Relationship between Utilization of Proline and Proline-Containing Peptides and Growth of Lactococcus lactis

Transcription

1 JOURNAL OF BACTERIOLOGY, Sept. 199, p /9/ $2./ Copyright 199, Americn Society for Microbiology Vol. 172, No. 9 Reltionship between Utiliztion of Proline nd Proline-Contining Peptides nd Growth of Lctococcus lctis EDDY J. SMID AND WIL N. KONINGS* Deprtment of Microbiology, University of Groningen, Kerkln 3, 9751 NN Hren, The Netherlnds Received 15 My 199/Accepted 3 July 199 Proline, which is the most bundnt residue in I-csein, stimultes growth of Lctococcus lctis in proline-requiring strin (Lctococcus lctis subsp. cremoris Wg2) nd in proline-prototrophic strin (Lctococcus lctis subsp. Ictis ML3). Both strins lck proline-specific uptke system, nd free proline cn enter the cell only by pssive diffusion cross the cytoplsmic membrne. On the other hnd, lctococci cn ctively tke up proline-contining peptides vi the lctococcl di- nd tripeptide trnsport system, nd these peptides re the mjor source of proline. Consequently, lctococcl growth on mino cid-bsed medi is highly stimulted by the ddition of proline-contining di- nd tripeptides. Growth of L. lctis subsp. ls M3 on chemiclly defined medi supplemented with csein does not pper proline limited. Addition of dipeptides (including proline-contining peptides) severely inhibits growth on csein-contining medium, which indictes tht the specific growth rte is determined by the blnced supply of different di- or tripeptides which compete for the sme di- nd tripeptide trnsport system. Lctococci (previously nmed group N lctic streptococci [13]) hve high nutritionl demnds, nd exogenous sources of nucleotides, vitmins, nd mino cids (12) re essentil for growth. The ctul number of mino cids required for growth is strin dependent. Generlly, strins belonging to Lctococcus lctis subsp. cremoris re more fstidious thn those of Lctococcus lctis subsp. lctis. A consequence of the multiple mino cid uxotrophy is tht growth of these bcteri depends on the uptke of the different essentil mino cids. Studies in our lbortory hve shown tht lctococci possess severl different mino cid trnsport systems (for review, see reference 7). Lctococci cn lso utilize the milk protein csein to stisfy the requirement for essentil mino cids. The combined ction of cell wll-ssocited cseinolytic proteinse nd different extrcellulrly locted peptidses results in the relese of free mino cids nd peptides from csein. In recent study, it hs been shown tht growth of Lctococcus lctis on csein cnnot sufficiently be supported by free mino cids relesed from csein but tht growth lso depends on the uptke of di- nd/or tripeptides (15). These peptides cn be tken up vi proton motive force-dependent di- nd tripeptide (di-tripeptide) trnsport system (14). Recently we hve shown tht proline, which is the most bundnt residue in,b-csein, is not ctively tken up by L. lctis subsp. lctis ML3 cells (15), nd we suggested tht 1-csein-derived proline most likely enters the cell in peptide-bound configurtion. In this study, we investigted the uptke of proline in strin of L. lctis which requires exogenous proline for growth (L. lctis subsp. cremoris Wg2) nd in strin which is prototrophic for proline (L. lctis subsp. lctis ML3). Furthermore, we compred the growth chrcteristics of both strins on mino cid-, peptide-, nd csein-contining medi. The results demonstrte tht growth of different strins of L. lctis on mino cidbsed medi is limited by the proline uptke cpcity of the cells. Proline-contining peptides pper superior to the free imino cid s source of proline. The results will be * Corresponding uthor. discussed in the context of the utiliztion of,-csein-derived proline. MATERIALS AND METHODS Culture conditions nd growth medi. Cultures of L. lctis subsp. lctis ML3 nd L. lctis subsp. cremoris Wg2, both proteolytic-positive vrints, were mintined in 1% (wt/ vol) reconstituted skim milk contining.1% (wt/vol) tryptone (Difco Lbortories, Detroit, Mich.) nd stored t -8 C. For growth experiments, the cultures were trnsferred from milk cultures to