INTRODUCTION

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Lactobacillus helveticus is used in the dairy industry with other lactic acid bacteria (LAB) as starter cultures to produce fermented milk, sour milk, and Swiss- and Italian-type cheeses. A useful guide to the suitability of these bacteria for cheese-making is their ability to coagulate autoclaved milk within 16 h at 42°C when used as a 1% freshly coagulated inoculum (35), a property which defines a fast milk-coagulating (Fmc⁺) strain (an Fmc strain requires more than 30 h of incubation for coagulating autoclaved milk at 42°C). Thus, the ideal starter culture should rapidly and dependably produce lactic acid during growth in milk. For this, LAB depend on their ability to metabolize lactose and on the presence of a complete proteolytic system which allows the efficient degradation and utilization of casein, the major milk protein. The specialized proteolytic system of these microorganisms consists of a cell envelope-associated proteinase, transport systems to allow uptake of the resultant amino acids and peptides, and several intracellular peptidases which degrade peptides to amino acids (17).

In Lactococcus, the conversion of an Fmc⁺ phenotype to an Fmc phenotype has mainly been attributed to loss of either a cell wall-bound proteinase (PrtP) or the enzyme(s) required for lactose utilization and can be due to loss of a plasmid coding for these enzymes (16, 20). Yu et al. (36) found that the Fmc phenotype in Lactococcus lactis could also be due to loss of a plasmid encoding an oligopeptide permease system. In addition, it was reported that an aminopeptidase (encoded by pepA) is required for optimal growth of L. lactis in milk (19). Recent findings have shown that Lactococcus lactis subsp. lactis C2 deficient in aspartate synthesis exhibited an Fmc phenotype (35). When any of these properties are lost from the cells, the ability to synthesize lactic acid is also slowed and the cells are no longer effective starter organisms. Despite the industrial importance of thermophilic lactobacilli (e.g., L. helveticus) as dairy starters, information about the nature of the Fmc phenotype in these microorganisms is limited.

L. helveticus strains show phenotypic and genotypic variability (7, 8, 10, 11). Fortina et al. (7) reported that a wide phenotypic variability exists among L. helveticus strains isolated from natural cheese starters. Cell heterogeneity affected different phenotypes such as resistance to lysozyme (7, 34), sugar fermentation, phage resistance, and proteolytic activity (7, 29). Morelli et al. (23) and Reinheimer et al. (29) reported the presence of spontaneous Fmc isolates within the L. helveticus strains HLM 1 and ATCC 15807, respectively, and have suggested the possibility of a linkage between casein hydrolysis and the Fmc phenotype. In previous studies, we have demonstrated that L. helveticus CRL 1062, a starter used for the manufacture of Argentinian hard cheeses, exhibits an Fmc⁺ phenotype (13). L. helveticus CRL 1062 is auxotrophic for aspartate (14); therefore, this strain may be able to utilize some of the casein-derived aspartate-containing oligopeptides. In this work, we describe the isolation and characterization of strain S1, a slow-milk-coagulation variant of L. helveticus CRL 1062. We show that, in addition to a functional proteolytic system, L. helveticus CRL 1062 requires an IMP dehydrogenase activity to exhibit an Fmc⁺ phenotype. This enzyme is necessary to provide sufficient GMP to allow the organism to grow to high cell densities in milk.

	MATERIALS AND METHODS

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Microorganisms, media, and growth conditions. L. helveticus CRL 1062 was obtained from CERELA (Centro de Referencia para Lactobacilos, Argentine). Cultures were stored at 70°C in 10% sterile reconstituted skim milk (RSM) containing 0.5% yeast extract and 10% glycerol and were reactivated in MRS (1) broth at 42°C for 16 h. Slowly milk-coagulating variants of L. helveticus CRL 1062 were isolated on casein medium (CM). CM is a modification of the casein-based medium described by Morelli et al. (23), where the concentrations of tryptic digest of casein, yeast extract, and MnSO₄ · H₂O were reduced five times. CM contains the following (in grams per liter): tryptic digest of casein, 1; yeast extract, 0.4; Tween 80, 1; cysteine, 0.2; MgSO₄ · 7H₂O, 0.2; MnSO₄ · H₂O, 0.04; sodium caseinate, 1; trisodium citrate, 4.4; glucose, 10; and agar, 12.

Coagulation tests. Washed cells, prepared as described above, were used to inoculate (1%) RSM, RSM supplemented with 1% glucose (RSM-G), and RSM supplemented with 0.25% yeast extract (RSM-YE). Cells were then incubated at 42°C for 16 h.

