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Microbiology (2002), 148, 3413-3421.
Research Paper

Cell-wall proteinases  PrtS and PrtB have  a different role  in Streptococcus thermophilus / Lactobacillus bulgaricus mixed cultures in milk

P. Courtin¹, V. Monnet¹ and F. Rul¹
 

Unité de Biochimie et Structure des Protéines, INRA, 78352 Jouy-en-Josas Cedex, France¹

Author for correspondence: F. Rul. Tel: +33 1 34 65 21 48. Fax: +33 1 34 65 21 63. e-mail: rul@jouy.inra.fr

 
The manufacture of yoghurt relies on the simultaneous utilization of two starters: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus). A protocooperation usually takes place between the two species, which often results in enhanced milk acidification and aroma formation compared to pure cultures. Cell-wall proteinases of Lactococcus lactis and lactobacilli have been shown to be essential to growth in milk in pure cultures. In this study, the role of proteinases PrtS from S. thermophilus and PrtB from Lb. bulgaricus in bacterial growth in milk was evaluated; a negative mutant for the prtS gene of S. thermophilus CNRZ 385 was constructed for this purpose. Pure cultures of S. thermophilus CNRZ 385 and its PrtS-negative mutant were made in milk as well as mixed cultures of S. thermophilus and Lb. bulgaricus: S. thermophilus CNRZ 385 or its PrtS-negative mutant was associated with several strains of Lb. bulgaricus, including a PrtB-negative strain. The pH and growth of bacterial populations of the resulting mixed cultures were followed, and the Lactobacillus strain was found to influence both the extent of the benefit of Lb. bulgaricus/S. thermophilus association on milk acidification and the magnitude of S. thermophilus population dominance at the end of fermentation. In all mixed cultures, the sequential growth of S. thermophilus then of Lb. bulgarius and finally of both bacteria was observed. Although proteinase PrtS was essential to S. thermophilus growth in milk in pure culture, it had no effect on bacterial growth and thus on the final pH of mixed cultures in the presence of PrtB. In contrast, proteinase PrtB was necessary for the growth of S. thermophilus, and its absence resulted in a higher final pH. From these results, a model of growth of both bacteria in mixed cultures in milk is proposed.

Keywords: bacterial growth, milk fermentation, thermophilic bacteria

Abbreviations: FSDA, Fast Slow Difference Agar; LAB, lactic acid bacterium/bacteria

 
Streptococcus thermophilus is a thermophilic lactic acid bacterium (LAB), widely used as a starter to produce fermented dairy products. It is generally used in association with other micro-organisms, in particular with Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) for the manufacture of yoghurt. For this application, the fast-growing capacity of these bacteria in milk is crucial to enable intense and rapid acidification of milk. LAB are fastidious micro-organisms, which have in particular several amino acid auxotrophies. Most S. thermophilus strains are stimulated by the supply of two to five amino acids (Bracquart & Lorient, 1977 ; Letort & Juillard, 2001 ; Shankar & Davies, 1977 ), whereas lactobacilli require between three and 14 amino acids (Hebert et al., 2000 ; Ledesma et al., 1977 ; Morishita et al., 1981 ). The optimal growth of LAB in milk thus depends on their proteolytic system, which hydrolyses milk caseins into peptides and amino acids (Thomas & Mills, 1981 ). The cell-wall proteinases of LAB are of major importance in this process, as they are responsible for the first step of casein breakdown. They belong to the same multi-domain proteinase family and show significant homologies, even though differences in specificity, bacterial anchor and domain organization have been described (Fernandez-Espla et al., 2000 ; Siezen, 1999 ). The cell-wall proteinase of Lactococcus lactis (PrtP), which is very frequent in this species, has been extensively studied. In milk, Lc. lactis PrtP-negative strains only reach 10% of the cell densities observed with PrtP-positive strains (Thomas & Mills, 1981 ). In S. thermophilus, the presence of a cell-wall proteinase, PrtS, recently characterized, is less common than in Lc. lactis. In this species, high cell-wall proteinase activities are associated with high milk-acidifying capacities (Shahbal et al., 1991 ). In Lb. bulgaricus, the cell-wall proteinase, PrtB, is also essential for optimal growth in milk; a proteinase-negative strain reaches only 22% of the final biomass of a proteinase-positive strain when grown in milk (Gilbert et al., 1997 ).