complex broth medium (2) t ph of 6.4 contining.5% (wt/vol) lctose s crbon nd energy source nd incubted overnight t 28 C. The overnight cultures were subsequently trnsferred to chemiclly defined medi contining.5% (wt/vol) lctose s crbon nd energy source. These cultures were used to inoculte the experimentl cultures. The composition of the chemiclly defined medium (CDM) ws s previously described (1). In ll cses, glutmine nd sprgine insted of glutmte nd sprtte were used. Unless stted otherwise in the legends to figures, the mino cids were dded to the medium t the following concentrtions (mm): glutmine, 3.5; sprgine, 2.6; leucine, 3.6; isoleucine, 1.6; vline, 2.8; methionine,.8; histidine,.7; glycine, 2.3; lnine, 2.7; serine, 3.2; threonine, 1.9; lysine, 2.4; rginine,.7; cysteine, 1.4; tyrosine, 1.6; phenyllnine, 1.7; nd proline, 5.9. All other relevnt ltertions in the medium composition re indicted in the legends to figures. The ph of the medium ws djusted to 6.5 with 1 N HCl. Where indicted in the legends to figures, CDM ws supplemented with.25% (wt/vol) csein (Sigm Chemicl Co., St. Louis, Mo.). To void precipittion of the milk protein in the exponentil growth phse, the medium ws buffered with 135 mm potssium phosphte. Growth experiments. When the proline concentrtion ws vried in the growth medium, overnight cultures were wshed three times in CDM which did not contin lctose nd proline. When the concentrtions of ll mino cids were vried, the cultures were wshed in CDM which ws not supplemented with lctose nd mino cids. The experimentl growth medi were subsequently inoculted with the Downloded from on My 1, 218 by guest 5286

2 VOL. 172, 199 RELATIONSHIP BETWEEN PROLINE AND L. LACTIS GROWTH 5287 wshed cell cultures to n opticl density t 66 nm of.2. Cells were cultured in screw-cp glss tubes which fit into Vittron UC2 spectrophotometer (Vittron Scientific Instruments, Dieren, The Netherlnds). The tubes lwys contined 1 ml of growth medium, nd growth ws monitored up to n opticl density t 66 nm of.4. All growth experiments were performed t 28 C. Trnsport ssys. Trnsport of rdioctively lbeled compounds in intct cells ws done s previously described (14). When [14C]proline trnsport ws mesured, the dense cell suspensions were filtered on glss microfiber filter (GF/F; Whtmn Interntionl Ltd., Midstone, United Kingdom). Trnsport of unlbeled peptides ws monitored by determining the intrcellulrly ccumulted mino cids. This ws done by seprting the cells from the buffer by silicon oil centrifugtion (18). Extrction of the cells ws performed s previously described (11). Amino cids nd peptides were nlyzed fter derivtiztion with dnsyl chloride by using methods essentilly described by Tpuhi et l. (17) nd Wiedmeier et l. (21). The dnsylted, neutrlized cell extrcts were seprted by reversed-phse high-performnce liquid chromtogrphy, s described previously (11). All trnsport ssys were performed t 3 C. Clcultions. The kinetic dt on leucyl-proline uptke were fitted with the Michelis-Menten eqution for sturtion kinetics. Chemicls. L-[U-_4C]proline (291 mci/mmol) ws obtined from Rdiochemicl Centre, Amershm, United Kingdom. Alnyl-[14C]glutmte ws synthesized s previously described (14). Pro-His-Vl, Pro-Vl-Asp, Pro-Pro-Gly, His- Pro-Vl, nd Ile-Pro-Ile were generous gift from R. Plpp, University of Kiserslutern, Kiserslutern, Federl Republic of Germny. All other peptides were obtined from Sigm Chemicl Co. nd from Bchem Feinchemiklien AG, Bubendorf, Switzerlnd. Peptides nd mino cids were in the L configurtion. All other chemicls were regent grde nd were obtined from commercil sources. RESULTS Effect of proline on growth of L. lctis. Both L. lctis subsp. lctis ML3 nd L. lctis subsp. cremoris Wg2 were grown on chemiclly defined medi supplemented with different concentrtions of proline. L. lctis subsp. lctis ML3 grows well in the bsence of proline in the medium (specific growth rte,.4/h), wheres L. lctis subsp. cremoris Wg2 does not (Fig. 1). However, growth of both strins is stimulted by incresing the concentrtion of proline. Hlf-mximl stimultion of growth of L. lctis