Cell suspensions and CE. Cells grown in the different media were harvested by centrifugation (10,000 × g, 15 min, 4°C) at the mid-exponential growth phase (OD₅₆₀ = 0.65), washed twice with 0.85% (wt/vol) NaCl supplemented with 10 mM CaCl₂, and resuspended to a final OD₅₆₀ of approximately 10 in 100 mM sodium phosphate buffer (pH 7.0). The washed whole cells were allowed to utilize the residual sugar and intracellular amino acids for 30 min at 42°C. Cell extracts (CE) were obtained by vortexing the bacterial cell suspensions with glass beads (0.15- to 0.25-mm diameter; Sigma) at a ratio of 1:1 and then kept on ice for 1 min each. This step was repeated seven times. Glass beads, cell debris, and unbroken cells were removed by centrifugation (10,000 × g, 10 min, 4°C).

Enzyme assays. Proteinase (PrtH) activity of whole-cell suspensions was measured in 50 mM phosphate buffer, pH 7.0, at 42°C with the chromogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroaniline (S-Ala; Sigma) as described by Exterkate (5). PrtH activity was measured from the initial part of the progress curve, where release of the p-nitroaniline was linear with time. One unit of proteinase was defined as the amount required to liberate 1 nmol of nitroanilide per min. Specific activity was expressed as proteinase units per milligram of protein. Cell lysis was determined by following the release of lactate dehydrogenase by the method of Thomas (33).

Casein hydrolysis. Washed cells, harvested from CDM, were suspended in 100 mM sodium phosphate buffer (pH 7.0) and allowed to utilize the residual intracellular amino acids for 30 min at 42°C. Casein degradation was conducted as described previously (12). Washed whole cells (OD₅₆₀ = 10) were incubated with 5 mg of substrate/ml dissolved in 100 mM phosphate buffer (pH 7.0) at a ratio of 1:1 (vol/vol). - or -casein (Sigma) was used as the substrate. The resulting mixtures were incubated at 42°C for 3 h, the samples were centrifuged, and the supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (18). Either Coomassie brilliant blue R-250 or silver staining (Bio-Rad Laboratories, Hercules, Calif.) was used to visualize the proteins after SDS-PAGE.

Protein determination. Protein concentration was determined by using a protein assay according to the manufacturer's instructions (Bio-Rad).

DNA isolation, amplification, and sequencing. Genomic DNA of L. helveticus was isolated as described previously (28). The primers (A2, 5'-GTTATCTCTGCTGGGAACTC-3', and A4, 5'-GAAAAAGCCCATGTATGG-3') were designed from an alignment of the conserved regions of the proteinase genes from different LAB, and they were used to amplify the region surrounding the active-site residues of the proteinase gene of L. helveticus. All primers were synthesized by Gibco-BRL Custom Primers (Grand Island, N.Y.). The PCR assay was performed with 50 µl containing 30 ng of bacterial DNA, 2.5 mM MgCl₂, 100 µM concentrations of each of four deoxynucleoside triphosphates, 1 µM concentrations of each primer in Taq buffer (Gibco BRL), and 2.5 U of Taq polymerase (Gibco BRL). PCR was carried out in a programmable heating incubator (Perkin-Elmer Corp., Norwalk, Conn.) for 30 cycles. The cycling program used was 30 cycles of 94°C (1 min), 50°C (1 min), and 72°C (1.5 min). Amplification products were analyzed by electrophoresis in 1% agarose gels containing 200 µg of ethidium bromide (Sigma)/liter. PCR products were purified with a Prep-A-Gene master kit (Bio-Rad), and DNA sequencing was performed by the BiorResource Center (Ithaca, N.Y.).

	RESULTS

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Isolation of an Fmc derivative from L. helveticus CRL 1062. Cell heterogeneity within L. helveticus strains HLM 1 (23) and ATCC 15807 (29) has been described. With L. helveticus CRL 1062, we initially failed to isolate Fmc variants using the casein-based medium described by Morelli et al. (23). In this medium, regardless of the size of single colonies of L. helveticus CRL 1062, all tested isolates (from large and small colonies) coagulated RSM in 16 h at 42°C. This could be due to the fact that this isolation medium contains yeast extract (2%) and tryptic digest of casein (5%) in addition to sodium caseinate (1%). In addition to serving as a nitrogen source, the first two compounds might also provide essential nutrients for optimal growth of the Fmc variants of CRL 1062 strain likely present in the culture. However, in the new CM, where the concentration of tryptic digest of casein and yeast extract was reduced fivefold, large (>2.5 mm) colonies, surrounded by a white precipitation halo, and pinpoint colonies could be ascribed to the Fmc⁺ and Fmc phenotypes, respectively. Fmc variants were observed with a frequency of 1%. An Fmc derivative, designated S1, was isolated and further characterized.

