Vol. 171, No. 11JOURNAL OF BACTERIOLOGY, Nov. 1989, p. 6135-61400021-9193/89/116135-06$02.00/0Copyright © 1989, American Society for Microbiology

Peptide Uptake Is Essential for Growth of Lactococcus lactis on theMilk Protein Casein

EDDY J. SMID,1 R. PLAPP,2 AND WIL N. KONINGS1*Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands,' and Department

of Microbiology, University of Kaiserslautern, 6750 Kaiserslautern, Federal Republic of Germany2

Received 8 May 1989/Accepted 23 August 1989

The chlorated dipeptide L-alanyl-j-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis. Spontane-ous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cellwall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di- andoligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type.Only the rates of di- and tripeptide transport were found to be significantly reduced in the diACA-resistantmutant ofL. lactis ML3. Since all other characteristics of this mutant were comparable to those of the wild type,the diACA-resistant mutant is most likely deficient in di- and tripeptide transport. Uptake of di- and tripeptidesby L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptidetransport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented withcasein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. Theseresults indicate that peptide transport in L. lactis ML3 is an essential component in the process of caseinutilization during growth in milk.

In the dairy industry, lactococci (previously named groupN lactic streptococci [19]) are commonly used as startercultures in milk fermentations. These gram-positive bacteriaare very fastidious and require exogenous sources of nucle-otides, vitamins, and amino acids (16). Several amino acidsare either stimulatory or essential for lactococcal growth (5,16). The concentrations of free amino acids in milk are,however, too low to support optimal starter growth (22). Forrapid acid production in milk fermentations, these organismspossess proteinases and a number of peptidases (see refer-ence 22 for a recent review) which hydrolyze the milkprotein casein and supply the cells with the required aminoacids. Most strains of Lactococcus lactis can utilize aminoacids and peptides with up to approximately four to sixresidues to satisfy their growth requirements (9, 18, 21, 24).Hugenholtz et al. (5) showed that the rate of growth of L.

lactis subsp. cremoris in milk is determined by the rate ofcasein hydrolysis and that casein limitation leads to a limi-tation of amino acids. These results raised the question ofwhether the casein-derived limiting amino acids were sup-plied as free amino acids or as small peptides.

Different studies were undertaken to elucidate the actualpathway of casein utilization by lactococci. Proteolyticattack of bovine P-casein by the cell wall proteinases of L.lactis (12) and L. lactis subsp. cremoris (25, 26) can occur atseven cleavage positions at the C-terminal stretch of the milkprotein. Proteolysis thus results in a mixture of peptidefragments which have to be further degraded by extracellularpeptidases before they can pass through the cytoplasmicmembrane. The information available about the extracellularbreakdown of these peptide fragments is limited. It is still notclear how many extracellular peptidases actually contributeto the catabolism of extracellular oligopeptides (22).We recently presented detailed information on the mech-

anism of dipeptide utilization by L. lactis ML3 (20). Dipep-tide transport was studied in L. lactis membrane vesicleswhich were fused with liposomes containing beef heart

* Corresponding author.

cytochrome c oxidase as a proton motive force-generatingsystem. With this experimental system, peptide transport inthe absence of peptidase activity was studied and found to bedriven by the proton motive force. Di- and tripeptide utili-zation by L. lactis takes place by a two-step process. Thefirst step is the translocation of the peptide across thecytoplasmic membrane via a specific peptide transport sys-tem. The second step is the hydrolysis of the peptide by anintracellularly located peptidase to free amino acids whichsubsequently are used for protein synthesis.The aim of this study was to determine the role of peptide

transport in the overall process of casein utilization by L.lactis. This was done by isolating a peptide transport-deficient mutant by selection for resistance to the toxicdipeptide L-alanyl-p-chloro-L-alanine (diACA). The mutantwas physiologically characterized and subsequently used ingrowth experiments on a chemically defined medium (CDM)containing different sources of organic nitrogen. The resultsdemonstrate that transport of di- and tripeptides is essentialfor the growth ofL. lactis ML3 on media containing casein asthe sole N source.