subsp. lctis ML3 nd L. lctis subsp. cremoris Wg2 is obtined t.6 nd.9 mm proline, respectively. Optiml growth of both strins is reched t 5 mm proline, nd n increse of the proline concentrtion to 5 mm hd no further stimulting effect on the growth rtes (dt not shown). No differences in growth rte were observed between L. lctis subsp. lctis ML3 cells tht were precultured in the presence or bsence of proline (dt not shown), which suggests tht the synthesis of proline is not strongly ffected by the presence of proline in the growth medium. The proline concentrtion in the growth medium did not influence significntly the finl opticl density of the cultures. The results show tht proline is growth stimulting, both in proline-uxotrophic nd in proline-prototrophic strins of L. lctis. Proline uptke in different strins of L. lctis. Proline ws found to be n essentil mino cid for L. lctis subsp. cremoris Wg2 but not for L. lctis subsp. lctis ML3. To proline concentrtion (mm) FIG. 1. Reltion between specific growth rtes nd proline concentrtions of L. lctis subsp. cremoris Wg2 () nd L. lctis subsp. lctis ML3 (). The cells were grown in CDM supplemented with n mino cid mixture in which ll mino cids were present t fixed concentrtion (see Mterils nd Methods), except for proline, which vried between nd 5 mm. All growth rtes were determined in duplicte. investigte whether this difference correltes with different trnsport properties of the strins, we studied the uptke of proline in cells of both strins. From previous study, we know tht cells of L. lctis subsp. lctis ML3 (prototrophic for proline) do not significntly tke up proline (15). The proline uptke ws mesured in cells cultured in broth medium nd in CDM with nd without proline (L. lctis subsp. lctis ML3) nd in cells grown on broth medium nd CDM with complete mino cid mixture (L. lctis subsp. cremoris Wg2). Furthermore, the uptke ws ssyed in different buffers such s 2-(N-morpholino)ethnesulfonic cid contining potssium nd/or sodium, phosphte buffers contining sodium nd/or potssium, nd CDM without unlbeled proline. All buffers contined.4% lctose-5 mm MgSO4 nd were djusted to ph of 6.5. None of the culture conditions or ssy buffers supported rpid proline ccumultion in L. lctis subsp. cremoris Wg2 or L. lctis subsp. lctis ML3 (dt not shown). Also, being cultured in broth medium supplemented with.5 M NCl nd ssyed in high-slt buffer (.5 M NCl) did not stimulte proline uptke (Molenr nd Hgting, unpublished dt). Under ll conditions, however, low but significnt proline influx could be observed. The concentrtion-dependence of the proline influx rte ws mesured in proline-depleted cells of L. lctis subsp. lctis ML3 (Fig. 2). The proline influx rte ws found to increse linerly with the proline concentrtion. In the concentrtion rnge tested, no sturtion of the influx rte ws observed, indicting tht the process is not enzyme ctlyzed. Also, in both strins, no exchnge or counterflow ctivity (dt not shown) ws observed, indicting tht proline trnsloction is not crrier medited. At 5 mm proline, the influx rte ws 6.4 nmol/min per mg Downloded from on My 1, 218 by guest

3 5288 SMID AND KONINGS J. BACTERIOL. *c CL E C.SI SE co x much lesser extent (Fig. 3A). A similr result ws obtined in growth experiment with L. lctis subsp. lctis ML3 (Fig. 3B). However, since L. lctis subsp. lctis ML3 is pro- * totrophic for proline, the effect ws less pronounced. This result shows tht for rpid growth, proline ws the only mino cid tht needs to be present t millimolr concentrtions, nd the result is in ccordnce with the conclusion tht L. lctis does not possess proline-specific uptke system. Di- nd tripeptides s proline source. The results presented thus fr show tht free proline is poor proline source. Therefore, we compred growth of L. lctis subsp. lctis ML3 nd L. lctis subsp. cremoris Wg2 in medi with free proline nd with proline-contining peptides s proline source. CDM supplemented with 5 ixm leucyl-proline s the sole proline source resulted in mximl growth rtes of both strins tested (Tble 1), wheres the sme concentrtion of the free imino