Proton-Motive-Force-Dependent and Non-Proton-Motive-Force-

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1 JOURNAL OF BACTRIOLOGY, Jn. 1989, p Vol. 171, No /89/128-5$2./ Copyright C 1989, Americn Society for Microiology Trnsport of Amino Acids in Lctocillus csei y Proton-Motive-Force-Dependent nd Non-Proton-Motive-Force- Dependent Mechnisms HRBRT J. STROBL, JAMS B. RUSSLL,t* ARNOLD J. M. DRISSN, AND WIL N. KONINGS Deprtment of Microiology, University of Groningen, 9751 NN Hren, The Netherlnds Received 2 June 1988/Accepted 8 Octoer 1988 Lctocilus csei 393 cells which were energied with glucose ( 6.) took up glutmine, sprgine, glutmte, sprtte, leucine, nd phenyllnine. Little or no uptke of severl essentil mino cids (vline, isoleucine, rginine, cysteine, tyrosine, nd tryptophn) ws oserved. Inhiition studies indicted tht there were t lest five mino cid crriers, for glutmine, sprgine, glutmte/sprtte, phenyllnine, or rnched-chin mino cids. Trnsport ctivities hd optim etween 5.5 nd 6., ut ll mino cid crriers showed significnt ctivity even t 4.. Leucine nd phenyllnine trnsport decresed mrkedly when the ws incresed to 7.5. Inhiitors which decresed proton motive force (Ap) nerly eliminted leucine nd phenyllnine uptke, nd studies with de-energied cells nd memrne vesicles showed tht n rtificil electricl potentil (A*) of t lest -1 mv ws needed for rpid uptke. An rtificil Ap ws unle to drive glutmine, sprgine, or glutmte uptke, nd trnsport of these mino cids ws sensitive to decline in intrcellulr. When intrcellulr ws greter thn 7.7, glutmine, sprgine, or glutmte ws trnsported rpidly even though the proton motive force hd een olished y inhiitors. Lctocilli re grm-positive, fermenttive cteri which cn rpidly lower (7), nd mny species re hlotolernt (2). They re importnt in the preservtion of foods such s cheese, susge, nd pickled vegetles; the vlue of these fermenttions exceeds $1 illion per yer (2). Lctocilli re lso le to colonie the digestive trct, nd they my ply therpeutic role in humn nd niml helth (5). The study of lctocillus genetics ws once stymied y the lck of nturl gene exchnge mechnisms, ut their economic importnce hs creted n interest in genetic mnipultion (2). Lctocilli hve nutritionl requirements for mino cids, vitmins, ftty cids, nd nucleic cids (2), ut there hve een few studies of solute trnsport. Homofermenttive species (e.g., Lctocillus csei) hve phosphotrnsferse system for glucose, ut heterofermenttive orgnisms pper to use proton-motive-force (Ap)-driven mechnisms of glucose uptke (18). Mny lctocilli require folte, nd the uptke involves ATP or some other phosphte-ond intermedite (6). Lctocilli cnnot utilie mmoni s nitrogen source (2), nd from 7 to 15 mino cids re essentil for growth (9). L. csei requires 1 mino cids, ut our results indicted tht only 4 of them, glutmte, sprtte, leucine, nd phenyllnine, re tken up rpidly. Glutmine nd sprgine, nonessentil mino cids, were tken up more quickly thn other mino cids. Studies with memrne vesicles indicted tht some mino cids re trnsported y Ap systems, while others re pprently tken up y mechnisms which do not require Ap. Memrne vesicles of lctocilli hd not een previously used to study solute trnsport. * Corresponding uthor. t Permnent ddress: Agriculturl Reserch Service, U.S. Deprtment of Agriculture, nd Cornell University, Ithc, NY MATRIALS AND MTHODS Orgnism nd growth conditions. L. csei 393 ws otined from A. H. Romno, Microiology Section, University of Connecticut, Storrs. The