In yoghurt, S. thermophilus and Lb. bulgaricus are grown in association, which often results in a positive interaction. This relationship, called protocooperation, has a beneficial effect on growth of both species and on acid and aroma production. S. thermophilus indeed produces pyruvic acid, formic acid and CO₂ (for reviews, see Tamine & Robinson, 1999 ; Zourari et al., 1992 ), which stimulate the growth of Lb. bulgaricus. In turn, Lb. bulgaricus produces peptides and amino acids that stimulate S. thermophilus growth (Accolas et al., 1971 ; Bautista et al., 1966 ; Higashio et al., 1977 ; Pette & Lolkema, 1950b ; Radke-Mitchell & Sandine, 1984 ), which correlates with a lower proteolytic capacity of S. thermophilus compared to Lb. bulgaricus (Hamdy et al., 1955 ; Hickey et al., 1983 ; Rajagopal & Sandine, 1990 ; Shankar & Davies, 1978 ).

In the present study, we wished to determine the role of the cell-wall proteinases PrtS from S. thermophilus and PrtB from Lb. bulgaricus in bacterial growth in milk. We therefore constructed a negative mutant for the cell-wall proteinase gene (prtS gene) of S. thermophilus CNRZ 385, which was recently sequenced and characterized in our laboratory (Fernandez-Espla et al., 2000 ). The latter mutant was used to study the role of PrtS on the growth of S. thermophilus in pure culture in milk. We also took advantage of the availability of a PrtB-negative mutant of Lb. bulgaricus (Gilbert et al., 1997 ) to evaluate the role of cell-wall proteinases PrtS and PrtB on growth and thus on acidification in S. thermophilus/Lb. bulgaricus mixed cultures.

 
Plasmids, bacterial strains, culture conditions and bacterial enumeration.
The bacterial strains and plasmids used for this study are presented in Table 1.

 
Strains of S. thermophilus and Lb. bulgaricus were grown in three different media: reconstituted skim milk (Nilac Low Heat Milk powder, NIZO) heated for 10 min at 95 °C, supplemented with yeast extract (3 g l^-1; Difco) when required, M17 medium (Terzaghi & Sandine, 1975 ) supplemented with 20 g lactose l^-1, and MRS medium (De Man et al., 1960 ) supplemented with 20 lactose g l^-1 and acidified at pH 5·2, supplemented with streptomycin (Sigma) (2 mg ml^-1) when required. The Escherichia coli strain was grown at 37 °C on Luria-Bertani (Difco) medium (Sambrook et al., 1989 ) with shaking, in the presence of erythromycin (Ery) (150 µg ml^-1) when required.

Stock cultures of each strain of S. thermophilus and Lb. bulgaricus were prepared after growth at 42 °C on skim milk, supplemented with yeast extract for proteinase-negative strains, from overnight skim milk cultures, supplemented with yeast extract when required. The pH was then measured, and bacterial numbers were estimated by plating, with an automatic spiral plater (AES Laboratory), appropriate dilutions of the culture on agar medium: M17Lac was used for specific enumeration of S. thermophilus cells, and MRSLac pH 5·2, supplemented with streptomycin when required, for specific enumeration of Lb. bulgaricus cells. For S. thermophilus strains and before dilution, chains of cells were disrupted for 30 s in a mechanical blender (Turax X620, Labo-Moderne). After 48 h incubation at 42 °C in anaerobic jars (Anaerocult A, Merck), cells were enumerated with the EC1 colony counter software (AES Laboratory). At the end of culture, bacteria were directly frozen in liquid nitrogen and kept at -80 °C.

Growth rates of S. thermophilus 385 and 385-PrtS strains were determined in M17 at 42 °C using a Microbiology Reader Bioscreen C (Labsystems) in 100-well, sterile, covered microplates. Each well, containing 200 µl M17Lac, was inoculated at 1% with overnight M17Lac cultures of S. thermophilus and covered with one drop of paraffin oil. The optical density was measured at 600 nm every 20 min, after gentle shaking. The apparent growth rate (µ_max) was defined as the maximum slope of semi-logarithmic representation of growth curves, assessed by optical density measurements.