Growth characteristics and -galactosidase and proteinase activities of S1. L. helveticus S1 required up to 30 h to coagulate RSM milk at 42°C; therefore, this strain exhibited an Fmc phenotype. As the ability of LAB to coagulate RSM depends mainly on their capacity to metabolize lactose and to hydrolyze casein, it was necessary to determine which, if any, of these components were missing in L. helveticus S1. Addition of yeast extract, but not glucose, to RSM restored the Fmc⁺ phenotype of S1 (Table 1). Wild-type strain CRL 1062 and its derivative S1 displayed similar -galactosidase activities (Table 1). Furthermore, these strains had similar levels of proteinase activity (evaluated on the chromogenic substrate S-Ala) (Table 1) and exhibited identical hydrolytic patterns with - and -casein as substrates (Fig. 1). PCR analysis also confirmed that S1 carried the proteinase genes of L. helveticus (data not shown). These data indicate that in S1, lactose metabolism and proteinase activity are functional.

View this table: [in this window] [in a new window]   TABLE 1.   Growth characteristics and -galactosidase and proteinase activities of L. helveticus CRL 1062 and its slowly milk-coagulating variant S1

View larger version (76K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE analysis of -casein (A) and -casein (B) hydrolysis by L. helveticus CRL 1062 (lane 2) and S1 (lane 3) after growth in CDM. Lane 1, starting substrates.

Peptide transport systems of L. helveticus S1. L. helveticus strains are auxotrophic for leucine (14, 24). To establish the role of di-, tri-, and oligopeptide transport systems in the utilization of casein-derived peptides, cell growth of L. helveticus CRL 1062 and its variant S1 was evaluated in leucine-free CDM supplemented with several leucine-containing peptides and compared to their growth in CDM. No differences in growth rate were observed between CRL 1062 and S1 in CDM (Table 2) and in leucine-deficient CDM containing either the dipeptide Leu-Pro, the tripeptide Leu-Gly-Gly, or the oligopeptide Leu-Leu-Val-Tyr-Ser. These results indicated the functionality of the amino acid and di-, tri-, and oligopeptide transport systems as well as the functionality of the intracellular peptidases which were able to utilize these peptides as a leucine source.

Nutritional requirements of L. helveticus S1. CRL 1062 and S1 (Fmc⁺ and Fmc, respectively) exhibited similar growth rates in CDM. However, the growth rate of S1 in SCDM was threefold lower than that of the parental strain (Table 2). CDM is an SCDM-based medium supplemented with a pool of bases (guanine, adenine, uracil, inosine, and orotic acid) and vitamins (folic acid, vitamin B₁₂, thiamine, biotin, and p-aminobenzoic acid). To characterize the specific base and vitamin requirements of S1 with respect to the wild-type strain, the nucleotides and vitamins were divided into six groups (Table 2). The strains were then grown on SCDM supplemented with each of the six groups of vitamins and nucleotides. No changes in the growth rate were observed when the variant S1 was cultured in SCDM supplemented with group A, C, D, or E (Table 2). However, the supplementation of SCDM with group B or F stimulated the growth of S1, and this growth rate was comparable to that obtained on CDM (Table 2). These results suggested that synthesis of guanine was affected in L. helveticus S1, since this base was present in groups B and F. This was confirmed when the growth rate of S1 was determined in SCDM supplemented with guanine, inosine, or adenine (Fig. 2). S1 grew at the same rate as the parental strain on SCDM containing guanine, but not on SCDM supplemented with adenine or inosine. Furthermore, the S1 growth rate in the presence of guanine was identical to that observed in SCDM supplemented with group B (Fig. 2).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Growth of L. helveticus CRL 1062 () and S1 () in SCDM (A), SCDM supplemented with adenine, guanine, and inosine (B), and SCDM plus adenine (C), guanine (D), or inosine (E). Cell growth was expressed as ln x/x0, where x0 is biomass produced at initiation of the experiment and x is biomass at the indicated time. Values are averages from three independent experiments.

