , cooled in an ice 2.2. Cell extracts bath and centrifugedŽ. 15 000=g 5 min, 108C before acetate determination. Control samples incubated in The cells were grown for 15 h and harvested under parallel and containing boiled enzyme extracts showed a 100% CO2 athmosphere. Cells were collected by slight absorbance variationsŽ possibly due to substrate centrifugationŽ. 12 500=g 12 min, 48C , washed in degradation at 1008C. that were subtracted from each reduced bufferŽ 50 mM potassium phosphate, 0.4% sample absorbance. Same concentration of acetate 54 C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 were obtained with or without addition of commer- NAD, 15 mU of diaphorase from pig heartŽ Boeh- cial acetokinaseŽ. EC 2.7.2.1 . Incubations of enzyme ringer. , and 30 ml of iodonitrotetrazolium chloride extracts under anaerobic conditions and with or with- Ž.INT solution Ž Boehringer kit for the determination out DTT were performed in parallel. Detection of of L-glutamic acid. and the enzyme extract. The phosphoketolase activity in Lactobacillus plantarum reactions were initiated by adding 10 mM sodium enzyme extracts formed a positive control. 6-phosphogluconate. The reduction of NAD was PyruÕate formate lyase Ž.EC 2.3.1.54 activity was measured as NADH-diaphorase-dependent reduction determined by spectrophotometric measurement of of INT, monitored at 492 nm. The detection of these formate formationŽ. Boehringer kit . The crude ex- activities in E. coli enzyme extracts formed a posi- tractsŽ 10 mg protein mly1. were pre-incubated 90 s tive control. at 378C with 0.57 mM CoA, 0.2 mM S-adenosyl Non-oxidatiÕe part of the pentose phosphate path- methionine, 2 mM benzyl viologen, 10 mM DTT, 0.3 way enzymes. Crude extracts diluted in a 50 mM mM FeŽ.Ž. NH42 SO 42 before addition of 10 mM sodium-potassium phosphate bufferŽ. pH 6.9 were pyruvate. The reaction was stopped at 2, 5, 10 and 15 incubated at 378C with 10 mM ribose-5-phosphate min, by heating for 15 min at 1008CŽ controls on a during 20 min. The reaction was stopped by addition standard solution of formate showed identical con- of trichloracetic acidŽ. 1.6% final concentration . centrations of formate before and after 15 min at Phosphorylated metabolites were detected by 31P- 1008C.Ž , cooled in an ice bath and centrifuged 15 000 NMR. =g 5 min, 108C. before formate determination. Con- Xylose and xylulokinase. Crude extracts trol samples incubated in parallel and containing diluted in a 50 mM sodium-potassium phosphate boiled crude extracts showed no significant varia- bufferŽ. pH 6.9 were incubated at 378C with 10 mM tions. xylose and 10 mM ATP or GTP. The reaction was 6-phosphogluconate dehydrase Ž.EC.4.2.1.12 plus stopped after 60 min, by addition of trichloracetic 2-keto-3-deoxy-6-phosphogluconate aldolase ŽKDPG acidŽ. 1.6% final concentration . Phosphorylated aldolase, EC.4.1.2.15. were assayed by measuring the metabolites were detected by 31P-NMR. 6-phosphogluconate-dependent formation of pyruvate wx11 . The assay mixture contained 10 mM FeCl2 , 30 2.5. NMR spectroscopy mM glutathione, and 10 mM 6-phosphogluconate. The reactions were initiated by adding enzyme ex- NMR spectra were recorded on a Bruker MSL 300 tract, and stopped by heating in a water-bath for 10 spectrometer at 300.13 MHz for 1H, 75.4 MHz for min at 1008C. NaOHŽ. 1 M, 18 ml was then added to 13C and 121.49 MHz for 31P. 1H were decoupled adjust the pH to 7.2. The pyruvate formed was using a Waltz decoupling scheme to avoid sample 2 assayed using a Boehringer kit for pyruvate determi- heating. The H resonance of D2 OŽ. 10% was used to nation. Detection of activity in Pseudomonas aerugi- lock the field and for shimming. nosa enzyme extracts formed a positive control. 