MATERIALS AND METHODSCulture conditions and growth media. Cultures of L. lactis

ML3 were maintained in 10% (wt/vol) reconstituted skimmilk containing 0.1% (wt/vol) tryptone (Difco Laboratories,Detroit, Mich.) and stored at -80°C. For growth experi-ments the cultures were transferred from milk to complexbroth medium (pH 6.4) (2) containing 0.5% (wt/vol) lactoseas a carbon and energy source and incubated overnight at30°C. The overnight cultures were subsequently transferredto different chemically defined media containing 0.5% (wt/vol) lactose as a carbon and energy source.The composition of the CDM was as previously described

(15). In all cases glutamine and asparagine instead of gluta-mate and aspartate were used. To avoid precipitation ofcasein during the exponential growth phase, we buffered themedium with 135 mM potassium phosphate. The pH of themedium was adjusted to 6.5 with 1 N HC1. When indicated in

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the legends to the figures, CDM was supplemented with0.5% (wt/vol) casein (Sigma Chemical Co., St. Louis, Mo.).

Isolation of mutants resistant to toxic peptides. Toxic pep-tide-resistant mutants were isolated from L. lactis ML3. Thetoxicity of different peptide analogs was tested as follows.Approximately 108 CFU of an overnight-grown MRS culturewere spread on a 1% (wt/vol) agar plate containing CDMsupplemented with 0.5% (wt/vol) lactose and a completeamino acid mixture. After plating, single crystals of the toxicpeptide analogs were placed in the center of the plate.Growth inhibition was observed after 48 h of incubation at30°C. Spontaneous resistant mutants were isolated fromplates containing CDM supplemented with lactose and acomplete amino acid mixture plus 0.25 mM concentrationsof different toxic peptides.

Determination of maximum specific growth rates. Maxi-mum specific growth rates were determined in screw-captubes from the increase in the A660 during exponentialgrowth. Growth rates were determined at 30°C. Whengrowth rates were determined on CDM-casein, the opticaldensity values were corrected for the initial optical densityof 0.5% (wt/vol) casein.

Preparation of vesicles, liposomes, and fused membranes.Membrane vesicles of L. lactis ML3 were prepared asdescribed by Otto et al. (13). Cytochrome c oxidase-con-taining liposomes were prepared by the procedure describedby Driessen et al. (3). Proteoliposomes were prepared withEscherichia coli L-ao-phosphatidylethanolamine type IX (Sig-ma), which was further purified by the method described byKawaga et al. (6). Fusion between the membrane vesiclesand the cytochrome c oxidase-containing liposomes wasperformed by the freeze-thaw-sonication procedure as de-scribed by Driessen et al. (3).

Casein hydrolysis. Cells were grown overnight in complexbroth medium (2) in the presence of 8 mM CaCI2. Cells werecollected by centrifugation, washed twice in 50 mM sodiummorpholineethanesulfonic acid (sodium MES) (pH 6.5) con-taining 10 mM CaCl2, and suspended to a final density of 0.5mg of cell protein per ml. Subsequently, ca-casein, P-casein,K-casein, or a mixture of all three types was added to thecells to a final concentration of 0.5% (wt/vol). After 2 h ofincubation at 30°C, the samples were boiled for 3 min insample buffer as described below and applied to a sodiumdodecyl sulfate (SDS)-polyacrylamide gel.SDS-PAGE. SDS-polyacrylamide gel electrophoresis

(PAGE) was performed as described by Laemmli (8) on gelscontaining 12.5% (wt/vol) acrylamide. The protein sampleswere diluted 4:1 in sample buffer (50 mM Tris [pH 6.8], 10%[wt/vol] SDS, 25% [vol/vol] glycerol, 0.1% [wt/vol] brom-phenol blue, 10% [vol/vol] P-mercaptoethanol), heated for 3min at 100°C, and applied to the gels. After electrophoresisthe gels were stained with Coomassie brilliant blue R-250.

