INTRODUCTION

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Lactococci are widely used as starters in cheese and other fermented milk products, and their rapid growth in milk is essential to yield an appropriate rate of lactic acid production, optimum curd formation, required flavor, and texture development in the final products and for the inhibition of undesirable microorganisms (28). The ability of a lactococcal strain to coagulate milk within 16 to 18 h at 21°C with a 1% inoculum defines a fast milk-coagulating (Fmc⁺) strain used in cheese manufacture (45). An important metabolic function that influences the rapid growth of lactococci in milk is their proteolytic system, which is required to obtain the amino acids needed for growth to high cell densities. If components of this caseinolytic system are missing, the result can be a slow milk coagulation (Fmc) phenotype requiring up to 48 h or longer to coagulate milk, because the limited availability of utilizable amino acids does not allow growth to reach the prerequisite high cell densities.

The hydrolysis of casein by lactococci is initiated by a cell wall-associated proteinase (PrtP) which is responsible for the degradation of casein into oligopeptides. PrtP is synthesized as an inactive pre-pro-protein and requires processing by a maturation protein (PrtM) to exhibit activity (18). Three distinct amino acid transport systems, two di- and tripeptide transport systems, and an oligopeptide permease system are available for the uptake of amino acids and peptides by the cell. Intracellular peptidases then degrade the peptides into amino acids for growth. Lactococcal strains without the Prt plasmid, encoding prtP and prtM, exhibit an Fmc phenotype. The oligopeptide transport (Opp) system is also essential for the Fmc⁺ phenotype (24, 38, 45). Although it has been speculated that an extracellular peptidase(s) may be involved in further hydrolysis of the large oligopeptides released by the action of PrtP on casein into smaller units utilizable by the available transport systems (19, 32), recent data indicate that the existence of an extracellular peptidase is not likely (14, 15). The latter studies found that a large fraction of the oligopeptides generated by PrtP degradation of casein were in the range of 4 to 8 amino acid residues, small enough to be transported into the cell through the Opp system. While an Opp mutant of L. lactis was unable to utilize oligopeptides and grew poorly in milk, a di- and tripeptide transport-deficient mutant (DtpT) grew as well as the wild-type strain in milk (15). It was further demonstrated that the wild-type strain and the DtpT mutant accumulated -casein-derived amino acids inside the cells when -casein served as a protein source, but no significant accumulation of amino acids occurred with Opp and DtpT Opp mutants (21). These observations indicated that oligopeptides are the main nitrogen source for lactococcal growth in milk and that there is no necessity to postulate the existence of an extracellular peptidase (15).

Based on these results and the intracellular locations of the peptidases identified so far, reasonable doubts have arisen concerning the existence of a putative extracellular peptidase in lactococci. There is also no evidence for the existence of other elements involved in a functional proteolytic system in lactococci. Previous work (44) in our laboratory on L. lactis C2 and its Fmc derivatives provided putative genetic evidence for the possible requirement of another component for the Fmc⁺ phenotype. In this communication, we demonstrate that, in addition to a functional proteolytic system, pyruvate carboxylase activity is required to supply the cell with sufficient aspartate for the Fmc⁺ phenotype in L. lactis C2.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and culture conditions. Lactococcal strains and the plasmid used in this study are listed in Table 1. Cultures were propagated at 32°C in M17 broth containing 0.5% glucose (M17-G) or 0.5% lactose (M17-L) as the sole carbohydrate source (36). Cultures were stored at 4°C and maintained by biweekly transfers in M17 broth. The Fmc⁺ phenotype was determined as previously desribed (45). Fmc (Prt⁺ Opp⁺) KB4 was originally isolated as a Lac⁺ Fmc variant by growth of the parental strain, L. lactis C2, in lactose broth containing 6 µg of acriflavine per ml at 32°C for 20 h. The treated culture was plated on lactose indicator agar, and Lac⁺ colonies were screened for the inability to coagulate milk within 16 to 18 h at 21°C (27, 45).