1H and 13C-NMR experiments and preparation of Glucose-6-phosphate dehydrogenase Ž.EC.1.1.1.49 cells were performed as previously describedwx 5 , was spectrophotometrically assayed by monitoring unless otherwise stated. The cells were at a final the glucose-6-phosphate dependent reduction of NAD concentration of 5 mg or 10 mg protein mly1 depend- or NADP at 365 nmwx 11 . The assay mixture con- ing on the experiments, they were incubated at 378C tained 10 mM MgCl2 , 10 mM glucose-6-phosphate with 32 mM or 64 mM glucose, respectively. and the enzyme extract. The reactions were initiated 31P-NMR experiments. Incubations of crude ex- by adding 0.4 mM NAD or NADP. Detection of tracts in the presence of ribose-5-phosphate or xylose activity in E. coli enzyme extracts formed a positive were centrifugedŽ. 15 000=g 10 min , and the super- control. natants were supplemented with trans-1,2-diamino- 6-phosphogluconate dehydrogenase Ž.EC 1.1.1.44 cyclohexane-N, N, N XX, N -tetraacetic acidŽ. CDTA , y2 y2 was spectrophotometrically assayed by monitoring Tris in D2 OŽ final concentration of 6.10 M, 4.10 the 6-phosphogluconate dependent reduction of NAD. M and 10%, respectively. wx 12 . The pH of the sam- The assay mixture contained 10 mM MgCl2 , 0.4 mM ples was adjusted to 7.4wx 13 , and 2 ml aliquots of the C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 55 samples were transferred to 10-mm diameter tubes. Table 1 1 1 At least 10 000 transients were collectedŽ 658 pulse, Phosphoketolase specific activities nmol miny mgy protein. 0.7 s recycling time, 6579 Hz spectral width, 8 K Strains Anaerobic conditions Aerobic conditions data points. . ribose-5-P Ribose-5-P Fructose-6-P qDTT yDTT 2.6. Chemicals F. succ. S85 143"60 0 0 F. succ. 095 140"5 0 nd nd 13 13 F. succ. HM2 62 0 nd nd C-Glucose and Na23 COŽ. 99% labelled were purchased from EurisotopŽ. Gif-sur-Yvette, France . F. intestinalis 127"31 0 nd nd L. plantarum nd nd 57 nd All enzymes and chemicals were purchased from Sigma or Boehringer. Enzyme extracts of Fibrobacter or Lactobacillus cells prepared under aerobic or anaerobic conditions were incubated at 378C with 0.6 mM TPP and 8 mM ribose-5-phosphate or 8 mM fructose-6-phosphate. 3. Results 0, no detectable activities; nd, not determined. a Aerobic conditions: 50 mM potassium phosphate, 3.3 mM

3.1. EÕidence of phosphoketolase actiÕity in Fi- MgCl2 bufferŽ. pH 7.1 . brobacter b Anaerobic conditions: 50 mM potassium phosphate, 0.4% Na23 CO , 0.05% cysteine, 10 mM DTT, 3.3 mM MgCl 2 buffer Ž.pH 7.1 under 100% CO athmosphere. Phosphoketolase converts xylulose-5-phosphate 2 into glyceraldehyde-3-phosphate and acetyl-phos- phatewx 14Ž. Fig. 2 . Ribose-5-phosphate was used as substrate as it was efficiently isomerized into xylu- measured when the enzyme extracts were prepared lose-5-phosphate in our extractsŽ. data not shown . A under aerobic conditions, even when DTTŽ 10 or 20 high ribose-5-phosphate phosphoketolase activity was mM. was added to the incubation mixture. Fructose- found in the presence of 5 mM DTT and 3 mM 6-phosphate was also tested as potential substrate but cysteineŽ. Table 1 , on enzyme extracts prepared un- no activity was found. Phosphoketolase activity was der strictly anaerobic conditions. No activity was also assayed in extracts prepared from other strains of