Miscellaneous. The transport assay with intact cells andfused membranes was carried out as previously described(20). Protein was determined by the method of Lowry et al.(10) with bovine serum albumin as a standard. An enzyme-linked immunosorbent assay and coating of intact cells wereperformed as described previously (7).

Chemicals. diACA was prepared from tert-butyloxy-car-bonyl-L-alanine-N-hydroxysuccinimide ester and P-chloro-L-alanine by the general method described by Anderson etal. (1). The synthesis of L-alanyl-L-1-aminoethylphosphonicacid was carried out as described by Atherton et al. (F. R.Atherton, M. J. John, C. H. Hassall, R. W. Lambert, andP. S. Stewart, British patent 3417-75, January 1975). L-Alanyl-L-[14C]glutamate (57 mCi/mmol) was synthesized as

TABLE 1. Growth -' the L. lactis ML3 wild type and theL. lactis ML3 diACA- ,istant mutant in the presence of diACAon CDM supplemented with a complete amino acid mixture

Maximum specific growth rate (h-') of:diACA

concn (mM) Wild type diACA-resistantmutant

0 0.69 0.710.05 0.25 0.530.1 <0.01 0.660.5 <0.01 0.601.0 <0.01 0.60

described previously (20). L-Leucyl-L['4C]leucine (0.2 mCi/mmol) was purchased from the Department of OrganicChemistry, University of Nijmegen, Nijmegen, The Nether-lands. L-[U-'4C]histidine (336 mCi/mmol), L-[U-14C]serine(171 mCi/mmol), L-[U-14C]phenylalanine (522 mCi/mmol),L-[U-14C]leucine (318 mCi/mmol), L-[U-14C]aspartic acid(232 mCi/mmol), L-[U-14C]glutamine (270 mCi/mmol), L-[U-14C]proline (291 mCi/mmol), L-[U-14C]alanine (174 mCi/mmol), L-[U- 4C]lysine (324 mCi/mmol), and L-[U-'4C]argi-nine (342 mCi/mmol) were obtained from the RadiochemicalCentre, Amersham, United Kingdom. All other chemicalswere reagent grade and obtained from commercial sources.

RESULTS

Isolation of a toxic peptide-resistant mutant. The toxicity ofthe peptide analog L-alanyl-3-chloro-L-alanine (diACA), L-alanine-L-aminoethylphosphonic acid, and triornithine wastested on confluently plated L. lactis ML3 cultures as de-scribed in Materials and Methods. Only diACA inhibited thegrowth of L. lactis ML3 completely. On plates containing0.25 mM diACA, spontaneous resistant mutants were iso-lated with a mutation frequency of about 5 x 10-5.The maximum specific growth rate of one mutant on media

containing different concentrations of the toxic dipeptidewas compared with that of the wild type. Table 1 shows thatthe growth of wild-type L. lactis ML3 was already severelyinhibited at a concentration of 50 ,uM diACA, while thegrowth of the diACA-resistant mutant was unaffected in thepresence of concentrations of up to 1 mM diACA, indicatingthat the mutant was resistant to high concentrations of thetoxic dipeptide. The amino acid analog P-chloro-L-alaninewas found to be toxic at 10 ,uM for both the L. lactis ML3wild type and the L. lactis ML3 diACA-resistant mutant(data not shown). These observations indicate that the toxicamino acid P-chloro-L-alanine moiety of diACA enters thecells in a peptide-bound configuration.