DNA manipulations. Plasmid DNA isolation, total cellular DNA purification, agarose gel electrophoresis, and electroporations were done as previously described (44). Southern transfer and probe hybridization were performed as described by Sambrook et al. (35). Genius system nonisotope digoxigenin labeling and detection were done according to the manufacturer's directions (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). PCR was used to make probe DNA with total cellular DNA or plasmid DNA of L. lactis C2 as a template. Oligoprimers were synthesized with a PCR-MATE EP DNA synthesizer (Applied Biosystems, Foster City, Calif.). These included oligonucleotides 5'-TCTCGCAGCAAACTAAGG-3' and 5'-GGAAAGATTGGAAACTGG-3' for amplifying a prtP subfragment (20), 5'-GTGTTGCCTTTCGGATTG-3' and 5'-TTTTTCCAAGCCTGACCT-3' for amplifying an oppD subfragment (44), and 5'-GGTTTCAGTCGCAGTAGC-3' and 5'-CTTAGCAGTGTCCTCGTC-3' for amplifying a prtM subfragment (39). PCR was conducted with Taq DNA polymerase according to the manufacturer's instructions (Perkin-Elmer Corp., Norwalk, Conn.). Probes were labeled via 3'-end labeling with digoxigenin-11-ddUTP (Genius; Boehringer). Hybridizations and washings were performed at 65°C.

Proteinase assay. Cells were harvested, washed, and fractionated with a combination of lysozyme and mutanolysin in sucrose-MgCl₂ (24% sucrose-10 mM MgCl₂ in 50 mM Tris-HCl [pH 7.0]) without differentiating among loosely associated cell material, cell wall material, and cell wall components (4, 5). Proteinase was monitored with the substrate fluorescein isothiocyanate (FITC)-labeled -casein (37) as described by Coolbear et al. (3). Fructose bisphosphate aldolase activity was determined by the method of Crow and Thomas (6).

Proteolytic assay. Reconstituted nonfat milk (NFM) (11% [wt/vol] with 1% glucose) was inoculated (1%) with an overnight culture in M17-L or M17-G and incubated at 32°C for 12 h. A second 1% transfer was made into 11% NFM, and the mixture was incubated at 32°C for 36 h. Proteolysis of milk proteins was determined by the method of Hull (12).

Defined media and culture conditions for growth studies. The chemically defined medium (CDM) described by Otto et al. (29) was used to study the utilization of casein, peptides, and amino acids by the lactococcal strains.

Assay for GOT. Cell extract (CE) was prepared as described by Wahls (40). The activity of glutamate-oxaloacetate transaminase (GOT) was determined as described by Yagi et al. (43).

Incubation of cells with sodium [¹⁴C]bicarbonate. L. lactis C2 and KB4 were grown in M17-L at 30°C for 18 h. Cells from 10 ml of each of the cultures were collected by centrifugation. The pellets were resuspended in 2.5 ml of M17 broth supplemented with 40 µl of sodium [¹⁴C]bicarbonate (0.5 mCi/ml; 2 to 10 mCi of crystalline solid per mmol) and incubated at room temperature for 1 h. The cells were then collected and washed twice with 1 volume of 0.85% NaCl. The pellets were suspended in 100 µl of saline, and 40 µl was used to determine fixed ¹⁴C activity by use of a model LS5000TD liquid scintillation spectrometer (Beckman Instruments, Inc., Fullerton, Calif.).

Assay for pyruvate carboxylase. The pyruvate carboxylase activity of L. lactis was determined by the method of Renner and Bernlohr (34) with a GOT-coupled [¹⁴C]bicarbonate fixation assay. One milliliter of the reaction cocktail contained the following: CE, 0.3 ml; Tris-HCl (pH 7.5), 100 µmol; MnCl₂, 5 µmol; sodium pyruvate, 10 µmol; ATP, 5 µmol; NaH¹⁴CO₃, 1 µCi (0.5 mCi/ml; 2 to 10 mCi of crystalline solid per mmol); acetyl coenzyme A, 0.5 µmol; glutamate, 20 µmol; and GOT, 10 U. The reaction was terminated by the addition of 0.1 ml of 10% trichloroacetic acid. The activity of acid-stable ¹⁴C in the supernatant was measured by liquid scintillation counting. Ten micromoles of OAA or aspartate or 4 U of avidin was added to the reaction mixture to study the effects of each on pyruvate carboxylase activity. Ten micromoles of phosphoenolpyruvate (PEP) instead of pyruvate was used in the reaction mixture to study the substrate specificity of the carboxylase.

Protein assay. Protein concentrations in CEs were determined by use of a protein assay according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, Calif.).

Paper electrophoresis. Pyruvate carboxylase assay products were analyzed by paper electrophoresis (41). Whatman no. 3 paper strips (1 cm wide) were used as a solid support, and electrophoresis was carried out with 2% pyridine-0.95% acetic acid (wt/wt) buffer (pH 5.2) for 60 min at 20 V/cm. About 20 µl of the reaction samples was subjected to electrophoresis, and unlabeled aspartate was added to the samples before loading as an internal control for detection. The amino acids were visualized by spraying with ninhydrin (41). The stained strip was then cut into pieces 1 cm long, and radioactivity was determined by scintillation counting.