cid hrdly stimulted growth. An increse of the Leu-Pro concentrtion to 1 mm did not further stimulte *+ growth. Comprble results were obtined when 5,uM of the dipeptides Pro-Met nd Met-Pro ws dded s proline source. Growth of L. lctis subsp. lctis ML3 ws lso stimulted 8 1 by the tripeptides Pro-His-Vl, Vl-Pro-Leu, nd 24681Ile-Pro-Ile. All other tripeptides tested did not stimulte proline concentrtion (mm) growth of L. lctis subsp. lctis ML3. Furthermore, growth of L. lctis subsp. cremoris Wg2 ws significntly stimulted inetics of proline influx in intct cells of L. lctis subsp. by Pro-His-Vl. All the other tripeptides tested could not FIG. 2. K lctis ML3 (P proline source nd subsequently wshed three times in 1 mm potssium 2-1(N-morpholino)ethnesulfonic cid buffer (ph 6.5) contining 5 mm[ MgSO4 nd 2 jxg of chlormphenicol per ml. For the tides were better proline sources thn the free imino cid. ['4C]proline influx mesurements, cells were suspended in the potssium 2'-(N-morpholino)ethnesulfonic cid buffer to finl Intrcellulr proline pools in cells growineg concentrtioi determined ftrom the uptke level in cells 2 min fter the ddition of ['4C]proline. mesuremenits.?h 6.5). Cells were cultivted in CDM in the bsence of replce the proline-contining dipeptides s proline source. The results show tht lctococci preferentilly utilized proline s dipeptide nd tht some proline-contining tripep- on different n of 1.8 mg of cell protein per ml. The influx rtes were proline sources. The observtion tht proline-contining dipeptides nd, to lesser extent, tripeptides re superior to All dtum points represent men vlues of triplicte free proline s proline source is lso reflected in the intrcellulr proline pool sizes of L. lctis subsp. lctis ML3 nd L. lctis subsp. cremoris Wg2 (Tble 2). When L. lctis of protein. This vlue is close to the clculted miniml subsp. lctis ML3 is cultivted in CDM without proline, proline trlnsport rte of 4.5 nmol/mg of protein, which is smller intrcellulr proline pool (.36 mm) is observed. necessry tto meet the growth requirements of L. lctis t Under these growth conditions, 14,uM extrcellulr proline mximum sspecific growth rte of.65/h. This rte hs been ws detected, which indicted significnt loss of the clculted 4on the bsis of the mino cid composition of intrcellulrly synthesized proline. In the presence of 1 mm totl cell h: ydrolyste of L. lctis subsp. lctis ML3 (1). proline (.96 mm detected), n intrcellulr pool of.52 mm These re! sults show tht under different growth conditions, ws observed. Tht mens tht under these conditions L. lctis dloes not tke up proline vi proline-specific, downhill grdient (low inside concentrtion versus high high-ffinit3y uptke system. Insted, free proline enters the outside concentrtion) of proline exists cross the cytopls- by pssive diffusion cross the cytoplsmic mic membrne. This ltter observtion ws lso mde in L. cells most Ilikely membrne. lctis subsp. cremoris Wg2 growing in CDM supplemented Proline-limited growth in mino-cid-contining medi. with 1 mm proline. When L. lctis subsp. lctis ML3 nd L. The chemiclly defined medi for the cultivtion of lcto- lctis subsp. cremoris Wg2 were cultivted on CDM supple- contin high (millimolr) concentrtions mented with 1 mm leucyl-proline, lrger internl proline cocci (1) routinely of ll minlo cids, including proline. The growth studies pools of 29.8 nd 2.5 mm, respectively, were detected. presented iln Fig. 1 justify the high proline concentrtions in Under these conditions n externl proline concentrtion of these medi;. Since the lctococcus uptke systems of most.25 mm ws present, indicting the presence of n uphill mino cid! s operte with ffinity constnts in the micromolr proline grdient. This result suggests tht leucyl-proline is rnge (7), iit is questionble whether millimolr concentr- ctively tken up by both Lctococcus strins. tions of thte other mino cids re necessry for optiml Trnsport of proline-contining peptides. To investigte growth. Tc test this, we mesured the growth rtes of L. whether proline-contining dipeptides re trnsported into lctis subs;. cremoris