Mixed cultures of S. thermophilus and Lb. bulgaricus strains were performed at 42 °C by inoculating skim milk with 5x10⁶ c.f.u. ml^-1 of stock cultures of each strain. For proteinase-negative strains, cells from stock culture were washed three times in 50 mM Tris buffer (pH 7) before inoculation to avoid peptides and/or amino acids being supplied in the mixed culture. Every 20 min, the pH of the culture was measured, and bacteria were enumerated as described above. Total bacterial populations were estimated by addition of data from enumerations of each bacterial species on specific medium to the others, as indicated above.

Proteinase assay.
The PrtS proteinase phenotype of S. thermophilus strains was determined on bacterial colonies in two ways. First, bacteria were grown on FSDA medium (Fast Slow Difference Agar) (Huggins & Sandine, 1984 ). This milk-based agar medium made it possible to differentiate bacteria exhibiting slow or limited growth in milk from those exhibiting rapid growth; in particular, bacteria possessing a cell-wall proteinase activity appeared as white, opaque, rounded colonies, whereas proteinase-negative colonies were small, flat and translucent. Second, bacteria from an overnight skim milk culture were diluted and plated on agar skim milk plates (cell culture dishes, 35 mm in diameter). After 24-48 h incubation at 42 °C in anaerobic jars, colonies were covered by a solution containing Tris buffer (50 mM, pH 7), a chromogenic substrate of proteinase PrtS (Suc-Ala-Ala-Pro-Phe-ßNA, 10 mg ml^-1; Novabiochem), 10 mg ml^-1 Fast-Garnet (GBC, Sigma) and 10-50 mM CaCl₂. PrtS-positive clones appeared as red colonies, whereas PrtS-negative clones remained white.

Proteinase activity was measured on cellular suspensions using [¹⁴C]casein as the substrate according to the method of Monnet et al. (1987) , modified as follows. Cell suspensions were prepared from 4 ml overnight M17 cultures; cells were recovered by centrifugation (20 min, 8000 g, 4 °C) and washed three times in Tris buffer (50 mM, pH 7). The last pellet was suspended in 150 µl Bistris buffer (50 mM, pH 6·5) containing 10 mM CaCl₂. Fifty microlitres of cell suspension was incubated with 50 µl of ¹⁴C casein solution (0·1%) at 37 °C for 15, 60 and 120 min. Enzyme reactions were stopped by the addition of 100 µl TCA (12%), left for 30 min at room temperature and centrifuged for 2 min at 10000 g, and the radioactivity was then measured in the supernatants. Protease activity corresponded to the percentage of casein hydrolysis in 10 min.

DNA manipulations and sequencing
Total DNA preparation.
Total DNA of S. thermophilus CNRZ 385 was prepared as described by Pospiech & Neumann (1995) .

Preparation of electrocompetent cells of S. thermophilus and Lc. lactis.
Electrocompetent cells of S. thermophilus CNRZ 385 and Lc. lactis MG1363 were prepared according to the method of Holo & Nes (1989) , modified as follows. From an overnight culture in M17Lac, a culture was performed at 37 °C (S. thermophilus) or at 30 °C (Lc. lactis) by 1% inoculation of M17Lac containing DL-Thr (100 mM) for S. thermophilus or Gly (1·5%) for Lc. lactis until the OD₆₀₀ reached 0·6-1. Cells were collected by centrifugation at 5000 g for 5 min and washed four times in 0·5 M sucrose/10% glycerol solution. They were then resuspended in 10% glycerol/30% PEG2000 solution for S. thermophilus or in 0·5 M sucrose/10% glycerol solution for Lc. lactis and immediately frozen in liquid N₂ and stored at -80 °C.

DNA sequencing.
The Sanger method of DNA sequencing was carried out on double-strand DNA plasmids and on PCR products with the BigDye Terminator cycle sequencing ready reaction kit (370A DNA sequencer, Applied Biosystems).