	RESULTS

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L. lactis KB4 is an Fmc derivative of L. lactis C2. L. lactis KB4 required up to 40 h to coagulate milk at 21°C, compared with 18 to 20 h for Fmc⁺ L. lactis C2, resulting in its classification as an Fmc derivative of C2. KB4 was also unable to grow in CDM with -casein as the sole nitrogen source (Fig. 1A).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Growth in CDM of Fmc+ (Prt+ Opp+) L. lactis C2 (), Fmc (Prt Opp+) L. lactis LM0230 (), and Fmc (Prt+ Opp+) L. lactis KB4 (). (A) CDM with casein as the sole amino acid source. (B) CDM with a pool of 20 amino acids. O.D., optical density.

Southern analysis and PCR detection of prtP, prtM, and oppD genes. As the absence of PrtP or Opp components involved in casein utilization can result in the Fmc phenotype, it was necessary to determine which, if any, of these components were missing in KB4. The prtP and prtM probes both hybridized to the 30-MDa Lac plasmid in KB4, indicating that these genes were located on this plasmid, as in L. lactis C2 (data not shown). PCR analysis also confirmed that KB4 carried the prtP and prtM genes. No corresponding prtM or prtP bands were detected in Fmc (Prt Opp⁺) L. lactis LM0230, the plasmid-free derivative of C2 (data not shown). PCR and Southern analyses of total cellular DNA indicated that the opp gene cluster was chromosomally located in KB4 (44).

L. lactis KB4 carries functional PrtP. As prtP and prtM were located on the Lac plasmid in KB4, it was necessary to determine if these genes were functional in this derivative. To examine the functionality of the prtP and prtM genes, the Lac plasmid from KB4 was isolated and electroporated into Prt Opp⁺ LM0230. The resulting transformants exhibited the Fmc⁺ phenotype, indicating that the prtP and prtM genes from KB4 were functional.

KB4 carries functional amino acid transport, di- and tripeptide transport, and functional oligopeptide permease systems. Growth studies showed that KB4 could grow in CDM containing a complete pool of amino acids (Fig. 1B) but not when leucine was omitted (Fig. 2). This result suggested that KB4 possessed functional amino acid transport systems and confirmed that Leu was an essential amino acid for the growth of KB4. When the leucine-deficient medium was supplemented with the leucine-containing dipeptide Leu-Gly or the tripeptide Leu-Gly-Gly, KB4 was also able to grow (Fig. 2). This result suggested that KB4 contained functional di- and tripeptide transport systems as well as functional intracellular peptidases to hydrolyze the peptides into free amino acids needed for growth.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Growth in CDM of Fmc (Prt+ Opp+) L. lactis KB4 with various leucine sources: CDM without leucine () and CDM without leucine but containing the dipeptide Leu-Gly (), the tripeptide Leu-Gly-Gly (), or the pentapeptide Phe-Leu-Glu-Glu-Leu (). O.D., optical density.

KB4 is an aspartate auxotrophic mutant. The amino acid requirements were compared for Fmc⁺ (Prt⁺ Opp⁺) C2, the Fmc (Prt⁺ Opp⁺) derivative KB4, and the Fmc (Prt Opp⁺) derivative LM0230. All three strains grew in SSM when supplemented with the complete amino acid pool (Fig. 3A). C2 and LM0230 were also able to grow in SSM containing just nine essential amino acids. KB4, however, was unable to initiate growth in this medium (Fig. 3B). To further characterize the specific amino acid requirements of KB4, the remaining 11 amino acids were divided into five groups according to common biosynthetic pathways (group 1, Gln and Pro; group 2, Asp, Asn, and Lys; group 3, Cys and Gly; group 4, Ala; and group 5, Phe, Tyr, and Trp). KB4 was inoculated into SSM supplemented with each of the five groups of amino acids. Supplementation with group 2 resulted in the growth of KB4 (Fig. 4A). Supplementation with aspartic acid or asparagine was then found to meet the nutritional requirement of KB4 (the cell density reached a level similar to that of the parental strain C2 in SSM) (Fig. 4B). Thus, KB4 appeared unable to meet its aspartate requirement from casein hydrolyzed by PrtP. This conclusion was further supported by the observation that KB4 was able to coagulate milk within 18 h when the milk was supplemented with aspartic acid or asparagine. The pH decrease for KB4 in aspartate-supplemented milk was comparable to that for L. lactis C2 with or without aspartate (Fig. 5).

