Fermentation of L-Aspartate by a Saccharolytic Strain of

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1 APPLIZD AND ENVIRONMENTAL MICROBIOLOGY, Jn. 1977, p Copyright 1977 Americn Society for Microbiology Vol. 33, No. 1 Printed in U.S.A. Fermenttion of L-Asprtte by Scchrolytic Strin of Bcteroides melninogenicus JASON C. WONG, JOHN K. DYER,* AD JACK L. TRIBBLE Deprtment of Orl Biology, College of Dentistry,* nd Section of Microbiology, Immunology, nd Plnt Pthology, School of Life Sciences, University of Nebrsk, Lincoln, Nebrsk Received for publiction 7 July 1976 Resting cells of Bcteroides melninogenicus fermented L-[14C]sprtte s single substrte. The 14C-lbeled products included succinte, cette, C02, oxlocette, formte, mlte, glycine, lnine, nd fumrte in the reltive percentges 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, nd 0.06, respectively, bsed on the totl counts per minute of the L_-[4C]sprtte fermented. Ammoni ws produced in high mounts, indicting tht 96% of the L-sprtte fermented ws deminted. These dt suggest tht L-sprtte is minly being reduced through number of intermedite rections involving enzymes of the tricrboxylic cid cycle to succinte. L_[14C]sprgine ws lso fermented by resting cells of B. melninogenicus to form -sprtte, which ws subsequently, but less ctively, fermented. Bcteroides melninogenicus represents heterogeneous group of microorgnisms tht cn vry widely from strin to strin with regrd to biochemicl nd serologicl chrcteristics (2, 9). These orgnisms utilize mino cids s sources of energy nd, in ddition, some strins ferment vriety of crbohydrtes. Studies with four strins ofb. melninogenicus showed tht they cn ferment mino cids when present s peptides, but my be limited in fermenting free mino cids (12). Recently, the influence of single mino cids on growth of either scchrolytic or scchrolytic strins of B. melninogenicus ws reported (6). Of ll the mino cids tested, L-sprtte nd L-sprgine induced mximl growth stimultion of both strins. Preliminry results indicted tht the growth-stimulting cpcity ws dependent upon fermenttion of these specific mino cids. The present study ws initited to determine both qulittively nd quntittively the products of i-sprtte fermenttion by the scchrolytic strin of B. melninogenicus. Crbon nd nitrogen blnces of these products were clculted. These dt suggest tht L-sprtte is minly reduced through number of intermedite rections involving enzymes of the tricrboxylic cid cycle to succinte. MATERIALS AND METHODS Orgnism nd culturl methods. B. melninogenicus subsp. melninogenicus ATCC 25261, scchrolytic strin obtined from the Americn Type Culture Collection, Rockville, Md., ws used in these studies. The culture medi ws Trypticse-yest extrct-hemin (TYH) medium of the following composition: Trypticse (BBL, Cockeysville, Md.), 3%; yest extrct (Difco, Detroit, Mich.), 0.3%; sodium thioglycolte, 0.05%; hemin (equine type III, Sigm Chemicl Co., St. Louis, Mo.), 5 ug per ml; nd NCl, 0.5%. Resting cells for the fermenttion studies were grown nerobiclly t 37 C in TYH medium supplemented with 0.5% L-sprtte (Sigm, regent grde). Cells were hrvested in the log phse (18 to 20 h) by centrifugtion t 12,000 x g t 5 C nd suspended to concentrtion of mg (dry weight) per ml in 0.1 M potssium phosphte buffer (ph 7.0). Growth responses to individul mino cids in combintion with L-sprtte were tested in T-YH medium with mino cids dded t 0.5% concentrtion. The mino cids (Sigm, regent grde) tested in combintion with L-sprtte included L-cysteine, L-serine, L-lnine, L-proline, L-ornithine, L-leucine, glycine, L-threonine, nd L-glutmte. The inocul for growth studies were grown nerobiclly t 37 C for 18 to 20 h. Cell suspensions were stndrdized to n bsorbnce of 0.7 to 0.8 in 13-mm tubes t 600 nm in buffer (0.05 M potssium phosphte, ph 7.0, with 0.05% sodium thioglycolte) using Busch nd Lomb Spectronic 20 colorimeter. For growth determintions (repeted four times), mtched test tubes (13 by 100 mm) contining 6 ml of medium were closed with rubber serum stoppers immeditely fter utoclving, cooled, nd inoculted to n bsorbnce of 0.03 using tuberculin syringe. The tubes were incubted t 37 C, nd bsorbnce redings were tken s indicted. Fermenttions. Fermenttions of rdioctive substrtes were conducted in test tubes (20 by