Wg2 nd L. lctis subsp. lctis ML3 in the cell vi highly specific peptide trnsport system or vi CDM whici h ws supplemented with different concentrtions the recently described lctococcl di-tripeptide trnsport of the com; )lete mino cid mixture. As control, the proline system (14), the effects of proline-contining peptides on the concentrtiion ws kept t 5 mm. The specific growth rte of uptke of rdioctively lbeled Al-Glu ws studied (Fig. 4). L. lctis suibsp. cremoris Wg2 decresed drmticlly with Uptke of Al-Glu vi the di-tripeptide trnsport system by decresing concentrtions of ll mino cids (Fig. 3A). glycolyzing L. lctis subsp. lctis ML3 cells ws inhibited by However,' when the concentrtion of proline ws kept t 5 excess unlbeled Al-Glu nd Pro-Met. Addition of proline mm, dilution of the mixture ffected the growth rte to did not inhibit Al-Glu uptke. This result shows tht Downloded from on My 1, 218 by guest

4 VOL. 172, 199 RELATIONSHIP BETWEEN PROLINE AND L. LACTIS GROWTH v- 4D.6 cw o I._.4 n L. : dilution fctor dilution fctor FIG. 3. Specific growth rtes of L. lctis subsp. cremoris Wg2 (A) nd L. lctis subsp. lctis ML3 (B) in CDM supplemented with different dilutions of the mino cid mixture. In one series, the complete mixture (including proline) ws diluted (). In the second series, the complete mixture with the exception of proline ws diluted (). In the second series, the proline concentrtion ws kept t 5 mm. The concentrtions of mino cids in the undiluted mino cid mixture (represented s dilution fctor 1) re given in Mterils nd Methods. All growth rtes were determined in duplicte. Pro-Met is trnsported vi the lctococcl di-tripeptide trnsport system (14). Kinetic prmeters of Leu-Pro uptke in proline-depleted, glycolyzing cells of L. lctis subsp. lctis ML3 hve been determined. The initil rtes of Leu-Pro uptke studied over wide concentrtion rnge (5 to 6 F.M) showed singlephsic Michelis-Menten-type sturtion kinetics (Fig. 5), which indicted the presence of one kineticlly distinguishble uptke system for Leu-Pro. At ph of 6.5, the TABLE 1. Specific growth rtes of L. lctis strins in CDM supplemented with n mino cid mixture Proline source (mm) Mximum specific growth rte (per h) of strin: Wg2 ML3 No proline NDb.4 Proline (.5).1.42 Proline (1) Leucyl-proline (.5) Leucyl-proline (1).64.9 Prolyl-methionine (.5) Methionyl-proline (.5) Pro-His-Vl (.5) Pro-Vl-Asp (.5).1.39 Pro-Pro-Gly (.5) <.1.39 Vl-Pro-Leu (.5).4.73 His-Pro-Vl (.5).4.47 Gly-Pro-Al (.5).1.44 Ile-Pro-Ile (.5).1.79 For concentrtions of mino cids, see Mterils nd Methods. Proline or proline-contining peptides were dded t the concentrtions indicted. All mesurements were performed in duplicte. b ND, Not determined. Michelis-Menten constnts (K,) for Leu-Pro uptke ws determined to be 59 FiM. A Vmx vlue of 113 nmol/min per mg of protein ws found. These kinetic prmeters cn explin the observed growth rtes of L. lctis subsp. lctis ML3 (Tble 1). Effects of dipeptides on the growth of lctococci in cseincontining medi. The mximl specific growth rte of lctococci in medi contining csein s the sole mino cid source is usully lower thn in medi contining free mino cids (3, 4, 15). One explntion for this observtion is tht uptke of di- or tripeptides contining essentil or growthstimulting mino cids limits growth. The significnt differences in nutritionl vlue between proline nd prolinecontining peptides (Tble 1) led us to investigte the effects of proline-contining peptides nd proline on cultures growing on csein s the sole orgnic nitrogen source. The ddition of 5 ViM Leu-Pro or 5 F.M Pro-Met inhibited rther thn stimulted growth of L. lctis subsp. lctis ML3 TABLE 2. Proline source (mm) Proline pools in L. lctis strins cultured in CDM' with proline dded Externl Proline (mm) Intrcellulr in strin: Wg2 ML3 No ddition.14 NDb.36 Proline (1.) Leucyl-proline (1.) ' CDM composition nd nlyticl procedures for determintion of proline pools re described in Mterils nd Methods. Pools were determined in exponentilly growing cultures t opticl densities t 66 nm between.55 nd.73. b ND, Not determined. Downloded from on My 1, 218 by guest