growth medium ws descried previously (17); the ws djusted to 6.7. Glucose ws dded s seprte solution (finl concentrtion, 4 g/ liter), nd cultures were grown t 39 C. Strin 393 ws unle to grow rpidly in medium (14) contining purified mino cids nd Tween 8 (. Merck AG, Drmstdt, Federl Repulic of Germny) insted of peptone (Difco Lortories, Detroit, Mich.). Glucose-energied cells. Btch cultures were grown overnight to n opticl density t 66 nm of 1. (16-mm tues) nd hrvested y centrifugtion (1,2 x g, 5 min, 2 C). Cells were wshed twice with 1 mm potssium phosphte ( 6.) contining 1 mm MgSO4. Cells (pproximtely 32,ug of protein) were energied with 2 mm glucose for 15 min, nd trnsport ssys were conducted t 28 C in 2,ul of uffer contining 1 to 3,uM 14C-mino cids (1 nci), 1 mm MgSO4, nd either 1 mm potssium phosphte ( 6. to 7.5) or 1 mm morpholineethnesulfonic cid ( 4. to 5.5). Trnsport ws terminted y dding 2 ml of ice-cold 1 mm LiCl to the rection mixture nd filtering this mixture through cellulose nitrte memrne filter (.45-,um pore sie). The filters were wshed once with 2 ml of 1 mm LiCl nd dried for 2 min t 12 C, nd rdioctivity ws mesured y liquid scintilltion. Metolic inhiitors nd ionophores were dded t the sme time s glucose, nd the finl ssy volume ws 21,ul. The Vmx nd K, for ech mino cid were estimted t sustrte concentrtions rnging from one-fifth to three times the K,. Initil rtes were determined over 3 s, nd previous experiments indicted tht the trnsport rte ws liner for t lest 6 s. Competing mino cids were dded t the sme time s unleled mino cid. All vlues reported re verges of two to four smples. Artificil potentils. Cells were incuted for 4 min with 2 mm 2 deoxyglucose (39 C) to de-energie them. Concentrted de-energied cell suspensions were then treted with 28 Downloded from on Octoer 29, 218 y guest

2 VOL. 171, 1989 AMINO ACID TRANSPORT IN LACTOBACILLUS CASI 281 vlinomycin (2,uM, 6 min, C) nd incuted in 1 mm potssium phosphte ( 6.) to lod them with K. K-loded cells (32,ug of protein) were diluted 1-fold into sodium phosphte ( 6.) which contined 1 mm MgSO4, nd mino cid trnsport ws conducted s descried ove. In some cses, cells were loded with 2 mm potssium phosphte nd extrcellulr KCl ws vried from to 2 mm to chnge the imposed rtificil electricl potentil (At. Memrne vesicles. The method of vesicle preprtion ws descried previously (19). As estimted from the decrese in opticl density, the formtion of osmoticlly sensitive protoplsts ws pproximtely 5%. Memrne vesicles were resuspended in 5 mm potssium phosphte ( 7.) contining 1 mm MgSO4 (finl protein concentrtion, 13 mg/ml) nd froen in liquid nitrogen until use. Memrne vesicles were loded with 2 mm potssium phosphte nd 8 mm potssium cette ( 6.) y vlinomycin tretment (2 p.m, 6 min, C), diluted into 1 mm sodium phosphte ( 6.) to crete n rtificil AiJ nd grdient, nd ssyed for trnsport ctivity s descried ove. Trnsport ssys contined pproximtely 5,ug of protein. Protein. Protein from NOH-hydrolyed cells (.2 N, 1 C, 15 min) ws mesured y the method of Lowry et l. (8) y using ovine serum lumin s stndrd. Proton motive force. Intrcellulr ws mesured y n cid distriution method tht is sed on the ssumption tht undissocited forms of wek cids cn diffuse freely through the cell memrne in response to grdient (16). Cells (15,ug of protein per 6 p,l) were incuted in the presence of [7-14C]enote (.2 RCi), [U-_4C]turine (.2,uCi), or 3H2 (5,uCi). After 1 min of incution t 28 C, the cells were centrifuged