213 13 Fig. 3. In vivo kinetics of Na CO3 utilization by resting cells of F. succinogenes incubated in 10-mm tubes. 50 mM Na23CO and 64 mM glucose were added at zero time to 10 mg proteinPmly1 cell suspension. A. Proton-decoupled 13C-NMR spectra collected every 9 Ž. 13 Ž. min; spectra shown were recorded before ts0 and after glucose and Na23 CO addition ts9 and ts36 min . Ref: pure benzene Ž. Ž. yŽ. capillary used as external reference. B. Time-dependent changes of signal integrals. ^ C1 succinate, ` HCO32 , v CO aq. 56 C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60

F. succinogenes Ž.095, HM2 and one strain of F. 3.2. EÕidence of phospho-enol pyruÕate carboxyl- intestinalis Ž.NR9 , using ribose-5-phosphate or fruc- ation tose-6-phosphate as substrateŽ. Table 1 . High activity 13 was found on ribose-5-phosphate for these four Glucose utilization when Na 23CO was added to strains. Phosphoketolase of Lactobacillus plantarum, resting cells was monitored in vivo by 13C-NMR known to possess this activity when grown on xylose, spectroscopyŽ. Fig. 3 . Resonances at 160.1 and 125.5 y1 13 y 13 was measured as a positive controlŽ 57 nmol min ppm correspond to H CO32aq and COwx 18,19 . y1 y mg protein. , and values compatible to that ob- The concentration of HCO3 decreased with time tained by others were foundwx 15 . during the incubation, while the concentration of CO2 As we have shown that anaerobic conditions for aq remained level. This can be explained by a shift of y preparing enzyme extracts were essential for some the equilibrium between HCO32aqand CO probably enzyme activities, we tested xylose isomerase and due to the acidification of the medium by the acids xylulokinase activities in crude extracts prepared un- producedŽ. pH decreased from 6.7 to 5.8 . Concomi- der strictly anaerobic conditions. Extracts were incu- tantly, the resonance at 182.8 ppm assigned tow 1- bated with xylose and either ATP or GTP as phos- 13Cx succinate increased, reflecting the incorporation phate donors and supernatants were analyzed by 31P- NMR. No resonance corresponding to pentose phos- phates sugars was detected on spectraŽ data not shown. . To test for a deviation towards the oxidative phase of the pentose phosphate pathway, the enzymatic activities implied had to be identified. Glucose-6- phosphate dehydrogenase and 6-phosphogluconate dehydrogenaseŽ. Fig. 2 were sought in enzyme ex- tracts but could not be evidenced under our experi- mental conditions. These activities were detected un- der the same experimental conditions in E. coli extracts. A high endogenous NADH-dehydrogenase activitywx 16 present in our extracts might have pre- vented the detection of these activities. The enzymes of the non-oxidative phase of pentose phosphate pathway, isomerase, transaldolase and transketolase Ž.Fig. 2 were functional in our extracts, as shown by enzyme activity measurements and 31P-NMR. Reso- nances corresponding to xylulose-5-phosphate, sedo- heptulose-7-phosphate, and dihydroxyacetone-phos- phate were identified after incubation of crude ex- tracts with ribose-5-phosphateŽ. data not shown . The key enzymes of the Entner-DoudoroffŽ. ED pathway, 6-phospho-gluconate dehydrase and KDPG aldolaseŽ. Fig. 2 , were tested in F. succinogenes S85 enzyme extracts. No activity was found even on changing extraction method or reaction conditions 13 Žbuffer, mineral cations: Fe2q , Mn2q , Mg2q , reduc- Fig. 4. In vivo proton-decoupled C-NMR spectra of resting 1 ing agents e.g. DTT or GSH. while both activities cells of F. succinogenes Ž5 mg proteinPmly . incubated in 10-mm tubes with 32 mM 1-13 C glucose. Spectra were collected were detected in extracts from Pseudomonas aerugi- w x every 4.5 min, an example is given for ts36 min. Ref: pure nosa, known to metabolize glucose via the ED path- benzene capillary used as external reference. A.w 1-13 Cx glucose. waywx 17 . B.w 1-13 Cx glucose and HQNOŽ 6P10y2 mmol mgy1 protein. . C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 57