Peptide transport and hydrolysis in the diACA-resistantmutant. Resistance to the toxic dipeptide can be based ontwo different defects: an inactive peptide transport system oran inactive intracellularly located peptidase. The secondpossibility implies that the peptide exerts its toxicity afterhydrolysis of the peptide bond.To discriminate between these two possibilities, we stud-

ied peptide transport and peptidase activity in the wild typeand the mutant. Dipeptide transport in intact cells wasassayed with the radioactively labeled dipeptides L-alanyl-L-glutamate at 19 ,uM (far below the K,) and L-leucyl-L-leucine at 478 FM (the saturating substrate concentration)(20, 23). The rates of uptake of L-alanyl-L-glutamate andL-leucyl-L-leucine were reduced by 96 and 80%, respec-tively, in cells of the diACA-resistant mutant as compared

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2 000.0

Ec

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0 J

0 2 4 6 0 2 4 6time (min)

FIG. 1. L-Alanyl-L-['4C]glutamate (A) and L-leucyl-L-[14C]leu-cine (B) uptake in glycolyzing cells of the L. lactis ML3 wild type(0) and the L. lactis ML3 diACA-resistant mutant (0). The assayswere started by the addition of 19 ,M L-alanyl-L-['4C]glutamate and478 ,uM L-leucyl-L-[14C]leucine, respectively.

with wild-type cells (Fig. 1A and B), suggesting that adipeptide transport system of L. lactis ML3 is inactivated inthe mutant. To obtain further support for this conclusion, weinvestigated L-alanyl-L-glutamate uptake in membrane vesi-cles derived from the wild type and the diACA-resistantmutant. The membrane vesicles were fused with liposomescontaining beef heart cytochrome c oxidase as a protonmotive force-generating device (3, 4). In this system, dipep-tide transport can be studied in the absence of peptidaseactivity (20). In contrast to the result with wild-type mem-brane vesicles, no significant accumulation of L-alanyl-L-glutamate could be detected in fused membrane vesiclesderived from the diACA-resistant mutant (Fig. 2B). As acontrol, the uptake of L-leucine was measured in both typesof fused membranes. Rapid accumulation of the amino acidcould be detected in both wild-type and mutant fused mem-branes. Surprisingly, the rate of L-leucine uptake in thevesicles derived from the diACA-resistant mutant was evenhigher than that in the wild-type vesicles. The resultsstrongly suggest that the diACA-resistant mutant is a dipep-tide transport-deficient mutant.

In addition, tripeptide uptake was strongly reduced in thediACA-resistant mutant, whereas the uptake of tetra-,penta-, and hexapeptides was unaffected (data not shown).To exclude the possibility that the diACA-resistant mutant

is a peptidase-negative mutant, we examined different pep-tidase activities in cell extracts of both the mutant and thewild-type cells. No differences were found between mutantand wild-type peptidase activities (data not shown), indicat-ing that the resistance of the mutant to diACA is not a resultof a defective peptidase.Amino acid transport in the diACA-resistant mutant. We

also investigated the activities of the different amino acidtransport systems of the L. lactis ML3 wild type and mutant(Table 2). All amino acid transport systems present in the

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time (min)FIG. 2. Uptake of ['4C]leucine (A) and L-alanyl-L-["4C]glutamate

(B) in fused membranes from the L. lactis ML3 wild type (O and L1)and the L. lactis ML3 diACA-resistant mutant (0 and U). The fusedmembranes were incubated in the presence (O and 0) or absence (Oand U) of a complete electron donor system. ['4C]leucine andL-alanyl-L-["4C]glutamate were added to final concentrations of 2.80and 2.3 ,iM, respectively. The broken lines indicate the levels ofL-leucine and L-alanyl-L-glutamate equilibration between the inter-nal and external media.

wild type were also present in the dipeptide transportmutant. Rates of uptake of asparagine, phenylalanine, andhistidine in the mutant and the wild type were comparable(Table 2). Surprisingly, rates of uptake of leucine and, to alesser extent, glutamine, alanine, and serine were higher inthe dipeptide transport mutant than in the wild type (Table2). A similar result was found for leucine uptake in fusedmembranes (Fig. 2). The rate of uptake of lysine appeared tobe somewhat reduced in the mutant as compared with that inthe wild type. The rates of arginine uptake given in Table 2do not reflect the activity of the transport system but reflectonly the initial pool size of ornithine (14). Interestingly, no