Construction of a negative mutant for PrtS.
A 3776 bp PCR product containing part of the prtS gene was amplified using oligonucleotides 1 (5' CAT CAC GGA AAG TCT AGG 3') and 2 (5' AAC GTA TTG ATA CTT ACC 3') from total DNA of S. thermophilus CNRZ 385 strain (Fig. 1). Streptococcal DNA (100 ng) was added to a PCR mixture containing 2·5 U of Taq polymerase (Quantum Appligene) and 0·26 µM of each oligonucleotide (Life Technology). After 5 min of denaturation at 94 °C, 30 cycles of 30 s annealing at 50 °C and 3 min of elongation at 72 °C were carried out using a Perkin-Elmer DNA thermal cycler (model 480). The amplified fragment was purified from 0·7% agarose gel with the QIAquick gel-extraction kit (Qiagen). It was then ligated to pCR-XL-TOPO vector (Invitrogen) and cloned by transformation of electrocompetent TOP10 E. coli cells (Invitrogen) according to the manufacturer's protocol. The recombinant vector, pCR-XL-TOPO-prtS-1, was purified with QIAprep Spin Miniprep Kit (Qiagen) from the recombinant cells and digested with BsgI (New England Biolabs). A 5·4 kb fragment containing the TopoXL vector and part of the prtS gene was then purified from 0·7% agarose gel with QIAquick gel extraction kit (Qiagen) and blunt-ended with T4 polymerase 3'5' exonuclease (Life Technologies) according to the supplier's protocol. It was then circularized by self-ligation with Fast-link DNA ligation kit (Epicentre Technology); the resulting plasmid, pCR®-XL-TOPO-prtS-2, was produced by transformation of electrocompetent TOP10 E. coli cells and purified as described above. It was then digested with NotI and SpeI (Eurogentec), and the resulting 2·078 kb fragment was purified as already described above. The 2·078 kb fragment (200 ng) was ligated to pGhost9 vector (100 ng) (Maguin et al., 1996 ), digested with NotI and SpeI. The ligation mix was used to electrotransform 100 µl of electrocompetent cells of Lc. lactis MG1363, as described by Holo & Nes (1989) . Recombinant clones were selected on M17Lac Ery plates after incubation at 28 °C. The recombinant vector, pG⁺h9::prtS, was purified as described above, and 20 µg was used to transform electrocompetent cells of S. thermophilus CNRZ 385, as previously described (Garault et al., 2000 ). Integration of pG⁺h9::prtS into the streptococcal chromosome was performed as described by Garault et al. (2000) with the following modification: to induce chromosomal integration of the plasmid, the culture was diluted and plated on M17Lac Ery plates. Finally, the mutant for PrtS was obtained by successive incubations of the culture containing the chromosomal integration at 37 °C to favour the excision of the pGhost9 plasmid.

 
PrtS is essential to S. thermophilus growth in milk
S. thermophilus PrtS-negative mutant construction. We have described here for the first time the construction of a targeted negative mutant for S. thermophilus cell-wall proteinase PrtS. This mutant of S. thermophilus CNRZ 385 was constructed by gene replacement using a truncated copy of prtS gene cloned in pGhost9 plasmid. DNA sequencing confirmed that this copy was inserted at the prtS locus and that pGhost9 was subsequently excised, resulting in a truncated prtS gene. As expected, the truncated gene was deprived of part of the signal sequence, all the pro-region (removed after maturation of the protein in the parental strain), and almost all of the region encoding the catalytic domain of the enzyme (Fig. 1). Only the region encoding the six C-terminal amino acids among the 495 constituting the catalytic domain (PR domain) was still present in the mutant and did not include the sequence encoding the residues involved in the catalytic activity of the proteinase (Fernandez-Espla et al., 2000 ). Furthermore, protein exportation signals were no longer present in the mutant; the signal sequence was truncated, and the expected peptide cleavage site was excised.