Guanine biosynthesis in L. helveticus CRL 1062 and S1. To further identify the defect in S1, the activities of two key enzymes directly linked to guanine biosynthesis were determined. GMP is derived from IMP in two steps (25, 37) (Fig. 3). The first step is NAD-dependent oxidation of IMP catalyzed by IMP dehydrogenase (encoded by guaB), and the product formed is XMP. In the second step, the amidotransferase GMP synthetase (encoded by guaA) catalyzes the amidation of XMP in a glutamine- or NH₃-dependent process requiring ATP. The values of GMP synthetase were similar in both strains (12 ± 0.4 and 11 ± 0.3 µmol/min/mg of protein for CRL 1062 and S1, respectively). In contrast, IMP dehydrogenase activity in CE of S1 strain was 14 ± 0.6 µmol/min/mg of protein, which is five times lower than that exhibited by the parental strain, CRL 1062 (66 ± 3.0 µmol/min/mg of protein), suggesting that S1 was defective in this enzyme. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc⁺ phenotype of L. helveticus S1 (Fig. 4). Thus, the pH decrease for S1 strain in RSM supplemented with xanthine or guanine was similar to that obtained for L. helveticus CRL 1062 with or without bases (Fig. 4). On the other hand, the addition of inosine or adenine to milk did not affect the coagulation rate of this strain (Fig. 4).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Metabolic pathways potentially involved in the biosynthesis and interconversion of purines in L. helveticus CRL 1062. Enzymes assayed in this study are identified by their gene symbols: guaB, IMP dehydrogenase; guaA, GMP synthetase. AICAR, aminoimidazolecarboxalamide ribonucleotide; APRT, adenine phosphoribosyltransferase; GPRT, guanine phosphoribosyltransferase; HPRT, hypoxanthine phosphoribosyltransferase. Data were adapted from reference 33.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Rates of acidification during cultivation of L. helveticus S1 in RSM without nucleotides (), L. helveticus S1 in RSM supplemented with inosine (), adenine (), xanthine (), or guanine (), and L. helveticus CRL 1062 in RSM ().

	DISCUSSION

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It has been reported that rapid growth and concomitant fast acid production of L. helveticus in milk, a substrate with a low content of free essential amino acids and peptides (22), depend mainly upon a proteolytic system which allows degradation of milk proteins (caseins) and on -galactosidase activity (15). In this sense, most of the Fmc variants of L. helveticus so far characterized have been putatively associated with a deficiency in proteolytic activity (2, 6, 23, 29). In the present study, however, strain S1, an Fmc variant of L. helveticus CRL 1062, possessed functional -galactosidase and proteinase activities. Furthermore, strain S1 was able to grow in leucine-free CDM supplemented with di-, tri-, or oligoleucine-containing peptides as the sole source of leucine, indicating the presence in L. helveticus S1 of functional di-, tri-, and oligopeptide transport systems as well as functional intracellular peptidases. L. helveticus CRL 1062 (and hence S1) is auxotrophic for aspartate (14). Therefore, and in contrast to L. lactis C2 (35), the deficiency in aspartate biosynthesis in strain S1 was not responsible for its Fmc phenotype.

Milk is also deficient in purine and pyrimidine compounds (3, 26, 31). The purine nucleotides can be formed by de novo biosynthesis, which requires in general 10 enzymatic steps leading to IMP, and by salvage reactions from purine nucleosides and bases (25, 37) (Fig. 3). A defective pathway of de novo purine biosynthesis seems to be common in most of the homofermentative lactobacilli (4). However, this study suggests that in CRL 1062, the de novo biosynthetic pathway for purine nucleotides is functional (CRL 1062 grew on SCDM) and is necessary to support fast acid production in milk and for the population to reach high cell densities. Like L. lactis (25), L. helveticus CRL 1062 was capable of converting adenine, guanine, and inosine to AMP, GMP, and IMP, respectively, indicating the existence of adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase activities. It also revealed that L. helveticus S1 lacks the ability to convert IMP into adequate amounts of XMP, a precursor of GMP, which is due to a reduced IMP dehydrogenase activity.

Plasmid instability has explained certain intrastrain variations of L. helveticus (2, 23, 30). However, both CRL 1062 and S1 are plasmid free (13). Therefore, the observed phenotypic variability does not appear to be linked to variations in plasmid DNA content. It has also been reported that spontaneous and phenotypically stable mutations in the -galactosidase locus of L. helveticus strains were caused by integration of ISL2 into the gene (38). L. helveticus CRL 1062 contains both IS1201 (32) and ISL2 sequences in its chromosomal DNA (unpublished data). Comparison of Southern blot patterns hybridized with an internal fragment of ISL2 revealed no visible differences between the S1 and wild-type strains (data not shown). These results suggest that the purine auxotrophy of S1 is due either to a point mutation or to small DNA rearrangements which were not detectable on the Southern blots. The alteration affecting the ability to synthesize guanine appears to result in the production of an altered IMP dehydrogenase with a partial loss of catalytic activity.

The simplified CM described in this work was also used to isolate Fmc derivatives from Lactobacillus delbrueckii subsp. lactis CRL 581. Preliminary characterization of these Fmc strains also showed that they were defective in the de novo synthesis of purine nucleotides, leading to IMP (unpublished data). We are currently investigating whether CM is suitable for isolating slowly milk-coagulating variants from other LAB.