5 529 SMID AND KONINGS J. BACTERIOL '2 c..15._ c 1 8._ E CL Id U C Time (min.) FIG. 4. Effects of excess unlbeled proline, prolyl-methionine, nd lnyl-glutmte on the uptke of lnyl-[14c]glutmte in glycolyzing cells of L. lctis subsp. lctis ML3. The trnsport ssy ws strted by the ddition of 1.75,uM lnyl-['4c]glutmte lone () or in the presence of 2 mm proline (A), 2 mm prolyl-methionine (A), or 2 mm lnyl-glutmte (). by 45 nd 4%o, respectively (Tble 3). A higher peptide concentrtion (1 mm) inhibited growth even further (55 nd 63%, respectively). Other dipeptides, such s Leu-Leu, inhibited growth of L. lctis subsp. lctis ML3 to pproximtely the sme extent (Tble 3). On the other hnd, ddition of proline t.5 nd 5 mm did not significntly influence the growth rte of L. lctis subsp. lctis ML3. Growth inhibition by 5,uM dipeptide ws observed only in the first 4 h of exponentil phse growth. Subsequently, the growth-inhibitory effect of the dipeptides decresed, probbly becuse the concentrtion of the peptides decresed below n effective level. This biphsic growth pttern ws not observed when the dipeptides were present t 1 mm (dt not shown). The results indicte tht growth of L. lctis subsp. lctis ML3 on csein-contining medium is not limited simply by proline. Since significnt growth inhibition is observed t low concentrtions of dipeptide, the supply of growth-stimulting di- nd tripeptides from csein hydrolysis by the cseinolytic proteinse nd one or more extrceliulr peptidses ppers very well blnced. DISCUSSION This report shows tht for lctococci, proline-contining peptides were nutritionlly superior to proline. The explntion ppers to be tht these bcteri do not possess proline-$pecific uptke system, wheres they do hve n efficient di-tripeptide uptke system vi which proline-contining di- nd tripeptides cn rpidly be tken up into the cell. Interestingly, the bsence of proline-specific trnsport system ws observed in both proline uxotroph (L. lctis subsp. cremoris Wg2) nd proline prototroph (L. lctis subsp. lctis ML3).._ CL 2co 4d C LeuPro concentrtion (mm) FIG. 5. Kinetics of leucyl-proline uptke in glycolyzing L. lctis subsp. lctis ML3 cells. The uptke ssy ws performed in 1 mm potssium 2-(N-morpholino)ethnesulfonic cid (ph 6.5) contining 5 mm MgSO4 nd 2,ug of chlormphenicol per ml. The cells which were cultured s described in the legend to Fig. 2 were, suspended in the buffer to finl concentrtion of.54 mg of protein per ml. The ssy ws strted 2 min fter the ddition of.4% (wt/vol) lctose by the ddition of Leu-Pro. After 3 s, 1 ml of the rection ws quenched by centrifugtion of 1 ml of the cell suspension through lyer of silicon oil, s described in Mterils nd Methods. The mount of ccumulted proline ws determined in the cell extrcts by dnsyltion followed by reversed-phse high-performnce liquid chromtogrphy. Despite the bsence of proline-specific uptke system, proline uxotrophs such s L. lctis subsp. cremoris Wg2 re ble to grow on chemiclly defined medi contining free proline s the sole proline source. Growth in these medi cn only be sustined by high concentrtion (5 mm) of proline (Fig. 1). Proline influx does not show sturtion kinetics (Fig. 2); therefore, the process most likely occurs vi pssive diffusion cross the cytoplsmic membrne. Since the slow influx rte of proline is not reduced when the uptke ssy is done in CDM ( medium contining high concentrtions of TABLE 3. Specific growth rtes of L. lctis subsp. lctis ML3 grown in CDM' supplemented with vrious dipeptides Mximum Substrte dded (nm) None... Proline (.5)... Proline (5)... Leucyl-proline (.5)... Leucyl-proline (1)... Prolyl-methionine (.5)... Prolyl-methionine (1)... Leucyl-leucine (.5)... Leucyl-leucine (1)... specific growth rte (per h) CDM ws supplemented with.25% (wt/vol) csein s the orgnic nitrogen source. Substrtes were dded t concentrtions indicted. All mesurements were performed in duplicte. Downloded from on My 1, 218 by guest