13 q of CO2 into succinateŽ. Fig. 3B . Resonances from cinate: NAD EC 1.3.1.6. in F. suc- 13 61.0 to 96.4 ppm and at 34.5 pm correspond to C cinogenes as in many other anaerobic bacteriawx 16,21 . natural abundance of respectively glucose and C2 of The resonance at 34.5 ppm for succinate was de- succinateŽ. Fig. 3A, ts9 min . At the end of the creasedŽ. inhibition of succinate synthesis compared incubation, while resonances corresponding to glu- to Fig. 4A by about 96%, and peaks corresponding to cose disappeared, resonances of 13C natural abun- malateŽ. 43.03 and 70.8 ppm from C3 and C2 , and dance glycogenŽ. 61.0 to 100.1 ppm were detected fumarateŽ. 135.7 ppm from C2 were observed. The Ž.Fig. 3A, ts36 minwx 20 . No other significantly assignments were confirmed by adding malate and labelled compound was observed, in particular no fumarate to acellular extracts. It was checked that labelled formate was detected. ethanolŽ. 58.3 and 17.7 ppm , the HQNO solvent, had This experiment performed directly on living cells no effect on metabolism of F. succinogenes at the clearly supports the carboxylation of phospho-enol concentration used. pyruvateŽ. PEP leading to final synthesis of succi- The detection of malate, fumarate and succinate, nate. all labelled fromw 1-13 Cx glucose, clearly evidenced the conversion of malate into fumarate by a fumarase 3.3. In ÕiÕo fumarase actiÕity Ž.or malate hydrolyase, EC 4.2.1.2 . As direct degra- dation ofw 1-13 Cx glucose leads to malate theoretically The utilization ofw 1-13 Cx glucose by resting cells of labelled only on the C3 positionŽ. Fig. 1 , the labelling F. succinogenes S85 was monitored in vivo by 13C- of the C2 position of malate implies the reversibility NMR spectroscopy. The utilization ofw 1-13 Cx glucose of the reaction catalysed by fumarase. led tow 2-13 Cxw succinate synthesis and to 1-13 Cx and Extracts performed at the end of the incubations of w6-13 Cx glycogen storageŽ. Fig. 4Awx 5 . In order to cells withw 1-13 Cx glucose in the absence of HQNO, evidence the intermediates of succinate synthesis in showed resonances ofw 2-13 Cxw acetate, 3-13 Cxw and 2- living cells, the electron-transport inhibitor HQNO 13Cxw malate and 2-13 Cx fumarateŽ. data not shown . Ž60 nmol mgy1 protein. was added to incubations of The presence of malate and fumarate was confirmed 1 resting cells withw 1-13 Cx glucoseŽ. Fig. 4B . HQNO is by the detection in H-NMR spectra of signals res- known to inhibit NADH: fumarate reductaseŽ suc- onating at 6.52 ppmŽ. fumarate and 2.66 ppm Ž malate,

Fig. 5. Proton-decoupled13 C-NMR spectra performed on acellular extracts of F. succinogenes Ž10 mg proteinPmly1. incubated with 64 mMw U6-13 Cx glucose, using an inverse-gated sequenceŽ AM400 Bruker spectrometer, 100.61 MHz, 908 pulse: 5.8 ms, relaxation delay: 60 s, data points: 32 K, 1200 scans, 5-mm diameter probe. . 