2 70 WONG, DYER, AND TRIBBLE mm) t 37 C. A glss vil contining 0.5 ml of 1 M hydroxide of hymine in methnol ws suspended bove the rection mixture. The tubes were fitted with n entry port covered with rubber serum stopper to llow ddition of 2 M H2SO4 to stop the rection nd relese 14CO2. The system ws flushed with rgon immeditely fter dding the components of the rection mixture. Anlyticl methods. Rection mixtures were quntittively trnsferred to centrifuge tubes, nd the cells were removed by centrifugtion. Voltile cids were seprted from the superntnt by stem distilltion. The cids were neutrlized with NOH nd concentrted under vcuum. The individul voltile cids from the concentrte were seprted nd identified using the chromtogrphic procedure of Seki (10) by use of column (1.2 by 70 cm) of Amberlite CG-50, 100 to 200 mesh (Mllinckrodt Chemicl Works, St. Louis, Mo.). Frctions (1 ml) were collected t flow rte of 6 ml/h. The totl rdioctivity for ech cid ws determined on subsmples of the frctions. Ammoni ws quntitted by stem distilltion from 1 M NOH, followed by titrtion with stndrd cid. The totl nions were seprted from the mino compounds with smll Dowex resin column (0.6 by 6 cm) s described by Mitruk nd Costilow (7). Individul nonvoltile nionic compounds were seprted by n ion-exchnge chromtogrphic procedure similr to tht of Busch et l. (1) using column (1.1 by 15.5 cm) of Dowex 1-formte form (Bio-Rd Lbortories, Richmond, Clif., AG 1- x 10, 100 to 200 mesh). A 6 N formic cid elution grdient ws used. Frctions (2 ml) were collected t flow rte of 24 ml/h. The totl rdioctivity for ech cid ws determined. The identity of the orgnic cids ws confirmed by thin-lyer chromtogrphy using cellulose pltes (Brinkmnn Instruments, Inc., Westbury, N.Y.) with lyer thickness of 0.10 mm. Diethyl ether-formic cid (90%)-wter (7:2:1) ws used s the solvent. Ctions were eluted from the column with 1 M NH40H concentrted to dryness nd mde to volume in wter. Amino cids were seprted nd identified by thin-lyer chromtogrphy (s bove) with four solvent systems: n-propnol-concentrted NH40H (7:3); chloroform-methnol-17% NH40H (40:40:20); phenol-wter (75:25); nd n-butnolcetic cid-wter (80:20:20, upper phse). For quntittion, duplicte smples of the totl ctions were pplied 2 cm prt on the cellulose pltes. For locting the individul mino cids, ninhydrin regent ws used s the indictor, with the spce bove the duplicte smple shielded by glss plte during sprying. The thin-lyer pltes were then heted t 100 C for severl minutes until blue-colored spots becme visible. Zones of the nonspryed smple corresponding to the visible cids were scrped off, nd rdioctivity ws determined. The scintilltion fluid Inst Gel (Pckrd Instrument Co., Inc., Downers Grove, Ill.) ws used in ll rdioctive ssys. Internl stndrds were used for quench corrections. A Tri-Crb liquid scintilltion spectrometer (Pckrd Instrument Co., Inc.), model 2002, ws used for counting the rdioctivity. Rdiochemicls. Uniformly lbeled L-['4C]spr- APPL. ENVIRON. MICROBIOL. tic cid nd uniformly lbeled L-[14C]sprgine were obtined from New Englnd Nucler Corp., Boston, Mss. RESULTS Quntittive nlysis of the rdioctive components in the superntnt showed tht resting-cell suspensions were ble to ferment L- sprtte s single substrte (Tble 1). The cells fermented 88.1% of the i_[14c]sprtte dded (120,umol) in 6-h period. The 14C-lbeled products included CO2, formte, cette, oxlocette, succinte, mlte, fumrte, lnine, nd glycine in the reltive percentges 9.9, 1.8, 15, 2.7, 68, 1, 0.06, 0.5, nd 0.7, respectively, bsed on the totl counts per minute of the L_['4C]sprtte fermented. Ammoni ws produced in high mounts, indicting tht 96.3% of the L-sprtte fermented ws deminted. In this experiment, the totl