through silicon oil (.4 ml, 2:1 mixture of AR2 nd AR2) in microcentrifuge (13, x g, 1 min, 1.5-ml tue) nd superntnt smples (5,ul) were removed for scintilltion counting. The tues were then froen, nd the ottoms contining the pellets were removed with pir of dog nil clippers. Pellets nd superntnts were then dissolved in scintilltion fluid comptile with queous smples. Intrcellulr volume ws clculted s the difference in specific ctivity etween 3H2 nd [U-14C]turine. The intrcellulr volume ws 3.7 pu/mg of protein. The memrne potentil (Ai) ws estimted from the distriution of [3H]tetrphenylphosphonium chloride (TPP+) y using the Nernst eqution. Cells (15 pug of protein per 6 pi) were incuted for 1 min with [3H]TPP+ (.2,uCi) nd processed s descried ove. Mesurements were corrected for nonspecific inding y sutrcting TPP+ ound to toluenetreted cells (6,ul of toluene per 6 pu1). Mterils. Rdioctively leled sustrtes were otined from the Rdiochemicl Centre, Amershm, United Kingdom. Only the L-mino cids were used. Silicon oils (AR2 nd AR2) were otined from Wcker Chemicls, Munich, Federl Repulic of Germny; yest extrct ws from BBL Microiology Systems, Cockeysville, Md. All other chemicls were of regent grde nd were otined from commercil sources. RSULTS Kinetics of trnsport. When sttionry-phse cultures of L. csei were incuted in potssium phosphte ( 6.) nd energied with glucose, uptke of glutmine, sprgine, glutmte, sprtte, leucine, nd phenyllnine ws rpid (Tle 1). All of the K, vlues were less thn 3 pum, nd Vmx vlues rnged from.66 to 6.52 nmol/mg of protein per min. Low rtes (<.2 nmol/mg of protein per min) of trnsport TABL 1. Kinetics of mino cid trnsport for glucose-energied L. csei cells t 6.' Amino cid Vmx K,c Glutmine Asprgine Glutmte Leucine Phenyllnine Cells were energied with 2 mm glucose for 15 min prior to mino cid ddition. Mximum trnsport rte (nnomoles per milligrm of protein per minute). c Affinity constnt (micromolr). were oserved for isoleucine nd vline, nd no uptke of rginine, cysteine, lysine, serine, tryptophn, or tyrosine could e detected. Within 35 min the glucose ws depleted, nd fter nother 5 min more thn 9% of the trnsport ctivity ws lost (dt not shown). Competition nd. Leucine uptke ws completely inhiited y 1-fold excess of unleled vline or isoleucine, nd similr excess of sprtte completely inhiited glutmte uptke. Glutmine uptke ws not inhiited y 1-fold excess of unleled glutmte, sprtte, or sprgine, nd glutmte trnsport ws not inhiited significntly y unleled sprgine. Glutmine, glutmte, nd sprgine uptke ws gretest t 6., nd there ws only modest decrese in trnsport ctivities when the ws c) C c- cn C/) cc c C-IL U CD 4hAS 3. : 2. - () 1. F_ FIG. 1. ffect of externl on the initil rte of mino cid trnsport y glucose-energied cells (mino cid concentrtions were 1 to 3,uM). Downloded from on Octoer 29, 218 y guest

3 282 STROBL T AL. TABL 2. ffect of DCCD, vlinomycin, nd uncouplers on mino cid trnsport y glucose-energied L. csei cells t 6. nd 7.5 % Inhiition of trnsport of : Tretment Glutmte t Glutmine t Asprgine : : t : DCCD CCCP Vlinomycin Nigericin vlinomycin DCCD ws dded t 1,M; CCCP nd the ionophores were ech dded t 5,uM. Leucine nd phenyllnine trnsport ws inhiited more thn 85% under ll conditions. vried from 4. to 7.5 (Fig. 1). Leucine trnsport ws most rpid t 5.5, ut neutrl vlues were inhiitory. The phenyllnine trnsport optimum ws 6.. Driving force for uptke. Leucine nd phenyllnine uptke ws lmost completely eliminted when glucoseenergied