6 VOL. 172, 199 RELATIONSHIP BETWEEN PROLINE AND L. LACTIS GROWTH 5291 mino cids nd vitmins), it is unlikely tht proline uptke is medited by one of the existing mino cid or vitmin trnsport systems. Becuse of the limited proline uptke cpcity, growth of L. lctis in mino-cid-bsed medi is clerly proline limited. For optiml growth, proline hs to be present t millimolr concentrtions, wheres ll other mino cids need only be present t micromolr concentrtions (Fig. 3). This is in greement with the observtion tht the ffinity constnts for trnsport of ll other mino cids re found to be in the micromolr rnge (7). These results cn be used to improve selective CDM for culturing of lctococci. Proline-contining di- or tripeptides significntly stimulte growth of L. lctis on mino-cid-bsed medi compred with medi in which proline is present t comprble concentrtion (Tble 1). Our explntion for this observtion is bsed on the difference between the trnsport mechnism of proline nd tht of proline-contining peptides. Proline trnsport occurs vi pssive diffusion cross the cytoplsmic membrne nd is reltively slow process, while trnsport of proline-contining peptides is medited by the previously described proton motive force-dependent di-tripeptide trnsport system (14). The Michelis-Menten constnts (K,) for Leu-Pro (Fig. 5) nd Al-Glu uptke (14) re 59 nd 69,iM, respectively. Furthermore, the Vmx vlues for Leu-Pro nd Al-Glu uptke in L. lctis subsp. lctis ML3 were found to be 113 nd 36 nmol/min per mg of protein, respectively. Although proline-contining peptides re nutritionlly superior to proline (Tble 1), these peptides do not stimulte growth of L. lctis subsp. lctis ML3 in CDM supplemented with csein. On the contrry, growth of L. lctis subsp. lctis ML3 in csein-contining medi is significntly inhibited by proline-contining peptides s well s by other dipeptides (Tble 3). In previous pper (15), we hve lredy shown tht uptke of peptides is essentil for growth of L. lctis on csein-contining medi. Thus, csein hydrolysis by lctococci results in the production of peptides contining essentil or growth-stimulting mino cids. Growth inhibition by 5,M dipeptide cn be explined by competition for uptke of csein-derived peptides vi the di-tripeptide trnsport system. Proline influx vi pssive diffusion cross the cytoplsmic membrne did not interfere with the uptke of peptides. Thus, high proline concentrtions (5 mm) did not influence significntly the specific growth rte of L. lctis subsp. lctis ML3 on CDM supplemented with csein. This observtion furthermore indictes tht growth on csein is, in contrst to growth on free mino cids, not proline limited. On the bsis tht proline utiliztion occurs essentilly vi uptke of proline-contining peptides, the overll scheme of the mechnism of 3-csein degrdtion by lctococci cn be drwn. The first degrdtion step is the hydrolysis of,-csein by the cell wll bound proteinse nd results in the formtion of 14 to 18 oligopeptides of vrious lengths (8, 9, 2). These oligopeptides cn serve s substrtes for extrcellulrly locted peptidses such s the recently described minopeptidse (16) nd n X-prolyl-dipeptidyl minopeptidse (6). The minopeptidse rpidly hydrolyzes oligopeptides nd, t lower rte, dipeptides. It does not hydrolyze peptides with proline residue t the N-terminl position. Consequently, Pro-X nd Pro-X-X peptides, which re excellent substrtes for the lctococcus di-tripeptide trnsport system, re produced by the ction of the minopeptidse. Furthermore, the X-prolyl-dipeptidyl minopeptidse (6) lso cts on oligopeptides which hve n X-Pro sequence t the N terminus. Action of this peptidse results in the formtion of X-Pro dipeptides, thereby gin supplying the di-tripeptide trnsport system with suitble substrtes for uptke. Free proline will not be produced by the ction of these peptidses. According to this scheme of,-csein utiliztion, peptidses which hydrolyze proline-contining di- nd tripeptides re locted intrcellulrly. The recently described tripeptidse from L. lctis subsp. cremoris Wg2 is n exmple of n enzyme tht cn hydrolyze Pro-X-X tripeptides. The intrcellulr dipeptidse described by vn Boven et l. (19) hydrolyzes Pro-Leu. Kminogw et l. (5) isolted prolidse, n enzyme tht specificlly hydrolyzes