Cell-wall proteinase activity of the wild-type and PrtS-negative mutant of S. thermophilus. Using two different methods, we checked that the S. thermophilus PrtS-negative mutant lacked cell-wall proteinase activity. First, using ¹⁴C-labelled casein as a substrate, we observed that cell suspensions of the PrtS-positive strain were capable of hydrolysing casein (12·5% of total casein hydrolysed within 10 min), whereas PrtS-negative cells had no detectable caseinolytic activity. Second, we set up a rapid test on colonies using a chromogenic substrate of PrtS. Three strains of S. thermophilus were used: the proteinase-negative strain CNRZ 302 as negative control and the two proteinase-positive strains CNRZ 385 and CNRZ 703, which have a high cell-wall proteinase activity (Shahbal et al., 1991 , 1993 ). After growing on milk agar plates, colonies were covered with a solution containing the substrate Suc-A-A-P-F-ßNA, Fast-Garnet and different concentrations of CaCl₂, the latter being an activator of PrtS proteinase (Fernandez-Espla et al., 2000 ). Whatever the CaCl₂ concentration (10, 20 or 50 mM), colonies of strains 703 and 385 rapidly became red, whereas those of the negative strain 302 remained white. Using this test, we confirmed that the mutant strain was PrtS-negative, as colonies remained white even after several hours of contact with the substrate solution. This test functioned on milk plates but not on rich medium M17 plates for strain 703, which confirmed a probable regulation of prtS expression by the growth medium as already observed for this strain (Shahbal et al., 1993 ). This test will be useful to screen for S. thermophilus PrtS-negative strains in milk and also for PrtS-deregulated strains in M17.

Growth characteristics of the wild-type and PrtS-negative mutant of S. thermophilus. By comparing the phenotypes of the parental strain 385 and its PrtS^- mutant on FSDA, and their growth curves in liquid M17 and milk, we showed that proteinase PrtS was essential to the growth of S. thermophilus in milk.

The PrtS^- mutant, plated on FSDA, appeared as flat and translucent colonies, as expected for PrtS^- bacteria, whereas the PrtS⁺ parental strain appeared as white, opaque, rounded colonies.

In M17, both strains had similar growth curves with a µ_max of 0·89 and 0·85 h^-1 for the parental strain and the mutant strain, respectively. In milk, streptococcal growth was determined indirectly by pH measurement. Growth of the PrtS^- mutant was severely impaired in milk, as indicated by the reduced acidification of milk by this strain compared to the parental strain 385 (Fig. 2). For the PrtS^- strain, milk acidification, and thus bacterial growth, was restored to the same extent as that for the wild-type strain, after the addition of yeast extract to milk (Fig. 2).

 
The Lactobacillus strain influences the extent of the positive effect of S. thermophilus/Lb. bulgaricus association
Mixed cultures of S. thermophilus and Lb. bulgaricus were made using two different strains of Lb. bulgaricus. To choose the last two strains, we first determined the effect of the co-culture of Lb. bulgaricus strains with the S. thermophilus CNRZ385 strain on milk acidification, compared to the pure culture of Lb. bulgaricus (Fig. 3). Among the three strains of lactobacilli tested, the effect of adding strain 385 on the acidification was greatest with strains Lb. bulgaricus 397 and 1038; indeed, for these two Lactobacillus strains, the addition of the Streptococcus highly enhanced the acidification rate compared to the Lb. bulgaricus strain alone (Fig. 3a, b). Furthermore, the positive effect of the bacterial association on milk acidification was more intense for strain 1038, as, for this strain, the acidification rate and the final pH were higher and lower, respectively, in the mixed culture than in the pure culture. In contrast, addition of S. thermophilus strain 385 had no significant effect on milk acidification by Lb. bulgaricus strain 752 (Fig. 3c). Thus, strains 397 and 1038 of Lactobacillus were kept for the following study. In addition, a proteinase-negative mutant of strain Lb. bulgaricus CNRZ 397 was available and was used for the following experiments.

 
In mixed cultures, proteinase PrtS has no effect on final pH and bacterial populations, but PrtB affects both
The effect of proteinases PrtS and PrtB on acidification of mixed cultures and bacterial populations was estimated by measuring the final pHs, final total bacterial populations and final individual populations of cultures performed with a strain of S. thermophilus PrtS⁺ (strain 385) or PrtS^- (strain 385-PrtS) and a strain of Lb. bulgaricus PrtB⁺ (strains 397 and 1038) or PrtB^- (strain 397-PrtB).

The presence of proteinase PrtS had no effect either on the final bacterial populations or on the final pHs of mixed cultures involving PrtB⁺ Lactobacillus strains. The final total populations were always similar in the presence or not of proteinase PrtS: 1·39 and 1·36x10⁹ c.f.u. ml^-1, respectively, for cultures involving Lb. bulgaricus strain 1038, and 7·05 and 6·27x10⁸ c.f.u. ml^-1, respectively, with strain 397. The absence of any differences in final total populations corresponded to similar final individual populations of S. thermophilus and Lb. bulgaricus, regardless of the presence of PrtS: 1·3x10⁹ c.f.u. ml^-1 for strains 385 and 385-PrtS and 8·9x10⁷ c.f.u. ml^-1 for strain 1038 for mixed cultures involving strain 1038, 5·5x10⁸ c.f.u. ml^-1 for strains 385 and 385-PrtS and 1·1x10⁸ c.f.u. ml^-1 for strain 397 in mixed cultures involving strain 397. This correlated well with the similar final pH obtained: 4·72 and 4·85 for mixed cultures involving, respectively, strain 1038 and strain 397.