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Growth of L. lactis C2 (), LM0230 (), and KB4 () in SSM with all 20 amino acids (A) or with nine essential amino acids (Arg, Glu, His, Ile, Leu, Met, Ser, Thr, and Val) (B). O.D., optical density.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Growth of L. lactis KB4 in SSM supplemented with different groups of amino acids. (A) Group 1, Gln and Pro (); group 2, Asp, Asn, and Lys (). For SSM supplemented with group 3 (Cys and Gly), group 4 (Ala), and group 5 (Phe, Tyr, and Trp), the growth of KB4 resembled that exhibited with group 1 amino acids. The growth of L. lactis C2 in SSM supplemented with group 2 amino acids () was included as a control. (B) Individual amino acid components from group 2: Asp (), Asn (), and Lys (). O.D., optical density.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Acid development by L. lactis C2 and KB4 in milk with or without added aspartate. Symbols: , KB4 in milk without added Asp; , KB4 in milk supplemented with Asp; , C2 in milk without added Asp; , C2 in milk supplemented with Asp.

KB4 uses various sources of aspartic acid. Because casein contains about 7% (wt/wt) aspartate or asparagine and KB4 was shown to possess a functional proteolytic system able to utilize casein as a nitrogen source, it was necessary to confirm that KB4 possessed the essential components to utilize aspartate-containing peptides, assuming that they were produced during PrtP hydrolysis of casein. KB4 was grown in SSM supplemented with an aspartate-containing dipeptide or oligopeptide. The results indicated that the aspartate-containing peptides Asp-Gly and Asp-Ser-Asp-Pro-Arg were able to support the growth of KB4 and implied that KB4 had functional peptide transport systems as well as intracellular peptidases necessary for their utilization (Table 3). KB4 could not use the oligopeptide Gly-Asp-Asp-Asp-Asp-Lys as an aspartate source (Table 3). This result could have been due to its inability to transport the oligopeptide into the cell or to hydrolyze the oligopeptide within the cell. Common biosynthetic precursors of aspartate were also added to SSM, and their effects on KB4 growth were examined (Table 3). The data suggested that none of these precursors was able to replace aspartate as a growth requirement for KB4.

KB4 is deficient in CO₂ fixation. To further identify the defect in KB4, activities were determined for two key enzymes directly linked to aspartate biosynthesis in lactococci. GOT is responsible for transferring the NH₂ group from glutamate to OAA to form aspartate. It was possible that this enzyme was defective in KB4. However, it was found that the GOT activities of L. lactis C2 and KB4 were similar (Table 4).

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Distribution of acid-stable 14C in pyruvate carboxylase activity assay products from L. lactis C2 () and KB4 (), as determined by paper electrophoresis.

	DISCUSSION

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Fast milk-coagulating ability is one of the characteristics of a successful cheese starter. Recent findings have shown that the loss not only of PrtP but also of Opp can cause the Fmc phenotype (22, 30, 45). In addition, it was reported that an aminopeptidase pepA mutant had a lower acidification rate in milk (13), which might also contribute to an Fmc phenotype. Evidence has also been presented that an extracellular peptidase is not required for the hydrolysis of oligopeptides into di- and tripeptides in order for lactococci to utilize casein as a nitrogen source (21).

During the examination of L. lactis C2 derivatives for the opp gene cluster, we observed that KB4 possessed a Lac⁺ Fmc phenotype. KB4 was unable to grow in CDM containing casein as the sole nitrogen source but grew if the medium was supplemented with a pool of 20 amino acids. This result was initially misleading, because it suggested that KB4 was missing an unidentified component necessary for the cell to further break down casein hydrolytic products into smaller peptides for the available transport systems. It is believed that casein contains all of the amino acids essential for lactococcal growth (22) and should be able to provide the nitrogen requirement for KB4. However, upon examining the amino acid requirements, we found that, unlike C2, KB4 required aspartate for growth and pyruvate carboxylase activity was defective. Being deficient in CO₂ fixation, OAA formation, and, hence, aspartic acid formation, KB4 became an aspartate auxotroph. KB4 was able to grow when SSM was supplemented with the dipeptide Asp-Gly or the pentapeptide Asp-Ser-Asp-Pro-Arg, demonstrating that KB4 carried functional transport systems and intracellular peptidases. KB4 was not able to grow in OAA-supplemented SSM, possibly because it lacked the proper transport system or because this compound was unstable in solution (26, 42).