TABLE 2. Amino acid uptake rates in the L. lactis ML3 wildtype and the diACA-resistant mutanta

Uptake (nmol/min per mg of protein)Amino acid

Wild type Mutant

Asn 12.1 10.4Gln 21.3 36.9Lys 33.4 19.4Arg 85.2 43.8Leu 12.5 32.2Ala 37.5 52.7Ser 28.1 51.1Phe 4.6 6.1Pro <0.1 <0.1His 6.6 4.7

a Uptake was assayed in duplicate as described in Materials and Methods.The initial transport rates were determined after incubation of the cellsuspension for 15 s in the presence of 0.1 mM concentrations of the differentradiolabeled amino acids.

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Ec

0( 0.4-

.~0.2-C

CL0

0.05

l l l l l l0 2 4 6 8

time (hours)FIG. 3. Growth of the L. lactis ML3 wild type (0 and *) and the

L. lactis ML3 diACA-resistant mutant (O and O) in CDM supple-mented with 0.5% (wt/vol) casein (O and *) or 0.5% casein plus acomplete amino acid mixture (O and 0). The complete amino acidmixture contained glutamine, asparagine, leucine, valine, isoleu-cine, glycine, alanine, threonine, serine, proline, arginine, lysine,histidine, methionine, tryptophan, tyrosine, and phenylalanine.

significant proline uptake could be detected in the mutant orin the wild type.Growth of the dipeptide transport mutant on casein-con-

taining media. The dipeptide transport-deficient mutant of-fers the possibility of studying the physiological role andsignificance of the lactococcal dipeptide transport system.During growth in milk, lactococci utilize the milk proteincasein as the major source of organic nitrogen. CDM sup-plemented with different sources of organic nitrogen wasused as an experimental model for milk.The L. lactis ML3 wild type reached a high growth rate

and cell density on CDM supplemented with 0.5% casein asthe sole source of organic nitrogen (Fig. 3), indicating thatthis strain possesses a functional proteolytic system. Thedipeptide transport mutant was unable to grow on thismedium (Fig. 3). Identical growth characteristics were foundfor the wild type and the mutant when the medium wassupplemented with a complete amino acid mixture.

Since similar growth properties were observed for prote-olysis-negative variants of lactococci (21), the presence andactivity of caseinolytic proteinases were studied in thedipeptide transport mutant and the wild type.

In a previous paper (7) we showed that the monoclonalantibodies Wg2-1 to Wg2-12, which are directed to the cellwall-associated proteinase of L. lactis subsp. cremoris Wg2,also interacted with different related strains, including L.lactis ML3. The monoclonal antibodies Wg2-1 and Wg2-10also cross-reacted with the dipeptide transport mutant, indi-cating the presence of immunologically related proteinasesin both strains (data not shown). The proteolytic activity ofthe dipeptide transport mutant was compared with that ofthe wild type. Washed cells were incubated in a buffercontaining a-, 1B-, or K-casein or a mixture of these caseins.The hydrolysis products were subsequently analyzed by

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FIG. 4. SDS-PAGE of ax-, and K-casein degradation bywashed cell suspensions of the L. lactis ML3 wild type (lanes 2, 5,8, and 11) and the L. lactis ML3 diACA-resistant mutant (lanes 3, 6,9, and 12). The cell suspensions were incubated with a mixture of ax-,1-, and K-caseins (lanes 1, 2, and 3), oa-casein (lanes 4, 5, and 6),1-casein (lanes 7, 8, and 9), and K-casein (lanes 10, 11 and 12). Thefirst of every three subsequent lanes represents the untreated caseinstandard. The molecular weights of the marker proteins are asindicated on the right (in thousands).

SDS-PAGE (Fig. 4). The same pattern of 1-casein hydrolysiswas observed with the dipeptide transport mutant and thewild type (Fig. 4, lanes 8 and 9). No significant breakdown ofot- or K-casein was detectable with either type of cell (Fig. 4,lanes 5, 6, 11, and 12).These experiments showed that 1-casein is the only

casein-type milk protein that L. lactis ML3 can utilize.Furthermore, they showed that the proteinase of the dipep-tide transport mutant is identical to the wild-type proteinase(HP-type proteinase; 25, 26).

After incubation of the diACA-resistant mutant ofL. lactisML3 for more than 10 h on CDM-casein, some growth wasobserved. When these cells were subsequently transferred tofresh medium containing 100 ,uM diACA, growth was againfully inhibited, indicating that revertants grew during thelong incubation time (data not shown).The inability of the dipeptide transport mutant to grow on

a medium containing casein as a sole source of organicnitrogen indicates that some of the amino acids only becomeavailable as di- (or tri-) peptides and implies that thesepeptide-bound amino acids are essential for growth.

DISCUSSION

The physiological role of the recently described dipeptidetransport system of L. lactis ML3 (20) in the overall processof casein utilization was investigated with the use of adipeptide transport-deficient mutant.For the isolation of the dipeptide transport-deficient mu-

tant, growth inhibition of L. lactis ML3 was assayed in thepresence of different peptides. The dipeptide diACA causedsignificant growth inhibition (Table 1), whereas L-alanine-L-aminoethylphosphonic acid and triornithine had no effecton the growth of L. lactis ML3 at all. Because diACA wasrapidly hydrolyzed by cell extracts of both the L. lactis ML3wild type and the L. lactis ML3 diACA-resistant mutant(data not shown), the peptide most likely exerts its toxicityafter intracellular hydrolysis, thereby liberating the toxicamino acid 13-chloro-L-alanine. In agreement with this con-clusion is the observation that the growth of the L. lactis

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ML3 wild type and the L. lactis ML3 diACA-resistant mutantwas strongly inhibited by P-chloro-L-alanine (data notshown).As in Streptococcus pyogenes, Diplococcus pneumoniae,

and E. coli, the target enzyme in L. lactis ML3 for 1B-chloro-L-alanine is most likely alanine racemase (EC 5.1.1.1)(11).

diACA-resistant mutants of L. lactis ML3 could be iso-lated at relatively high mutation frequencies (5 x 10-5) onplates containing a sufficient amount of the toxic dipeptide.Revertants were selected when the mutants were cultivatedunder extreme selection pressure (i.e., on CDM containingcasein. This result suggests that the spontaneous diACAresistance probably results from a point mutation in the genecoding for the peptide transport system.

Since L-leucyl-L-leucine and L-alanyl-L-glutamate trans-port activity in the plasmid-cured strain L. lactis MG1363 iscomparable to that in wild-type L. lactis ML3 (E. J. Smid,unpublished results), the gene coding for the dipeptidetransport system is most likely not plasmid linked butlocated on the chromosome. Consequently, the high muta-tion frequency cannot be explained by the loss of a plasmid.

In a recent publication we showed that the uptake ofnutritional dipeptides such as L-alanyl-L-glutamate and L-leucyl-L-leucine is mediated by one kinetically distinguish-able peptide transport system (20). Our present resultsconfirm this conclusion, since the uptake of both L-alanyl-L-glutamate and L-leucine-L-leucine was severely reduced inthe diACA-resistant mutant.The diACA-resistant mutant was physiologically charac-

terized. Relevant features, such as the presence of all aminoacid transport systems, peptidolytic activity, and casei-nolytic activity, of the mutant were compared with those ofthe wild type. Evidence was presented that the diACA-resistant mutant of L. lactis ML3 is a dipeptide transport-negative mutant.An interesting observation was that the rate of uptake of

different amino acids (L-glutamine, L-leucine, L-alanine, andL-serine) was significantly increased in the dipeptide trans-port mutant as compared with that in the wild type (Table 2).This result suggests that dipeptide transport plays a role inthe regulation of some of the amino acid transport systems.Another indication that the presence of peptides in thegrowth medium affects the expression of amino acid trans-port systems in L. lactis ML3 was given by Poolman andKonings (15), who showed that the expression of thebranched-chain amino acid transport system of L. lactis ML3is 10-fold higher in organisms cultivated on CDM supple-mented with amino acids than in organisms cultivated incomplex broth medium.The dipeptide transport-negative mutant that we have

isolated offers the possibility of studying the physiologicalrole and significance of the dipeptide transport system. Theinvolvement of the dipeptide transport system in the proteo-lytic pathway of casein was investigated by comparing thespecific growth rates of the wild type and the dipeptidetransport mutant on CDM supplemented with different ni-trogen sources. In sharp contrast to the wild type, thedipeptide transport mutant was unable to grow on CDMsupplemented with casein (Fig. 4). This result suggests thatduring the process of extracellular ,B-casein degradation, oneor more essential or stimulatory amino acids are suppliedmainly as di- and tripeptides.Hugenholtz et al. (5) demonstrated that restricted casein

hydrolysis leads to amino acid limitation during the growthof L. lactis subsp. cremoris in milk. In that study it was

shown that L-phenylalanine and L-leucine stimulated thegrowth of L. lactis subsp. cremoris HP and E8. In view of theresults presented in this paper, the growth limitation in milkis most probably caused by a limited supply of L-phenylala-nine-containing di- or tripeptides. Because L. lactis ML3 isunable to take up proline (Table 2), this amino acid, which isthe most abundant amino acid residue in ,B-casein (17), mostlikely enters the cell exclusively in a peptide-bound config-uration. This conclusion is supported by the observation thatexcess unlabeled L-prolyl-L-methionine inhibits the uptakeof radioactively labeled L-alanyl-L-glutamic acid, indicatingthat prolyl peptides can be transported via the dipeptidetransport system (E. J. Smid, unpublished results).The results in this paper show that the synthesis of

proteolytic enzymes is not the only fundamental requirementfor rapid acid production in milk fermentations but thatdipeptide transport is also an essential component in theprocess of casein utilization by lactococci during growth inmilk.

ACKNOWLEDGMENTS

We thank T. Abee, D. Molenaar, R. Hogendorp, and H. Laan forvaluable suggestions throughout this work.

This investigation was supported by the Foundation for Funda-mental Biological Research, which is subsidized by The NetherlandsOrganization for Scientific Research.

LITERATURE CITED1. Anderson, G. W., J. E. Zimmermann, and F. M. Callahau. 1964.

The use of esters of N-hydroxysuccinimide in peptide synthesis.J. Am. Chem. Soc. 86:1839-1842.

2. De Man, J. C., M. Rogosa, and M. E. Sharpe. 1960. A mediumfor the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130-135.

3. Driessen, A. J. M., W. de VrU, and W. N. Konings. 1985.Incorporation of beefheart cytochrome c oxidase as a protonmotive force generating system in bacterial membrane vesicles.Proc. Natl. Acad. Sci. USA 82:7555-7559.

4. Driessen, A. J. M., W. de Vrij, and W. N. Konings. 1986.Functional incorporation of beefheart cytochrome c oxidaseinto membranes of Streptococcus cremoris. Eur. J. Biochem.154:617-624.

5. Hugenholtz, J., M. Dikstra, and H. Veldkamp. 1987. Aminoacid limited growth of starter cultures in milk. FEMS Microbiol.Ecol. 45:191-198.

6. Kawaga, Y., A. Kandrach, and E. Racker. 1973. Partial resolu-tion of the enzymes catalyzing oxidative phosphorylation. J.Biol. Chem. 248:676-684.

7. Laan, H., E. J. Smid, L. de LeU, E. Schwander, and W. N.Konings. 1988. Monoclonal antibodies to the cell wall-associ-ated proteinase of Lactococcus lactis subsp. cremoris Wg2.Appl. Environ. Microbiol. 54:2250-2256.

8. Laemmli, U. K., and K. Faure. 1973. Maturation of the head ofbacteriophage T4. I. DNA packaging events. J. Mol. Biol.80:575-599.

9. Law, B. A. 1978. Peptide utilization by group N streptococci. J.Gen. Microbiol. 105:113-118.

10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

11. Manning, J. M., N. E. Merrifield, W. M. Jones, and E. C.Gotschlich. 1974. Inhibition of bacterial growth by P-chloro-D-alanine. Proc. Natl. Acad. Sci. USA 71:417-421.

12. Monnet, V., D. Le Bars, and J.-C. Gripon. 1986. Specificity of acell wall proteinase from Streptococcus lactis NCDO763towards bovine ,-casein. FEMS Microbiol. Lett. 36:127-131.

13. Otto, R., R. G. Lageveen, H. Veldkamp, and W. N. Konings.1982. Lactate efflux-induced electrical potential in membranevesicles of Streptococcus cremoris. J. Bacteriol. 149:733-738.

14. Poolman, B., A. J. M. Driessen, and W. N. Konings. 1987.Regulation of arginine-ornithine exchange and the arginine

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deiminase pathway in Streptococcus lactis. J. Bacteriol. 169:5597-5604.

15. Poolman, B., and W. N. Konings. 1988. Growth of Streptococ-cus cremoris in relation to amino acid transport. J. Bacteriol.170:700-707.

16. Reiter, B., and J. D. Oram. 1962. Nutritional studies on cheesestarters. 1. Vitamin and amino acid requirements of single strainstarters. J. Dairy Res. 29:63-77.

17. Ribadeau Dumas, B., G. Brignon, F. Grosclaude, and J.-C.Mercier. 1972. Structure primaire de la casdine a bovine;sdquence complete. Eur. J. Biochem. 25:505-514.

18. Rice, G. H., F. H. C. Stewart, A. J. Hillier, and G. R. Jago. 1978.The uptake of amino acids and peptides by Streptococcus lactis.J. Dairy Res. 45:93-107.

19. Schleifer, K. H., and R. Kilpper-Bfilz. 1987. Molecular andchemotaxonomic approaches to the classification of strepto-cocci, enterococci and lactococci: a review. Syst. Appl. Micro-biol. 10:1-19.

20. Smid, E. J., A. J. M. Driessen, and W. N. Konings. 1989.Mechanism and energetics of dipeptide transport in membrane

vesicles of Lactococcus lactis. J. Bacteriol. 171:292-298.21. Thomas, T. D., and 0. E. Mills. 1981. Proteolytic enzymes of

starter bacteria. Neth. Milk Dairy J. 35:255-273.22. Thomas, T. D., and G. G. Pritchard. 1987. Proteolytic enzymes

of dairy starter cultures. FEMS Microbiol. Rev. 46:245-268.23. Van Boven, A., and W. N. Konings. 1987. A phosphate bond-

driven dipeptide transport system in Streptococcus cremoris isregulated by the internal pH. Appl. Environ. Microbiol. 53:2897-2902.

24. Van Boven, A., and W. N. Konings. 1988. Utilization of dipep-tides by Lactococcus lactis ssp. cremoris. Biochimie 70:535-542.

25. Visser, S., F. A. Exterkate, C. J. Slangen, and G. J. C. M. deVeer. 1986. Comparative study of the action of cell wall protein-ase from various strains of Streptococcus cremoris on bovineas1-, P-, and K-casein. Appl. Environ. Microbiol. 52:1162-1166.

26. Visser, S., C. J. Slangen, F. A. Exterkate, and G. J. C. M. deVeer. 1988. Action of a cell wall proteinase (PI) from Strepto-coccus cremoris HP on bovine ,Q-casein. Appl. Microbiol.Biotechnol. 29:61-66.

J. BACTERIOL.

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