rdioctivity recovery ws 99.6%. The high recoveries of crbon nd nitrogen (99.6 nd 98.2%, respectively) indicted tht essentilly ll products were recovered. Resting-cell suspensions were lso ble to ferment L_['4C]sprgine s single substrte (Tble 2). Essentilly ll L-sprgine dded (60,umol) ws fermented in 6-h period with high ccumultion of sprtte (33.1% of the totl counts per minute of the L-sprgine fermented). The other lbeled products were similr to those found for L-sprtte fermenttion nd quntittively proportionl bsed on the mount of L-sprgine utilized. Anlysis of the mmoni produced showed tht 160.8,umol of mmoni ws formed from 100 umol of substrte fermented. The totl rdioctivity recovery ws 97%. The growth responses of the orgnism to the ddition of individully selected mino cids, together with L-sprtte, re shown in Tble 3. A moderte increse in growth rte nd totl cell yield ws observed with the ddition of i- cysteine, L-serine, L-threonine, or L-lnine to TYH medium, supplemented with L-sprtte, s compred with the growth rte in unsupplemented TYH medium with L-sprtte. Addition of L-proline, L-ornithine, L-leucine, glycine, nd L-glutmte hd no effect on growth. The results of L-['4C]sprtte fermenttion in the presence of individully selected mino cids or glucose by resting cells re shown in Tble 4. The products detected in ll cses were qulittively nd quntittively similr to those found in the L-sprtte fermenttion. However, in the presence of glucose, less mmoni nd CO2 were produced, wheres more lnine ws detected. Recovery of dded rdioctivity ws essentilly complete.

3 VOL. 33, 1977 TABLE 1. L-ASPARTATE FERMENTATION 71 Crbon nd nitrogen blnces of L-sprtte fermenttion Amt per 100,umol of L-sprtte fermented Determintion Rdioctivity % of totl cpm (cpm) Product Crbon Nitrogen (,umol) (j,utoms) (,utoms) Substrte L-['4C]sprtte Added x Fermented x Products Co x b Formte x Acette x Oxlocette x Succinte x Mlte x Fumrte x Alnine x Glycine x NH d Totl in productsc x Rection mixtures in tubes (20 by 150 mm) contined 400,umol of potssium phosphte buffer (ph 7.0), 120 Atmol of L-['4C]sprtte (specific ctivity, 18,260 cpm/4mol), 84 mg (dry weight) of cells, nd wter to 5.6 ml. To trp 14CO2, glss vil contining 0.4 ml of 1 M hydroxide of hymine in methnol ws suspended bove the rection mixture. The rection ws incubted under rgon t 37 C for 6 h nd ws stopped by ddition of 0.4 ml of 2 M H2SO4. b Vlues represent percentges of totl counts per minute of the L-['4C]sprtte fermented. c Crbon recovery = 99.6%; nitrogen recovery = 98.2%. d Vlues for NH3 in the rection mixture without substrte were subtrcted. TABLE 2. Products of L-sprgine fermenttion Substrte Product Rdioctivity (cpm) % of totl cpm L-['4C]sprgine Added x Fermented x CO x " Anionic compounds x Asprtte x Glycine x Alnine x NH3C Totl in products x Rection mixtures in tubes (20 by 150 mm) contined 200,umol of potssium phosphte buffer (ph 7.0), 60 /Imol of L-[14C]sprgine (specific ctivity, 18,550 cpm/,umol), 33.4 mg (dry weight) of cells, nd wter to 2.8 ml. To trp '4CO2, glss vil contining 0.4 ml of 1 M hydroxide of hymine in methnol ws suspended bove the rection mixture. The rection ws incubted under rgon t 37 C for 6 h nd ws stopped by ddition of 0.2 ml of 2 M H2SO4. b Vlues represent percentges of totl counts per minute of the L-['4C]sprgine fermented. c A totl of 160.8,umol of NH3 ws formed from 100 /.mol of substrte fermented. DISCUSSION The present study shows tht resting cells of B. melninogenicus re ble to ferment L-sprtte s single substrte with succinte s the mjor product (Tble 1). This finding is significnt becuse succinte hs never been demonstrted s mjor product of mino cid fermenttion by B. melninogenicus. Since detectble mounts of oxlocette, mlte, nd fumrte were lso present mong the products, the mjor pthwy of L-sprtte fermenttion by B. melninogenicus my be tht L- sprtte first undergoes oxidtive demintion, forming oxlocette. Trnsmintion my be unlikely, since ll of the mmonium from L-sprtte ws recovered in the form of mmoni, except for trce mounts found in