cells were treted with dicyclohexylcrodiimide (DCCD), cronyl-cynide m-chlorophenyl-hydrone (CCCP), vlinomycin, or nigericin plus vlinomycin (Tle 2). These decreses were correlted with declines in Ap (Tle 3). In de-energied cells (Fig. 2) nd memrne vesicles (Fig. 2), rtificil memrne potentils were needed for leucine nd phenyllnine trnsport. When the mgnitude of K diffusion potentil (Atf) ws decresed from -12 to -6 mv, there ws drmtic decline in the rte of leucine trnsport (Fig. 3). DCCD, vlinomycin, nd uncouplers lso decresed the trnsport of glutmine, sprgine, nd glutmte t 6., ut little or no decrese ws oserved when the extrcellulr ws incresed to 7.5 (Tle 2) even though Ap ws gretly decresed (Tle 3). Arsente (1 mm), n inhiitor of ATP formtion, inhiited glutmine, sprgine, nd glutmte uptke more thn 6% (dt not shown). Intrcellulr. DCCD, CCCP, nd the ionophores decresed intrcellulr when the extrcellulr ws 6. TABL 3. ffect of DCCD, vlinomycin, nd uncouplers on intrcellulr (i) nd proton motive force (Ap) of L. csei cells t n externl (e) of 6. or 7.5" Tretment i t e of: Ap (mv) t r of: De-energied with deoxyglucose Nonenergied Glucose energied Glucose energied, DCCD Glucose energied, CCCP Glucose energied, vlinomycin Glucose energied, nigericin + vlinomycin DCCD ws dded t 1,uM; CCCP nd the ionophores were ech dded t 5 ptm. -.5 CL : ~ cn cc.5 CL c r_ cc U) r TIM (min) TIM (sec) FIG. 2. () Trnsport of leucine nd phenyllnine y de-energied cells which were loded with 1 mm potssium phosphte nd diluted into 1 mm sodium phosphte ( 6.) to crete n rtificil A*i of -1 mv. () Memrne vesicles were loded with 2 mm potssium phosphte nd 8 mm potssium cette nd diluted into 1 mm sodium phosphte ( 6.) to crete memrne Ap of -2 mv. (Tle 3), nd this decrese ws correlted with reduction in glutmine, sprgine, nd glutmte trnsport (Tle 2). At 7.5, the inhiitors did not cuse decrese in either intrcellulr or trnsport ctivity. Since there ws no grdient in the presence of nigericin plus vlinomycin, it ws possile to exmine the effect of the intrcellulr on the C -* w w -i J. BACTRIOL ARTIFICIAL POTNTIAL (mv) FIG. 3. ffect of imposed rtificil potentil on the initil rte of leucine trnsport y de-energied cells which were loded with potssium phosphte (2 mm) nd diluted into sodium phosphte (1 mm) contining from 2 to mm KCI ( 6.). The inset shows the logrithmic reltionship etween initil velocity (V) nd potentil (mv). Downloded from on Octoer 29, 218 y guest

4 VOL. 171, 1989 AMINO ACID TRANSPORT IN LACTOBACILLUS CASI ~~~~~~~~~~ X od 3.U _ N It~G VAL >.5 >. X > ~ npq FIG. 4. () ffect of intrcellulr on the rte of glutmine trnsport y glucose-energied cells which were treted with 5,uM nigericin nd 5,uM vlinomycin. () Hill plot of the dt, where v is the rte of glutmine trnsport (nnomoles per milligrm of protein per minute) nd npp is the slope. glutmine crrier. When extrcellulr ws decresed to 6.25, the velocity of glutmine trnsport ws reduced y 5% (Fig. 4). A Hill plot of the dt (Fig. 4) indicted tht the glutmine crrier hd pproximtely one (npp =.9) inding site for protons. DISCUSSION L. csei ws le to trnsport severl mino cids t rpid rte, ut some essentil mino cids were tken up very slowly (Tle 1). Since the cells were grown in peptonecontining medium, it is possile tht peptide trnsport systems lso provided essentil mino cids. Asprtte nd glutmte were tken up y the sme crrier, ut glutmine, glutmte, nd sprgine were tken up y seprte systems. Incresing the extrcellulr hd little effect