It is noteworthy that both the final total bacterial populations and the acidification rates varied according to the Lactobacillus strain associated with S. thermophilus strain 385. The final total population when using Lactobacillus strain 1038 (1·38x10⁹ c.f.u. ml^-1) was twice as high as that of strain 397 (6·67x10⁸ c.f.u. ml^-1), because the Streptococcus populations were more than twice as high with strain 1038, Lactobacillus populations remaining constant. Final pHs were not significantly different, but the time required to reach these pHs was shorter for mixed cultures, including strain 1038, than those including strain 397 (4·66 h with strain 1038 versus 5·66 h with strain 397).

In contrast, the presence of proteinase PrtB affected both the final bacterial populations and the final pHs. Final total bacterial populations were threefold higher in the presence of PrtB than in its absence (7·05x10⁸ versus 2·8x10⁸ c.f.u. ml^-1). This resulted from higher final populations of S. thermophilus in the presence of PrtB (6·1x10⁸ versus 2·06x10⁸ c.f.u. ml^-1) and led to a significantly better acidification in the presence of PrtB (final pH 4·86 versus 5·42).

In our conditions of inoculation (Streptococcus/Lactobacillus ratio of 1:1), S. thermophilus was systematically predominant in the total final populations, regardless of the strain of Lactobacillus and the presence of proteinases PrtS and PrtB. The magnitude of this predominance depended on the Lb. bulgaricus strain used: with strain 1038, S. thermophilus populations were 15-fold higher than Lactobacillus populations and fivefold higher with strain 397, when PrtB was present. This predominance was less marked in the absence of PrtB, as the S. thermophilus populations were threefold lower (6·1x10⁸ c.f.u. ml^-1) than populations reached in the presence of PrtB (2·06x10⁸ c.f.u. ml^-1).

Variation of individual populations of S. thermophilus and Lb. bulgaricus throughout mixed cultures in milk
Proteinase PrtS had no significant effect on the variation of pH and of individual populations throughout the culture and, regardless of the mixed culture considered (except that involving strain PrtB^-), the variation of these two parameters remained similar. Fig. 4a gives an example of this variation (a mixed culture made of S. thermophilus 385 and Lb. bulgaricus 1038); for mixed cultures including Lb. bulgaricus strain 397, we observed the same behaviour (data not shown). During the first 60-90 min, which corresponded to the first acidification phase, S. thermophilus grew exponentially, whereas Lb. bulgaricus did not grow significantly. Then, as the pH remained constant, the streptococcal population stabilized for about 60-90 min, whereas Lb. bulgaricus started to grow regularly and continuously. Finally, during the last 2 or 3 h of fermentation, when the acidification rate was the highest, both the Lactobacillus and the Streptococcus grew regularly.

 
In contrast, proteinase PrtB was clearly involved in the variation of bacterial populations and of pH, as demonstrated with mixed cultures involving strain 397-PrtB (Fig. 4b). In fact, regardless of the presence of PrtB, the first two phases of acidification corresponding to the sequential growth of S. thermophilus and Lb. bulgaricus were similar. However, during the third acidification phase, the growth of S. thermophilus slowed down in the absence of PrtB, the bacterial populations remaining almost constant during the last 2 h of fermentation. This reduced growth resulted in a reduced acidification rate and an increased final pH (pH 5·42 in the absence of PrtB and 4·86 in the presence of PrtB).

 
The present work aimed at evaluating the role of proteinase PrtS from S. thermophilus in the growth in milk of S. thermophilus in a pure culture. We also determined the effect of the presence of both PrtS and PrtB from Lb. bulgaricus on S. thermophilus/Lb. bulgaricus mixed cultures. For this purpose, we constructed a targeted negative mutant of proteinase PrtS from S. thermophilus CNRZ 385 and performed pure cultures of S. thermophilus and mixed cultures with Lb. bulgaricus in milk.