58 C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 bCH2 ; parts A of ABX. . The unexpected labelling 4. Discussion on the C2 position of malate proved that the reversal of this branch takes also place under normal incuba- New features of glucose catabolism in F. succino- tion conditions. Furthermore, the almost equivalent genes were evidenced using NMR spectroscopy and intensities of the C2 and C3 signals of malate indi- enzymatic assays. cated a very efficient reversion. These results were A high pentose phosphates phosphoketolase activ- confirmed byw 2-13 Cx glucose utilization by resting ity was evidenced in the presence of DTT, when the cells, leading also to the synthesis of malate equiva- enzyme extracts were prepared under anaerobic con- lently enriched on the C2 and C3 positionsŽ data not ditionsŽ anaerobic buffer containing 3 mM cysteine shown. . and 5 mM DTT. , whereas no activity was found when the extracts were prepared under aerobic condi- 3.4. EÕidence of pyruÕate-formate lyase tions, even if DTT was added to the incubation mixture. The phosphoketolase was not active on fruc- When the cells were incubated with uniformly tose-6-phosphate. enrichedw U6-13 Cx glucoseŽ. 64 mM , resonances at Matte et al.wx 6 did not measure any significant 170.8 and 160.1 ppm were detected on quantitative pentose phosphates phosphoketolase activity on F. 13C-NMR spectra, performed on acellular extracts, succinogenes extractsŽ below 1 nmolPmgy1 .proteinP indicating the presence of 13C enriched formate and miny1. . This is probably due to differences in the - HCO3 Ž. Fig. 5 . As this spectrum was recorded under extracts preparation, as we have shown that phospho- quantitative conditions, it can be assumed that the ketolase activity could be irreversibly inhibited under 13 amount of H CO2 H is much lower than that of aerobic conditions. Pentose phosphates phosphoketo- 13 y 13 y H CO33. This is specially true as H CO is under- lase is usually found in yeasts or bacteria grown on 13 estimated as it is in equilibrium with CO2 . The xylosewx 15,25,26 where it accounts for the very presence of formate was confirmed by the detection efficient pentose metabolism. For these microorgan- of a resonance at 8.46 ppm in 1H-NMR spectra isms the enzyme can be active on ribose-5-phosphate, corresponding to formate moleculeŽ. data not shown . fructose-6-phosphate or both substrate. However, F. In parallel, formate was assayed enzymatically and succinogenes, while degrading xylans very effi- was found to be 0.22 mM. These results indicate that ciently, is not able to use xylose, due to the lack of formate is produced from glucose. Resonance corre- xylose permease, xylose isomerase and xylulokinase 13 sponding to H CO2 H was never observed when the wx6 . We checked xylose isomerase and xylulokinase cells were incubated withw 1-13 C or 2- 13 Cx glucose. in crude extracts of F. succinogenes prepared under 13 This means that the C of HCO2 H produced from strictly anaerobic conditions and also with GTP as glycolysis ofw U6-13 Cx glucose originated from the C1 phosphate donor. GTP was not tested as substrate for of pyruvate and thus from the C3 or C4 of glucose xylulokinase by Matte et al.wx 6 while it was shown Ž.Fig. 1 . Furthermore labelled formate was not de- that hexokinase of F. succinogenes was GTP depen- 13 tected when cells were incubated with Na23 COŽ Fig. dentwx 27 . However we confirm, using our extracts, 3. , indicating that formate was not produced via CO2 that xylose isomerase and xylulokinase were not pre- reduction. Pyruvate-formate lyase was assayed in sent in F. succinogenes. Pentose phosphates, needed crude extracts of F. succinogenes incubated with for nucleic acid biosynthesis are thus formed in a pyruvateŽ. 