on the rte of glutmte trnsport, nd since the trnsport ssys routinely contined concentrtion of glutmte (1.75,uM) tht ws less thn the K, (2.99,uM), it ws likely tht dissocited nd undissocited species were trnsported. In Streptococcus cremoris nd Streptococcus lctis, glutmine nd glutmte re tken up y the sme crrier, the ffinity for glutmte decreses drmticlly s the increses from 5.1 to 7., nd it ppers tht only the neutrl species is tken up (13). The high-ffinity glutmte trnsport system of Streptococcus feclis is lso unle to trnsport glutmine, nd it is not seriously ffected y (21). A distinct crrier for sprgine is lso found in S. lctis (W. N. Konings, B. Poolmn, nd A. J. M. Driessen, Crit. Rev. Microiol., in press). While L. csei ppers to hve common crrier for rnched-chin mino cids, leucine ws tken up more rpidly thn vline or isoleucine. Since leucine nd phenyllnine trnsport in memrne vesicles ws driven y K nd cette diffusion potentil (Fig. 2), it is likely tht these mino cids were tken up y Ap-driven mechnism. Similr results were otined with S. cremoris (4). Nonenergied cells were unle to trnsport leucine nd phenyllnine t significnt rte ( 6.), even though the Ap ws 8 mv (Tle 3), nd there ws logrithmic reltionship etween trnsport rte nd the mgnitude of n imposed rtificil potentil (Fig. 3). A similr reltionship etween the initil rte of leucine uptke nd Ap ws lso noted in S. cremoris memrne vesicles (3), ut the threshold ws not s low s the one oserved for L. csei. DCCD, CCCP, nd vlinomycin did not completely olish the Ap of glucose-energied cells, ut the Ap ws similr to the one in nonenergied cells (Tle 3). Since cells which were treted with either 2-deoxyglucose or nigericin plus vlinomycin hd virtully no Ap t either, the Ap of nonenergied cells ws not solely due to Donnn potentil. An rtificil Ap ws unle to drive the trnsport of glutmine, glutmte, or sprgine in de-energied cells or memrne vesicles, nd trnsport y glucose-energied cells ws inhiited y rsente (dt not shown). These results suggested tht phosphte-ond energy ws needed for uptke. However, rsente lso decresed the trnsport of leucine nd phenyllnine, mino cids which were tken up y Ap-driven mechnisms. Becuse ATP nd Ap re interrelted, effects of inhiitors cn e miguous. Mechnistic studies of phosphte-ond-driven trnsport hve een confounded y the inility of memrne vesicles to generte ATP (1). Becuse D)CCD (n ATPse inhiitor), vlinomycin, nd uncouplers such s CCCP nd nigericin plus vlinomycin cn cuse decrese in Ap, their effects hve often een tken s evidence for Ap involvement in inding-proteindependent trnsport of grm-negtive cteri. Plte (1) noted tht vlinomycin decresed glutmine trnsport in scherichi coli nd suggested tht oth ATP nd memrne potentil were necessry for uptk'e. DCCD nd uncouplers inhiited glutmine, sprgine, nd glutmte trnsport in L. csei ut only when the externl ws less thn 7.5 (Tle 2). Since nigericin plus vlinomycin eliminted Ap even t 7.5 (Tle 3), it ppered tht Ap ws not essentil for uptke. Recent studies hve demonstrted the importnce of intrcellulr s regultor of certin trnsport systems (11). In S. lctis, the initil rte of glutmte uptke incresed more thn sixfold when the intrcellulr ws incresed from 6. to 6.7 (12, 13). Becuse vlinomycin is uniporter which does not directly fcilitte proton movement, previous workers hd ssumed tht it would not ffect intrcellulr (15). However, in L. csei vlinomycin cused decrese of intrcellulr when extrcellulr ws 6,. The effects of the inhiitors on glutmte, glutmine, nd sprgine trnsport re consistent with the negtive impct of low intrcellulr on phosphte-onddriven trnsport crriers. ACKNOWLDGMNT J. B. Russell ws supported y the U.S. Diry Forge Reserch Center, Mdison, Wis. LITRATUR CITD 1. Ames, G. F.-L Bcteril periplsmic trnsport systems: structure, mechnism, nd evolution. Annu. Rev. Biochem. 55: Downloded from on Octoer 29, 218 y guest

