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 metabolism in Fibrobacter succinogenes, using NMR spectroscopy and enzymatic assays. Evidence for pentose phosphates phosphoketolase and pyruvate formate lyase 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 enzyme 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 enzymes 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.