4 72 WONG, DYER, AND TRIBBLE lnine nd glycine. Subsequently, oxlocette could be reduced through number of intermedite rections involving enzymes of the tricrboxylic cid cycle vi mlte nd fumrte to succinte. Also, L-sprtte my be directly converted to fumrte, ctlyzed by the enzyme sprtse, which ws demonstrted in mny fculttive nerobic bcteri (11). Metbolism of B. melninogenicus ppers to require the presence of functionl membrne-bound electron trnsport system (8). This respirtory system includes cytochrome c, crbon monoxide-binding pigment, nd possibly flvoproteins. Rizz et l. (8) lso reported tht the pigments could be reversibly reduced by reduced nicotinmide denine dinucleotide (NADH) or endogenous metbolism nd could be oxidized nerobiclly by fumrte or by shking in ir. C. Reddy nd M. P. Brynt (Bcteriol. Proc., p. 40, 1967) observed tht one strin of B. melninogenicus contined cytochrome c nd second strin contined cytochromes b nd o. Another heme-requiring nerobe, Bcteroides ruminicol, contins membrne-bound electron trnsport system tht is reduced in the presence of NADH nd is TABLE 3. Effect of single mino cid dditions to TYH medium supplemented with 0.5% L-sprtte on the growth of B. melninogenicus ATCC Amino cid d- Amt of growth tb ded 10 h 20 h 30 h 40 h 50 h None L-Cysteine L-Serine L-Threonine L-Alnine Ech mino cid ws dded in 0.5% concentrtion. b Opticl density t 600 nm. oxidized in the presence of fumrte (13). Mcy et l. (5) found type b cytochrome, possibly type c cytochrome, nd very ctive fumrte reductse in the cells of strin of Bcteroides frgilis grown with hemin present in glucosebsed medium. However, this orgnism ws unble to synthesize cytochromes nd functionl fumrte reductse when grown in the bsence of hemin. These uthors suggested tht hemin is required by B. frgilis to induce synthesis of functionl fumrte reductse nd tht the hemin-dependent increse of the growth yield my be due to denosine 5'-triphosphte production during reduction of fumrte to succinte. Bsed upon the evidence tht functionl electron trnsport system with cytochromes is present in vrious Bcteroides species, it is tempting to speculte tht similr electron trnsport system is lso present in the strin of B. melninogenicus investigted in this study nd tht hemin is required for synthesizing the cytochrome-contining enzyme system. If this postultion is correct, L-sprtte fermenttion by B. melninogenicus my be ssocited with the membrne-bound electron trnsport system by which denosine 5'-triphosphte is synthesized vi nerobic electron trnsport phosphoryltion during the reduction of fumrte to form succinte. Since CO2, cette, formte, nd trce mounts of glycine nd lnine were lso found s products of L-sprtte fermenttion (Tble 1), n oxidtive pthwy of L-sprtte fermenttion must lso be present. However, there is not enough evidence from the present study to postulte the oxidtive pthwy becuse there re too mny probble pthwys by which these products could be formed. Bsed on the fcts tht NADH cn only be prtly furnished by the oxidtive demintion of L-sprtte TABLE 4. Effect of vrious compounds on L-[14C]sprtte fermenttion L-[4C]sprtte fermented Products (% of totl cpme) Amt of NH3 Additive Anionic formed cpm (x 10-5) %b c compoundo Alnine Glycine Totl (,.mol) None L-Alnine L-Serine L-Threonine '-(+)-Glucose Rection mixtures in tubes (20 by 150 mm) contined 200,umol of potssium phosphte buffer (ph 7.0); 60 gmol of L_[44C]sprtte (specific ctivity, 17,495 cpm/,umol); 60 Amol of L-lnine, L-serine, or L- threonine, or 0.1% D-(+)-glucose; 35.7 mg (dry weight) of cells; nd wter to 2.8 ml. To trp 14C02, glss vil contining 0.4 ml of 1 M hydroxide of hymine in methnol ws suspended bove the rection mixture. The rection ws incubted under rgon t 37 C for 6 h nd ws stopped by ddition of 0.2 ml of 2 M H2SO4. b Vlues represent percentges oftotl counts per minute ofthe L-[14C]sprtte dded ( x 106 cpm). c All vlues re bsed on the mount of L_['4C]sprtte fermented. APPL. ENVIRON. MICROBIOL.