5 284 STROBL T AL. 2. Chssy, B. M Prospects for the mnipultion of lctocilli, FMS Microiol. Rev. 46: Crielrd, W., A. J. M. Driessen, D. Molenr, K. J. Heilingwerf, nd W. N. Konings Light-driven mino cid uptke in Streptococcus cremoris or Clostridium cetoutylicum memrne vesicles fused with liposomes contining cteril rection centers, J. Bcteriol. 17: Driessen, A. J. M., S. de Jong, nd W. N. Konings Trnsport of rnched-chin mino cids in miemrne vesicles of Streptococcus cremoris. J. J3cteriol. 169: Ferqndes, C. F., K. M. Shhni, nd M. A. Amer Therpeutic role of dietry lctocilli nd lctocilli fermented diry products. FMS Microiol. Rev. 46: Henderson, G. B.,. M. Zevely, nd F. M. Huennekens Coupling of energy to folte trnsport in Lctocillus csei. J. Bcteriol. 139: Kndler, O., nd N. Weiss Genus Lctocillus Beijerinck 191, 212AL, p In P. H. A. Sneth, N. S. Mir, M.. Shrpe, nd J. G. Holt (ed.), Bergey's mnul of systemtic cteriology, vol. 2. The Willims & Wilkins Co., Bltimore. 8. Lowry,. H., N. J. Roserough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: Morishit, T., Y. Deguchi, M. Yjim, T. Skuri, nd T. Yur Multiple nutritionl requirements of lctocilli: genetic lesions ffecting mino cid iosynthetic pthwys. J. Bcteriol. 148: Plte, C. A Requirement for memrne potentil in ctive trnsport of glutmine y scherichi coli. J. Bcteriol. 137: Poolmn, B., A. J. M. Driessen, nd W. N. Konings Regultion of solute trnsport in streptococci y externl nd internl vlues. Microiol. Rev. 51: Poolmn, B., K. J. Helingwerf, nd W. N. Konings J. BACTRIOL. Regultion of the glutmte-glutmine trnsport system y intrcellulr in Streptococcus lctis. J. Bcteriol. 169: Poolmn, B.,. J. Smid, nd W. N. Konings Kinetic properties of phosphte-ond-driven glutmte-glutmine trnsport system in Streptococcus lctis nd Streptococcus cremoris. J. Bcteriol. 169: Rgos, M., J. G. Frnklin, nd K. D. Perry Correltion of the vitmin requirements with culturl nd iochemicl chrcteristics of Lctocillus sp. J. Gen. Microiol. 25: Rmos, S., nd H. R. Kck The electrochemicl proton grdient in scherichi coli memrne vesicles. Biochemistry 16: Reieling, V., R. K. Thuer, nd K. Jungermnn The internl-lkline grdient, sensitive to uncoupler nd ATPse inhiitor, in growing Clostridium psteurinum. ur. J. Biochem. 55: Rhee, S. K., nd M. Y. Pck ffect of environmentl on fermenttion lnce of Lctocillus ulgricus, J. Bcteriol. 144: Romno, A. H., J. D. Trifone, nd M. Brutilone Distriution of the phosphoenolpyruvte:glucose phosphotrnsferse system in fermenttive cteri. J. Bcteriol. 139: Russell, J. B., H. J. Stroel, A. J. M. Driessen, nd W. N. Konings Sodium-dependent trnsport of neutrl mino cids y whole cells nd memrne vesicles of Streptococcus ovis, ruminl cterium. J. Bcteriol. 17: Shrpe, M The genus Lctocillus, p In M. P. Strr, H. Stolp, H. Truper, A. Blous, nd H. G. Schlegel (ed.), The prokryotes, vol. 2. Springer-Verlg KG, Berlin. 21. Utech, N. M., K. G. Reid, nd J. T. Holden Properties of dicroxylic mino cid trnsport-deficient mutnt of Streptococcus feclis. J. Biol. Chem. 245: Downloded from on Octoer 29, 218 y guest