In milk, the extent of the beneficial effect of the S. thermophilus/Lb. bulgaricus association varies
We observed that the effect of the co-culture of Lb. bulgaricus strains with S. thermophilus strain 385 on the acidification of milk, and thus the benefit of the bacterial association, depends on the strain of Lb. bulgaricus used. In fact, with Lb. bulgaricus strain 752, we did not obtain a marked beneficial effect of the association with S. thermophilus 385 as already observed by several authors with other strains (Accolas et al., 1977 ; Bautista et al., 1966 ; Sodini et al., 2000 ). In contrast, mixed cultures of strains 1038 and 397 resulted in a higher acidification than pure cultures. Acidification was higher with strain 1038 than with strain 397 due to higher S. thermophilus populations, Lb. bulgaricus populations being similar. These higher S. thermophilus populations probably resulted from a better peptide and/or amino acid supply by one Lactobacillus strain compared to the other as these nitrogen compounds are growth-limiting for S. thermophilus in milk. The two strains of Lb. bulgaricus thus probably differ in their proteolytic potential, which is in agreement with the differences observed in the final quantities of free amino acids and free NH₂ groups in the supernatants of mixed cultures including these two strains (data not shown). Some authors have also reported a variability in the Lb. bulgaricus proteolytic potential (El-Soda et al., 1986 ; Rajagopal & Sandine, 1990 ; Singh & Sharma, 1983 ). This variability could be related to the presence of one cell-wall proteinase in Lb. bulgaricus, which is the case of strain 397 (Gilbert et al., 1997 ), or of two proteinases, as reported for other strains (Pederson et al., 1999 ; Stefanisti et al., 1995 ).

PrtS is essential to the growth of S. thermophilus in milk in pure culture but not in mixed culture
Proteinase PrtS is essential to the growth of S. thermophilus growth in milk as its PrtS^- mutant was unable to grow efficiently in milk until a nutritional complement [yeast extract or bactotryptone (data not shown)] was added. This indicated that proteinase PrtS was involved in nitrogen supply to the cell, via casein hydrolysis, which is consistent with data previously obtained with cell-wall proteinases of other lactic acid bacteria (Exterkate, 1990 ; Gilbert et al., 1997 ; Thomas & Pritchard, 1987 ).

However, we demonstrated here that proteinase PrtS had no significant effect on the growth of S. thermophilus in mixed cultures in milk with Lb. bulgaricus; the growth of the parental strain 385 and of the PrtS^- mutant in mixed culture was similar when Lb. bulgaricus proteinase PrtB was present. This indicates that assimilable nitrogen compounds necessary for S. thermophilus growth are supplied by PrtB, as confirmed by the fact that the absence of PrtB led to lower streptococcal populations. Furthermore, as the streptococcal population was higher in the presence of PrtB than in the presence of PrtS, we can assume that PrtB is more efficient in the supply of peptides to S. thermophilus than PrtS. This can be explained by a more active proteinase PrtB compared to PrtS, as previous studies reported that the global proteolytic activities of Lb. bulgaricus strains were 25-70 times higher than that of S. thermophilus strains (Rajagopal & Sandine, 1990 ; Shankar & Davies, 1978 ). We cannot rule out the possibility that PrtS and PrtB have different substrate specificity, which leads to the production of different peptides, some being more assimilable than others. Indeed, PrtS is capable of hydrolysing MS-Arg-Pro-Tyr-pNA (Fernandez-Espla et al., 2000 ), a substrate also hydrolysed by lactococcal proteinase PrtP (Exterkate, 1990 ) but not by PrtB (Laloi et al., 1991 ). Furthermore, when comparing the substrate-binding region of proteinases PrtS and PrtB, in particular the residues 138, 166, 748, which have been identified as being directly involved in substrate specificity in lactococci (Siezen et al., 1993 ), we noticed that they are totally different in PrtS (Thr, Ala, Asp) (Fernandez-Espla et al., 2000 ) and PrtB (Gly, Val, Thr) (Gilbert et al., 1996 ).