Surprisingly, LM0230 grew to a higher cell density in SSM containing nine amino acids than did C2 or KB4. The reason for this phenomenon remains to be elucidated. However, supplementing SSM with aspartate or asparagine complemented the growth defect in KB4 and allowed this mutant to grow to cell densities comparable to those of the parental strain C2.

It is interesting that milk casein contains approximately 7% aspartate or asparagine but is still insufficient to meet the aspartate growth requirement of KB4. Several possibilities could account for this phenomenon. KB4 may be unable to utilize some of the casein-derived aspartate-containing oligopeptides due to the absence of appropriate transport systems (the transport systems may have preference for certain types of peptides) or may lack the intracellular peptidases needed to hydrolyze these peptides. The inability of KB4 to utilize aspartate from the oligopeptide Gly-Asp-Asp-Asp-Asp-Lys (Table 3) agrees with the assessment given above. Recently, Kunji et al. (23) also demonstrated that some peptidase-deficient mutants could not use certain Leu-containing tripeptides or oligopeptides because of the inability to hydrolyze these peptides intracellularly. This finding also supports the assumptions made above.

Another possibility is that the amount of aspartate derived from casein hydrolysis is insufficient to support lactococcal growth. As aspartate can be deaminated in vivo to form OAA, the amount of exogenous aspartate needed for KB4 actually reflects the sum of requirements for both aspartate and OAA in vivo. Further studies are needed to determine the quantity of aspartate required for the growth of lactococci. Previous reports demonstrated that aspartate may not be actively transported into L. lactis (10) or Leuconostoc mesenteroides (25). The mechanism by which KB4 is able to take up aspartate for growth needs to be investigated. The influence of pH on the transport of aspartate and asparagine in C2 and KB4 should also be considered (31).

It is known that CO₂ is required for the optimum growth of lactococci in milk (16, 33). During a study of [¹⁴C]bicarbonate incorporation into L. lactis C10, Hillier and Jago (8) demonstrated that radioactivity was incorporated into the protein and nucleic acid fractions of the cell as well as into compounds which were excreted by the organism into the medium. The fixation of [¹⁴C]bicarbonate by L. lactis C10 was achieved by the combined reactions of pyruvate carboxylase and GOT to form aspartate. Resting cells of L. lactis C10 were able to synthesize aspartate de novo but could not actively transport aspartate into the cell (8-10). Our data on the pyruvate carboxylase-deficient L. lactis KB4 further illustrate the importance of this in vivo OAA biosynthesis pathway in lactococci. We are interested in KB4 because of the possible role of pyruvate carboxylase in coordinating metabolism in lactococci. OAA and aspartate not only are precursors for five other amino acids and thus critical for protein synthesis but also are involved in the biosynthesis of purines and pyrimidines. OAA is also a common intermediate for carbon metabolism, although lactococci do not have a complete tricarboxylic acid cycle. Thus, theoretically, pyruvate carboxylase is in a key position for these critical metabolic activities. Further studies of the pyruvate carboxylase gene and its expression are currently under way and might provide insights into metabolic cooperation and regulatory mechanisms in lactococci.

Study of the pyruvate carboxylase-deficient mutant L. lactis KB4 may also lead to information having industrial significance. Pyruvate is a key metabolic intermediate in lactic acid bacteria, and the flow of pyruvate into various pathways is tightly controlled. Because of the central position of pyruvate in sugar metabolism and especially its involvement in the production of flavor compounds such as diacetyl, studies of pyruvate metabolism recently have been greatly expanded (2). However, among these studies of pyruvate metabolic pathways, the drainage of pyruvate through OAA biosynthesis has been ignored. Thus, knowledge of the significance of pyruvate carboxylase will further benefit our understanding of the pyruvate pool in lactococci and may stimulate new ideas for bioengineering starter strains with enhanced diacetyl production. The unusual property of KB4 is that it is defective in pyruvate carboxylase yet can take up sufficient aspartate from the medium to fulfill its growth requirements. Thus, genetically it provides a model for generating a bioengineered strain in which one of the pathways for pyruvate drainage is blocked but which is still able to maintain other regular physiological properties in the presence of aspartate. Continuing the study of the biochemistry and molecular biology of pyruvate carboxylase in lactococci could have potential industrial applications.