10 mM and CoA under strictly anaerobic reversal of the pentose phosphate pathway by the conditions in the presence of reduced benzyl viologen action of transketolase and transaldolase on fructose- as electron donor and of Fe2q, DTT and S-adeno- 6-phosphate and glyceraldehyde-3-phosphate. The sylmethionine as effectors. Under these conditions, presence of a pentose phosphates phosphoketolase in 29 nmol mgy1 proteinPminy1 of formate were pro- a bacterium unable to use exogenous pentoses is very duced. Activity was barely detectableŽ 3 nmol mgy1 surprising. As the F. succinogenes strain S85 used in protein P miny1 . in the absence of S-adeno- this study has been in artificial laboratory culture for sylmethionine. No formate was produced in the ab- many years, it is possible that genotypic and pheno- sence of pyruvate. typic changes have occurred during this time. Thus C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60 59 we looked for the presence of phosphoketolase activ- channelled to fumarate, giving rise through the activ- ity in other strains more recently isolated or originat- ity of fumarate reductase to succinate as the end ing from different animals Ž F. succinogenes 095, productwx 24 . The presence of a formate-hydrogen HM2 and F. intestinalis.. Pentose phosphates phos- lyase can be excluded as H2 was never detected as a phoketolase activity was evidenced in enzyme ex- product of F. succinogenes metabolismwx 1 . tracts of all the strains, suggesting that phosphoketo- In addition, this study brought experimental evi- lase is a characteristic enzyme of the genus Fi- dence of hypothetical steps in F. succinogenes brobacter and not a particularity of the S85 strain of metabolism. First, in vivo 13C-NMR spectroscopy in F. succinogenes. We suggest that the role of such an the presence of HQNO evidenced the nature of the enzyme in a bacterium unable to use pentoses could intermediate metabolitesŽ. malate and fumarate in the be to allow the synthesis of ATP when endogenous production of succinate from phospho-enol pyruvate phosphorylated pentoses, obtained by reversal of the and thus the presence of a fumarase ŽL-malate hy- pentose phosphate pathway, are accumulating, possi- drolyase EC 4.2.1.2. . The equivalent labelling of the bly under conditions of slow or no growth. C2 and C3 atoms of the malate molecule showed the Incubation of cells withw U6-13 Cx glucose showed reversibility of the fumarase under anaerobic condi- that F. succinogenes can produce low amounts of tions, even in the absence of HQNO, suggesting that 13C formate from pyruvate cleavage while no labelled the fumarate reductase might be limiting in this path- 13 13 formate was observed during Na23 CO orw 1- C or way. Second, incubation of living cells of F. succino- 13 13 2- Cx glucose utilization. In fact, it is generally ac- genes with Na 23CO confirmed the incorporation of 13 cepted that F. succinogenes produces formate as CO2 in the carboxylic group of succinate. minor metabolitewx 2 . In most anaerobic bacteria as Finally, we did not detect the activity of the en- well as in facultatively anaerobic bacteria such as E. zymes of the Entner-Doudoroff pathway, nor of those coli, formate is generally produced via the CoA-de- of the oxidative part of the pentose phosphate path- pendent, non-oxidative cleavage of pyruvate to way. Thus, in spite of the presence of phosphoketo- acetyl-CoA catalyzed by pyruvate-formate lyasewx 22 . lase, that connected with the oxidative part of the In E. coli, under anaerobic conditions, pyruvate-for- pentoses phosphates pathway would produce unla- mate lyase is converted into an active form through belled acetate fromw 1-13 Cx glucose, the low percent- the action of an activating enzyme. This interconver- age of enrichment of acetate measured remains unex- sion is a reductive process which uses a reducing plained. This implies the presence of a different agent such as reduced benzyl viologen for electron pathway not yet identified leading