5 VOL. 33, 1977 nd tht NADH from endogenous metbolism of the resting cells is not sufficient for the reductive pthwy of L-sprtte fermenttion to be operble, NADH must be supplied by the oxidtive pthwy of L-sprtte ctbolism. The ccumultion of sprtte resulting from the exposure of L-['4C]sprgine to resting cells of B. melninogenicus (Tble 2) indictes tht L-sprgine is ctively deminted to L- sprtte, which is subsequently, but less ctively, fermented. The formtion of sprtte nd mmoni from L-sprgine my by ctlyzed by the enzyme L-sprginse, which hs been demonstrted in Escherichi coli (4). L-Alnine, L-serine, nd L-threonine were shown to be growth stimulting when dded to TYH medium supplemented with L-sprtte (Tble 3). It ws decided to determine whether the enhncement effect of these mino cids ws due to fermenttion by coupled oxidtionreduction rection with L-sprtte fermenttion. However, these stimultory mino cids did not show ny effect on L[-4C]sprtte fermenttion (Tble 4). In ddition, very little mmoni other thn tht from L-sprtte fermenttion ws produced in ll three cses, which indictes tht these three mino cids pprently re not fermented by resting cells of B. melninogenicus. This is similr to the observtion of Whren nd Gibbons (12) tht most mino cids tested were not fermented by the resting-cell suspensions of their strins of B. melninogenicus. Glucose is fermented to form succinte, with phosphoenolpyruvte, pyruvte, oxlocette, mlte, nd fumrte s intermedites, by E. coli under nerobic conditions (3) nd B. frgilis grown nerobiclly in the presence of hemin (5). Results of L-[4Clsprtte fermenttion in the presence of glucose (Tble 4) show tht glucose hs no effect on L-sprtte fermenttion, which indictes tht the pthwy of glucose fermenttion by B. melninogenicus L-ASPARTATE FERMENTATION 73 my not be similr to tht by E. coli nd B. frgilis. ACKNOWLEDGMENTS This investigtion ws supported by Public Helth Service Grnt DEO-3672 from the Ntionl Institute of Dentl Reserch. We pprecite the ssistnce of Donn Dinges in the preprtion of the mnuscript. LITERATURE CITED 1. Busch, H., R. B. Huribert, nd V. Potter Anion exchnge chromtogrphy of cids of the citric cid cycle. J. Biol. Chem. 196: Cournt, P. R., nd R. J. Gibbons Biochemicl nd immunologicl heterogeneity of Bcteroides melninogenicus. Arch. Orl Biol. 12: Dvis, B. D Biosynthesis, p In B. D. Dvis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg, nd W. B. Wood (ed.), Microbiology, 2nd ed. Hrper nd Row Publishers, Hgerstown, Md. 4. Jckson, R. C., nd R. E. Hndschumcher Escherichi coli L-sprginse. Ctlytic ctivity nd subunit nture. Biochemistry 9: Mcy, J., I. Probst, nd G. Gottschlk Evidence for cytochrome involvement in fumrte reduction nd denosine 5'-triphosphte synthesis by Bcteroides frgilis grown in the presence of hemin. J. Bcteriol. 123: Miles, D. O., J. K. Dyer, nd J. C. Wong Influence of mino cids on the growth of Bcteroides melninogenicus. J. Bcteriol. 127: Mitruk, B. M., nd R. N. Costilow Arginine nd ornithine ctbolism by Clostridium botulinum. J. Bcteriol. 93: Rizz, V., P. R. Sinclir, D. C. White, nd P. R. Cournt Electron trnsport system of the protoheme-requiring nerobe Bcteroides melninogenicus. J. Bcteriol. 96: Swyer, S. J., J. B. Mcdonld, nd R. J. Gibbons Biochemicl chrcteristics ofbcteroides melninogenicus. Arch. Orl Biol. 7: Seki, T Chromtogrphic seprtion of lower ftty cid. J. Biochem. Tokyo 45: Virtnen, A. I., nd N. Elifolk Asprtse, p In S. P. Colowick, nd N. 0. Kpln (ed.), Methods in enzymology, vol. 2. Acdemic Press Inc., New York. 12. Whren, A., nd R. J. Gibbons Amino cid fermenttion by Bcteroides melninogenicus. Antonie vn Leeuwenhoek J. Microbiol. Serol. 36: White, D. C., M. P. Brynt, nd D. R. Cldwell Cytochrome-linked fermenttion in Bcteroides ruminicol. J. Bcteriol. 84: