1 JOURNAL OF BACTRIOLOGY, Nov. 1989, p /89/ $02.00/0 Copyright 1989, American Society for Microbiology Vol. 171, No. 11 Peptide Uptake Is ssential for Growth of Lactococcus lactis on the Milk Protein Casein DDY 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. Spontaneous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di- and oligopeptide 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-resistant mutant 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 tripeptides by L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. These results indicate that peptide transport in L. lactis ML3 is an essential component in the process of casein utilization during growth in milk. In the dairy industry, lactococci (previously named group N lactic streptococci ) are commonly used as starter cultures in milk fermentations. These gram-positive bacteria are very fastidious and require exogenous sources of nucleotides, vitamins, and amino acids (16). Several amino acids are 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). For rapid acid production in milk fermentations, these organisms possess proteinases and a number of peptidases (see reference 22 for a recent review) which hydrolyze the milk protein casein and supply the cells with the required amino acids. Most strains of Lactococcus lactis can utilize amino acids and peptides with up to approximately four to six residues 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 of casein hydrolysis and that casein limitation leads to a limitation of amino acids. These results raised the question of whether the casein-derived limiting amino acids were supplied as free amino acids or as small peptides. Different studies were undertaken to elucidate the actual pathway of casein utilization by lactococci. Proteolytic attack of bovine P-casein by the cell wall proteinases of L. lactis (12) and L. lactis subsp. cremoris (25, 26) can occur at seven cleavage positions at the C-terminal stretch of the milk protein. Proteolysis thus results in a mixture of peptide fragments which have to be further degraded by extracellular peptidases before they can pass through the cytoplasmic membrane. The information available about the extracellular breakdown of these peptide fragments is limited. It is still not clear how many extracellular peptidases actually contribute to the catabolism of extracellular oligopeptides (22). We recently presented detailed information on the mechanism of dipeptide utilization by L. lactis ML3 (20). Dipeptide transport was studied in L. lactis membrane vesicles which were fused with liposomes containing beef heart * Corresponding author cytochrome c oxidase as a proton motive force-generating system. With this experimental system, peptide transport in the absence of peptidase activity was studied and found to be driven by the proton motive force. Di- and tripeptide utilization by L. lactis takes place by a two-step process. The first step is the translocation of the peptide across the cytoplasmic membrane via a specific peptide transport system. The second step is the hydrolysis of the peptide by an intracellularly located peptidase to free amino acids which subsequently 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 transportdeficient mutant by selection for resistance to the toxic dipeptide L-alanyl-p-chloro-L-alanine (diaca). The mutant was physiologically characterized and subsequently used in growth experiments on a chemically defined medium (CDM) containing different sources of organic nitrogen. The results demonstrate that transport of di- and tripeptides is essential for the growth of L. lactis ML3 on media containing casein as the sole N source. MATRIALS AND MTHODS Culture conditions and growth media. Cultures of L. lactis ML3 were maintained in 10% (wt/vol) reconstituted skim milk containing 0.1% (wt/vol) tryptone (Difco Laboratories, Detroit, Mich.) and stored at -80 C. For growth experiments the cultures were transferred from milk to complex broth medium (ph 6.4) (2) containing 0.5% (wt/vol) lactose as a carbon and energy source and incubated overnight at 30 C. The overnight cultures were subsequently transferred to 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 glutamate and aspartate were used. To avoid precipitation of casein during the exponential growth phase, we buffered the medium with 135 mm potassium phosphate. The ph of the medium was adjusted to 6.5 with 1 N HC1. When indicated in
2 6136 SMID T AL. the legends to the figures, CDM was supplemented with 0.5% (wt/vol) casein (Sigma Chemical Co., St. Louis, Mo.). Isolation of mutants resistant to toxic peptides. Toxic peptide-resistant mutants were isolated from L. lactis ML3. The toxicity of different peptide analogs was tested as follows. Approximately 108 CFU of an overnight-grown MRS culture were spread on a 1% (wt/vol) agar plate containing CDM supplemented with 0.5% (wt/vol) lactose and a complete amino acid mixture. After plating, single crystals of the toxic peptide analogs were placed in the center of the plate. Growth inhibition was observed after 48 h of incubation at 30 C. Spontaneous resistant mutants were isolated from plates containing CDM supplemented with lactose and a complete amino acid mixture plus 0.25 mm concentrations of different toxic peptides. Determination of maximum specific growth rates. Maximum specific growth rates were determined in screw-cap tubes from the increase in the A660 during exponential growth. Growth rates were determined at 30 C. When growth rates were determined on CDM-casein, the optical density values were corrected for the initial optical density of 0.5% (wt/vol) casein. Preparation of vesicles, liposomes, and fused membranes. Membrane vesicles of L. lactis ML3 were prepared as described by Otto et al. (13). Cytochrome c oxidase-containing liposomes were prepared by the procedure described by Driessen et al. (3). Proteoliposomes were prepared with scherichia coli L-ao-phosphatidylethanolamine type IX (Sigma), which was further purified by the method described by Kawaga et al. (6). Fusion between the membrane vesicles and the cytochrome c oxidase-containing liposomes was performed by the freeze-thaw-sonication procedure as described by Driessen et al. (3). Casein hydrolysis. Cells were grown overnight in complex broth medium (2) in the presence of 8 mm CaCI2. Cells were collected by centrifugation, washed twice in 50 mm sodium morpholineethanesulfonic acid (sodium MS) (ph 6.5) containing 10 mm CaCl2, and suspended to a final density of 0.5 mg of cell protein per ml. Subsequently, ca-casein, P-casein, K-casein, or a mixture of all three types was added to the cells to a final concentration of 0.5% (wt/vol). After 2 h of incubation at 30 C, the samples were boiled for 3 min in sample buffer as described below and applied to a sodium dodecyl sulfate (SDS)-polyacrylamide gel. SDS-PAG. SDS-polyacrylamide gel electrophoresis (PAG) was performed as described by Laemmli (8) on gels containing 12.5% (wt/vol) acrylamide. The protein samples were diluted 4:1 in sample buffer (50 mm Tris [ph 6.8], 10% [wt/vol] SDS, 25% [vol/vol] glycerol, 0.1% [wt/vol] bromphenol blue, 10% [vol/vol] P-mercaptoethanol), heated for 3 min at 100 C, and applied to the gels. After electrophoresis the gels were stained with Coomassie brilliant blue R-250. Miscellaneous. The transport assay with intact cells and fused 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 enzymelinked immunosorbent assay and coating of intact cells were performed as described previously (7). Chemicals. diaca was prepared from tert-butyloxy-carbonyl-l-alanine-n-hydroxysuccinimide ester and P-chloro- L-alanine by the general method described by Anderson et al. (1). The synthesis of L-alanyl-L-1-aminoethylphosphonic acid was carried out as described by Atherton et al. (F. R. Atherton, M. J. John, C. H. Hassall, R. W. Lambert, and P. S. Stewart, British patent , January 1975). L- Alanyl-L-[14C]glutamate (57 mci/mmol) was synthesized as TABL 1. Growth -' the L. lactis ML3 wild type and the L. lactis ML3 diaca-,istant mutant in the presence of diaca on CDM supplemented with a complete amino acid mixture Maximum specific growth rate (h-') of: diaca concn (mm) Wild type diaca-resistant mutant < < < described previously (20). L-Leucyl-L['4C]leucine (0.2 mci/ mmol) was purchased from the Department of Organic Chemistry, University of Nijmegen, Nijmegen, The Netherlands. 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]arginine (342 mci/mmol) were obtained from the Radiochemical Centre, Amersham, United Kingdom. All other chemicals were reagent grade and obtained from commercial sources. RSULTS J. BACTRIOL. Isolation of a toxic peptide-resistant mutant. The toxicity of the peptide analog L-alanyl-3-chloro-L-alanine (diaca), L- alanine-l-aminoethylphosphonic acid, and triornithine was tested on confluently plated L. lactis ML3 cultures as described in Materials and Methods. Only diaca inhibited the growth of L. lactis ML3 completely. On plates containing 0.25 mm diaca, spontaneous resistant mutants were isolated with a mutation frequency of about 5 x The maximum specific growth rate of one mutant on media containing different concentrations of the toxic dipeptide was compared with that of the wild type. Table 1 shows that the growth of wild-type L. lactis ML3 was already severely inhibited at a concentration of 50,uM diaca, while the growth of the diaca-resistant mutant was unaffected in the presence of concentrations of up to 1 mm diaca, indicating that the mutant was resistant to high concentrations of the toxic dipeptide. The amino acid analog P-chloro-L-alanine was found to be toxic at 10,uM for both the L. lactis ML3 wild type and the L. lactis ML3 diaca-resistant mutant (data not shown). These observations indicate that the toxic amino acid P-chloro-L-alanine moiety of diaca enters the cells in a peptide-bound configuration. Peptide transport and hydrolysis in the diaca-resistant mutant. Resistance to the toxic dipeptide can be based on two different defects: an inactive peptide transport system or an inactive intracellularly located peptidase. The second possibility implies that the peptide exerts its toxicity after hydrolysis of the peptide bond. To discriminate between these two possibilities, we studied peptide transport and peptidase activity in the wild type and the mutant. Dipeptide transport in intact cells was assayed 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 and L-leucyl-L-leucine were reduced by 96 and 80%, respectively, in cells of the diaca-resistant mutant as compared
3 VOL. 171, 1989 ROL OF PPTID TRANSPORT IN L. LACTIS _ ' ' L CL c ~~~~~~~~~~ J time (min) FIG. 1. L-Alanyl-L-['4C]glutamate (A) and L-leucyl-L-[14C]leucine (B) uptake in glycolyzing cells of the L. lactis ML3 wild type (0) and the L. lactis ML3 diaca-resistant mutant (0). The assays were started by the addition of 19,M L-alanyl-L-['4C]glutamate and 478,uM L-leucyl-L-[14C]leucine, respectively. with wild-type cells (Fig. 1A and B), suggesting that a dipeptide transport system of L. lactis ML3 is inactivated in the mutant. To obtain further support for this conclusion, we investigated L-alanyl-L-glutamate uptake in membrane vesicles derived from the wild type and the diaca-resistant mutant. The membrane vesicles were fused with liposomes containing beef heart cytochrome c oxidase as a proton motive force-generating device (3, 4). In this system, dipeptide transport can be studied in the absence of peptidase activity (20). In contrast to the result with wild-type membrane vesicles, no significant accumulation of L-alanyl-Lglutamate could be detected in fused membrane vesicles derived from the diaca-resistant mutant (Fig. 2B). As a control, the uptake of L-leucine was measured in both types of fused membranes. Rapid accumulation of the amino acid could be detected in both wild-type and mutant fused membranes. Surprisingly, the rate of L-leucine uptake in the vesicles derived from the diaca-resistant mutant was even higher than that in the wild-type vesicles. The results strongly suggest that the diaca-resistant mutant is a dipeptide transport-deficient mutant. In addition, tripeptide uptake was strongly reduced in the diaca-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 peptidase activities in cell extracts of both the mutant and the wild-type cells. No differences were found between mutant and wild-type peptidase activities (data not shown), indicating that the resistance of the mutant to diaca is not a result of a defective peptidase. Amino acid transport in the diaca-resistant mutant. We also investigated the activities of the different amino acid transport systems of the L. lactis ML3 wild type and mutant (Table 2). All amino acid transport systems present in the C. 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 fused membranes were incubated in the presence (O and 0) or absence (O and U) of a complete electron donor system. ['4C]leucine and L-alanyl-L-["4C]glutamate were added to final concentrations of 2.80 and 2.3,iM, respectively. The broken lines indicate the levels of L-leucine and L-alanyl-L-glutamate equilibration between the internal and external media. wild type were also present in the dipeptide transport mutant. Rates of uptake of asparagine, phenylalanine, and histidine in the mutant and the wild type were comparable (Table 2). Surprisingly, rates of uptake of leucine and, to a lesser extent, glutamine, alanine, and serine were higher in the dipeptide transport mutant than in the wild type (Table 2). A similar result was found for leucine uptake in fused membranes (Fig. 2). The rate of uptake of lysine appeared to be somewhat reduced in the mutant as compared with that in the wild type. The rates of arginine uptake given in Table 2 do not reflect the activity of the transport system but reflect only the initial pool size of ornithine (14). Interestingly, no TABL 2. Amino acid Amino acid uptake rates in the L. lactis ML3 wild type and the diaca-resistant mutanta Uptake (nmol/min per mg of protein) Wild type Mutant Asn Gln Lys Arg Leu Ala Ser Phe Pro <0.1 <0.1 His a Uptake was assayed in duplicate as described in Materials and Methods. The initial transport rates were determined after incubation of the cell suspension for 15 s in the presence of 0.1 mm concentrations of the different radiolabeled amino acids.
4 6138 SMID T AL. J. BACTRIOL ' -44Onj3 c 0 ( 0.4-3,-- p - c.3i 3-1, 2-1.~0.2- C -14,4 CL l l l l l l 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 supplemented with 0.5% (wt/vol) casein (O and *) or 0.5% casein plus a complete amino acid mixture (O and 0). The complete amino acid mixture contained glutamine, asparagine, leucine, valine, isoleucine, glycine, alanine, threonine, serine, proline, arginine, lysine, histidine, methionine, tryptophan, tyrosine, and phenylalanine. significant proline uptake could be detected in the mutant or in the wild type. Growth of the dipeptide transport mutant on casein-containing media. The dipeptide transport-deficient mutant offers the possibility of studying the physiological role and significance of the lactococcal dipeptide transport system. During growth in milk, lactococci utilize the milk protein casein as the major source of organic nitrogen. CDM supplemented with different sources of organic nitrogen was used 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 as the sole source of organic nitrogen (Fig. 3), indicating that this strain possesses a functional proteolytic system. The dipeptide transport mutant was unable to grow on this medium (Fig. 3). Identical growth characteristics were found for the wild type and the mutant when the medium was supplemented with a complete amino acid mixture. Since similar growth properties were observed for proteolysis-negative variants of lactococci (21), the presence and activity of caseinolytic proteinases were studied in the dipeptide transport mutant and the wild type. In a previous paper (7) we showed that the monoclonal antibodies Wg2-1 to Wg2-12, which are directed to the cell wall-associated proteinase of L. lactis subsp. cremoris Wg2, also interacted with different related strains, including L. lactis ML3. The monoclonal antibodies Wg2-1 and Wg2-10 also cross-reacted with the dipeptide transport mutant, indicating the presence of immunologically related proteinases in both strains (data not shown). The proteolytic activity of the dipeptide transport mutant was compared with that of the wild type. Washed cells were incubated in a buffer containing a-, 1B-, or K-casein or a mixture of these caseins. The hydrolysis products were subsequently analyzed by 2 i(;~~~~~~~~1 FIG. 4. SDS-PAG of ax-, and K-casein degradation by washed 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). The first of every three subsequent lanes represents the untreated casein standard. The molecular weights of the marker proteins are as indicated on the right (in thousands). SDS-PAG (Fig. 4). The same pattern of 1-casein hydrolysis was observed with the dipeptide transport mutant and the wild type (Fig. 4, lanes 8 and 9). No significant breakdown of ot- 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 dipeptide transport mutant is identical to the wild-type proteinase (HP-type proteinase; 25, 26). After incubation of the diaca-resistant mutant of L. lactis ML3 for more than 10 h on CDM-casein, some growth was observed. When these cells were subsequently transferred to fresh medium containing 100,uM diaca, growth was again fully inhibited, indicating that revertants grew during the long incubation time (data not shown). The inability of the dipeptide transport mutant to grow on a medium containing casein as a sole source of organic nitrogen indicates that some of the amino acids only become available as di- (or tri-) peptides and implies that these peptide-bound amino acids are essential for growth. DISCUSSION The physiological role of the recently described dipeptide transport system of L. lactis ML3 (20) in the overall process of casein utilization was investigated with the use of a dipeptide transport-deficient mutant. For the isolation of the dipeptide transport-deficient mutant, growth inhibition of L. lactis ML3 was assayed in the presence of different peptides. The dipeptide diaca caused significant growth inhibition (Table 1), whereas L-alanine- L-aminoethylphosphonic acid and triornithine had no effect on the growth of L. lactis ML3 at all. Because diaca was rapidly hydrolyzed by cell extracts of both the L. lactis ML3 wild type and the L. lactis ML3 diaca-resistant mutant (data not shown), the peptide most likely exerts its toxicity after intracellular hydrolysis, thereby liberating the toxic amino acid 13-chloro-L-alanine. In agreement with this conclusion is the observation that the growth of the L. lactis
5 VOL. 171, 1989 ML3 wild type and the L. lactis ML3 diaca-resistant mutant was strongly inhibited by P-chloro-L-alanine (data not shown). As in Streptococcus pyogenes, Diplococcus pneumoniae, and. coli, the target enzyme in L. lactis ML3 for 1Bchloro-L-alanine is most likely alanine racemase (C ) (11). diaca-resistant mutants of L. lactis ML3 could be isolated at relatively high mutation frequencies (5 x 10-5) on plates containing a sufficient amount of the toxic dipeptide. Revertants were selected when the mutants were cultivated under extreme selection pressure (i.e., on CDM containing casein. This result suggests that the spontaneous diaca resistance probably results from a point mutation in the gene coding for the peptide transport system. Since L-leucyl-L-leucine and L-alanyl-L-glutamate transport activity in the plasmid-cured strain L. lactis MG1363 is comparable to that in wild-type L. lactis ML3 (. J. Smid, unpublished results), the gene coding for the dipeptide transport system is most likely not plasmid linked but located on the chromosome. Consequently, the high mutation frequency cannot be explained by the loss of a plasmid. In a recent publication we showed that the uptake of nutritional dipeptides such as L-alanyl-L-glutamate and L- leucyl-l-leucine is mediated by one kinetically distinguishable peptide transport system (20). Our present results confirm this conclusion, since the uptake of both L-alanyl-Lglutamate and L-leucine-L-leucine was severely reduced in the diaca-resistant mutant. The diaca-resistant mutant was physiologically characterized. Relevant features, such as the presence of all amino acid transport systems, peptidolytic activity, and caseinolytic activity, of the mutant were compared with those of the wild type. vidence was presented that the diacaresistant mutant of L. lactis ML3 is a dipeptide transportnegative mutant. An interesting observation was that the rate of uptake of different amino acids (L-glutamine, L-leucine, L-alanine, and L-serine) was significantly increased in the dipeptide transport mutant as compared with that in the wild type (Table 2). This result suggests that dipeptide transport plays a role in the regulation of some of the amino acid transport systems. Another indication that the presence of peptides in the growth medium affects the expression of amino acid transport systems in L. lactis ML3 was given by Poolman and Konings (15), who showed that the expression of the branched-chain amino acid transport system of L. lactis ML3 is 10-fold higher in organisms cultivated on CDM supplemented with amino acids than in organisms cultivated in complex broth medium. The dipeptide transport-negative mutant that we have isolated offers the possibility of studying the physiological role and significance of the dipeptide transport system. The involvement of the dipeptide transport system in the proteolytic pathway of casein was investigated by comparing the specific growth rates of the wild type and the dipeptide transport mutant on CDM supplemented with different nitrogen sources. In sharp contrast to the wild type, the dipeptide transport mutant was unable to grow on CDM supplemented with casein (Fig. 4). This result suggests that during the process of extracellular,b-casein degradation, one or more essential or stimulatory amino acids are supplied mainly as di- and tripeptides. Hugenholtz et al. (5) demonstrated that restricted casein hydrolysis leads to amino acid limitation during the growth of L. lactis subsp. cremoris in milk. In that study it was ROL OF PPTID TRANSPORT IN L. LACTIS 6139 shown that L-phenylalanine and L-leucine stimulated the growth of L. lactis subsp. cremoris HP and 8. In view of the results presented in this paper, the growth limitation in milk is most probably caused by a limited supply of L-phenylalanine-containing di- or tripeptides. Because L. lactis ML3 is unable to take up proline (Table 2), this amino acid, which is the most abundant amino acid residue in,b-casein (17), most likely enters the cell exclusively in a peptide-bound configuration. This conclusion is supported by the observation that excess unlabeled L-prolyl-L-methionine inhibits the uptake of radioactively labeled L-alanyl-L-glutamic acid, indicating that prolyl peptides can be transported via the dipeptide transport system (. J. Smid, unpublished results). The results in this paper show that the synthesis of proteolytic enzymes is not the only fundamental requirement for rapid acid production in milk fermentations but that dipeptide transport is also an essential component in the process of casein utilization by lactococci during growth in milk. ACKNOWLDGMNTS We thank T. 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