X-Pro peptides, from L. lctis subsp. cremoris H61. The results presented on the utiliztion of proline nd proline-contining peptides emphsize the importnt role of the di-tripeptide trnsport system in the overll pthwy of csein utiliztion. Mnipultion of the process of 1-csein degrdtion requires not only knowledge bout the extrcellulr proteolytic nd peptidolytic ctivity but lso detiled informtion bout ll mino cid nd peptide trnsport systems in L. lctis. ACKNOWLEDGMENTS We thnk T. Abee, D. Molenr, nd B. Poolmn for vluble suggestions throughout this work. The investigtions were supported by the Foundtion for Fundmentl Biologicl Reserch, which is subsidized by The Netherlnds Orgniztion for Scientific Reserch. LITERATURE CITED 1. Bosmn, B. W., P. S. T. Tn, nd W. N. Konings Purifiction nd chrcteriztion of tripeptidse from Lctococcus lctis subsp. cremoris Wg2. Appl. Environ. Microbiol. 56: De Mn, J. C., M. Rogos, nd M. E. Shrpe A medium for the cultivtion of lctobcilli. J. Appl. Bcteriol. 23: Hugenholtz, J., M. Dijkstr, nd H. Veldkmp Amino cid limited growth of strter cultures in milk. FEMS Microbiol. Ecol. 45: Hugenholtz, J., nd H. Veldkmp Competition between different strins of Streptococcus cremoris. FEMS Microbiol. Ecol. 31: Kminogw, S., N. Azum, I.-K. Hwng, Y. Suzuki, nd K. Ymuchi Isoltion nd chrcteriztion of prolidse from Streptococcus cremoris H61. Agric. Biol. Chem. 48: Kiefer-Prtsch, B., W. Bockelmnn, A. Geis, nd M. Teuber Purifiction of n X-prolyl-dipeptidyl minopeptidse from the cell wll proteolytic system of Lctococcus lctis subsp. cremoris. Appl. Microbiol. Biotechnol. 31: Konings, W. N., B. Poolmn, nd A. J. M. Driessen Bioenergetics nd solute trnsport in lctococci. Crit. Rev. Microbiol. 16: Monnet, V., W. Bockelmnn, J. C. Gripon, nd M. Teuber Comprison of cell wll proteinses from Lctococcus lctis subsp. cremoris AC1 nd Lctococcus lctis subsp. lctis NCDO 763. II. Specificity towrds bovine,-csein. Appl. Microbiol. Biotechnol. 31: Monnet, V., D. Le Brs, nd J.-C. Gripon Specificity of cell wll proteinse from Streptococcus lctis NCDO763 towrds bovine,-csein. FEMS Microbiol. Lett. 36: Poolmn, B., nd W. N. Konings Reltion of growth of Streptococcus lctis nd Streptococcus cremoris to mino cid trnsport. J. Bcteriol. 17: Poolmn, B., E. J. Smid, H. Veldkmp, nd W. N. Konings Bioenergetic consequences of lctose strvtion for continuously cultured Streptococcus cremoris. J. Bcteriol. 169: Reiter, B., nd J. D. Orm Nutritionl studies on cheese strters. 1. Vitmin nd mino cid requirements of single strin strters. J. Diry Res. 29: Downloded from on My 1, 218 by guest

7 5292 SMID AND KONINGS J. BACTERIOL. 13. Schieifer, K. H., nd R. Kilpper-Bilz Moleculr nd chemotxonomic pproches to the clssifiction of Streptococci, Enterococci nd Lctococci: review. Syst. Appl. Microbiol. 1: Smid, E. J., A. J. M. Driessen, nd W. N. Konings Mechnism nd energetics of dipeptide trnsport in membrne vesicles of Lctococcus lctis. J. Bcteriol. 171: Smid, E. J., R. Plpp, nd W. N. Konings Peptide uptke is essentil for growth of Lctococcus lctis on the milk protein csein. J. Bcteriol. 171: Tn, P. S. T., nd W. N. Konings Purifiction nd chrcteriztion of n minopeptidse from Lctococcus lctis subsp. cremoris Wg2. Appl. Environ. Microbiol. 56: Tpuhi, Y., D. E. Schmidt, W. Lindner, nd B. L. Krger Dnsyltion of mino cids for high-performnce liquid chromtogrphy nlysis. Anl. Biochem. 115: Ten Brink, B., R. Otto, U.-P. Hnsen, nd W. N. Konings Energy recycling by lctte efflux in growing nd nongrowing cells of Streptococcus cremoris. J. Bcteriol. 162: vn Boven, A., P. S. T. Tn, nd W. N. Konings Purifiction nd chrcteriztion of dipeptidse from Streptococcus cremoris Wg2. Appl. Environ. Microbiol. 54: Visser, S., C. J. Slngen, F. A. Exterkte, nd G. J. C. M. de Veer Action of cell wll proteinse (P,) from Streptococcus cremoris HP on bovine,-csein. Appl. Microbiol. Biotechnol. 29: Wiedmeier, W. T., S. P. Porterfield, nd C. E. Hendrich Quntittion of Dns-mino cids from body tissues nd fluids using high-performnce liquid chromtogrphy. J. Chromtogr. 231: Downloded from on My 1, 218 by guest