to the synthesis of donation and Fe2q, DTT and S-adenosylmethionine unlabelled acetate. as positive effectorswx 22,23 . As formate synthesis was dependent on the presence of these effectors in F. succinogenes extracts, we suggest that formate is Acknowledgements produced during the non-oxidative cleavage of pyru- vate to acetyl-CoA catalysed by a pyruvate-formate This work was supported by a grant from the lyase. Miller proposed that in F. succinogenes, pyru- CNRS and the Region Auvergne. vate is oxidized via a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and COwx 4 . These 2 References two activities are both likely to contribute to pyruvate degradation in F. succinogenes. It can be noticed that wx1 Stewart C.S. and Flint H.J.Ž. 1989 Appl. Microbiol. Biotech- the amount of formate is low compared to that of nol. 30, 433±439. y Ž. HCO3 . Two interpretations can be given, depending wx2 Bryant, M.P. and Burkey, L.A. 1953 J. Dairy Sci. 36, on the origin of CO : i. the activity of pyruvate-for- 205±217. 2 Ž. mate lyase is low compared to that of pyruvate wx3 Joyner Jr., A.E. and Baldwin, R.L. 1966 J. Bacteriol. 92, 1321±1330. flavoprotein oxido-reductase, ii. the major part of wx4 Miller, T.L.Ž. 1978 Arch. Microbiol. 117, 145±152. q formate is converted to CO2 q2H by a formate wx5 Gaudet, G., Forano, E., Dauphin, G. and Delort, A.-M. dehydrogenase, and from there, electrons could be Ž.1992 Eur. J. Biochem. 207, 155±162. 60 C. Matheron et al.rBiochimica et Biophysica Acta 1355() 1997 50±60

wx6 Matte, A., Forsberg, C.W. and Verrinder, A.M.Ž. 1992 Can. wx18 Santos, H., Fareleira, P., Toci, R., Legal, J., Peck Jr., H.D. J. Microbiol. 38, 370±376. and Xavier, A.V.Ž. 1989 Eur. J. Biochem. 180, 421±427. wx7 Halliwell, G. and Bryant, M.P.Ž. 1963 J. Gen. Microbiol. wx19 Narbad, A., Hewlins, M.J.E. and Callely, A.G.Ž. 1989 J. 32, 441±448. Gen. Microbiol. 135, 1469±1477. wx8 Man, J.C., de Rogosa, M. and Sharpe, M.E.Ž. 1960 J. Appl. wx20 Canioni, P., Alger, J.R. and Shulman, R.G.Ž. 1983 Biochem- Bacteriol. 23, 130±135. istry 22, 4974±4980. wx9 Maniatis, T., Fritsch, F. and Sambrook, J.Ž. 1989 Molecular wx21 Stouthamer, A.H.Ž. 1980 in: Anaerobes and Anaerobic In- cloning: a laboratory manual. Cold Spring Harbor Labora- fectionsŽ. Gottschalk, et al., eds. , pp. 17±29, Gustav Fischer tory, Cold Spring Harbor, New York. Verlag, Stuttgart New York. wx10 Lowry, O.M., Rosebrough, N.J., Farr, A.L. and Randall, wx22 Knappe, J., Elbert, S., Frey, M. and Wagner, A.F.V.Ž. 1993 A.J.Ž. 1951 J. Biol. Chem. 193, 265±275. Biochem. Soc. Trans. 21, 731±734. wx11 Eidels, L. and Preiss, J.Ž. 1970 Arch. Biochem. Biophys. wx23 Wong, K.K., Murray, B.W., Lewisch, S.A., Baxter, M.K., 140, 75±89. Ridky, T.W., Ulissi-DeMario, L. and Kozarich, J.W.Ž. 1993 wx12 Dorne, A.J., Bligny, R., Rebeille, F., Roby, C. and Douce, Biochemistry 32, 14102±14110. R.Ž. 1987 Plant Physiol. Biochem. 25, 589±595. wx24 Gottschalk, G.Ž. 1986 in: Bacterial metabolism. 2nd ed. pp. wx13 Mendz, G.L. and Hazell, S.L.Ž. 1991 FEMS Microbiol. Lett. 240±241, Springer-Verlag, New York. 84, 331±336. wx25 Ratledge, C. and Holdsworth, J.E.Ž. 1985 Appl. Microbiol. wx14 Evans, C.T. and Ratledge, C.Ž. 1984 Arch. Microbiol. 139, Biotechnol. 22, 27±221. 48±52. wx26 Veiga-da-Cunha, M., Santos, H., Van Schaftingen, E.Ž. 1993 wx15 Marounek, M. and Petr, O.Ž. 1995 Lett. Appl. Microbiol. J. Bacteriol. 175, 3941±3948. 21, 272±276. wx27 Glass, T.L. and Sherwood, J.S.Ž. 1994 Arch. Microbiol. wx16 Meinhardt, S.W. and Glass, T.L.Ž. 1994 Curr. Microbiol. 162, 180±186. 28, 247±251. wx17 Meloche, P. and Wood, W.A.Ž. 1966 Methods Enzymol. 9, 653±656.