Model of growth of S. thermophilus associated with Lb. bulgaricus and effect on acidification
In all the mixed cultures performed in milk, we observed the sequential development of S. thermophilus and then of Lb. bulgaricus, which is in agreement with previous studies (Beal & Corrieu, 1991 ; Pette & Lolkema, 1950a ; Puhan & Banhegyi, 1974 ; Tamine & Robinson, 1999 ). Recently, the growth of S. thermophilus in pure culture in milk has been characterized, in particular with regard to nitrogen nutrition (Letort et al., 2002 ); it consists of two exponential growth phases, interrupted by a non-exponential growth phase. From these latter results and those of the present work, we propose the following model of growth of S. thermophilus in mixed cultures with Lb. bulgaricus with three S. thermophilus growth phases corresponding to three acidification steps.

During the first acidification step, characterized by a small decrease in pH (<0·5 pH units), S. thermophilus grows exponentially, whereas Lb. bulgaricus does not grow; S. thermophilus is thus responsible for this first acidification, as first observed by Pette & Lolkema (1950c ). The preferential growth of S. thermophilus can be explained first by the fact that S. thermophilus has fewer nutritional requirements than lactobacilli in milk (Desmazeaud, 1983 ). In particular, S. thermophilus requires few amino acids and is capable of synthesizing branched-chain amino acids (Garault et al., 2000 ); its growth can probably be supported by free amino acids and peptides present in milk, as previously demonstrated in pure culture, regardless of the presence of PrtS (Letort et al., 2002 ). In contrast, Lb. bulgaricus is much more demanding from a nutritional point of view than S. thermophilus (Letort, 2001 ); its optimal growth relies on the supply of essential factors (CO₂, pyruvate, formate) produced by S. thermophilus (for reviews, see Tamine & Robinson, 1999 ; Zourari et al., 1992 ). Second, in our study, mixed cultures were performed at 42 °C, a temperature more favourable for S. thermophilus, whose optimal growth temperature ranges between 40 and 45 °C, versus 45-50 °C for Lb. bulgaricus.

Then, the S. thermophilus exponential growth pauses and, concomitantly, the acidification, while Lb. bulgaricus begins to grow slowly and regularly until the end of fermentation. This pause probably corresponds to depletion of amino acids and peptides in milk, due to their consumption by S. thermophilus, as shown recently by Letort et al. (2002) in pure culture, and the absence of compensatory production by cell-wall proteinases. These authors actually demonstrated that proteinase PrtS synthesis starts in the middle of this phase and is maximal during the second exponential growth phase in pure culture. Concerning the growth of Lb. bulgaricus, we assume that as S. thermophilus reaches a high cellular density during its first growth phase, it probably produces enough growth-stimulating factors to favour the growth of Lb. bulgaricus.

Finally, during the following acidification phase, which leads to a high pH decrease (about 1·5 pH units), Lb. bulgaricus continues to grow; at the same time, S. thermophilus starts a second exponential growth phase. We suggest that this acidification results not only from the growth of Lb. bulgaricus but also from that of S. thermophilus. This acidification phase is indeed greatly improved by the addition of S. thermophilus to a Lb. bulgaricus culture; furthermore, in the absence of PrtB, acidification is reduced, while only S. thermophilus populations significantly decrease. The growth of S. thermophilus probably occurs because of the utilization of peptides produced by PrtS (when PrtB is absent) but also mainly by PrtB. No differences in the growth of S. thermophilus were observed in the presence or absence of PrtS when PrtB was present, and S. thermophilus populations were significantly reduced in the absence of PrtB, i.e. when PrtS was the sole source of peptide production.

In conclusion, we have determined the role of cell-wall proteinases PrtS and PrtB in the growth of S. thermophilus and Lb. bulgaricus in mixed cultures. We have shown that PrtB is involved in the optimal growth of S. thermophilus, whereas PrtS does not play a significant role when PrtB is present. Studies of the effect of these proteinases on the free amino acid and peptide contents as well as on the aroma profiles of mixed cultures are in progress. As precursors, amino acids are involved in the formation of aroma in dairy products, and variations in their composition can affect aroma development. However, the different pH values observed in the present study at the end of fermentation, when varying the presence of proteinase PrtB, can modify the yoghurt flavour (Ott et al., 2000 ).

 
We gratefully acknowledge D. Atlan and M. van de Guchte for the supply of Lb. bulgaricus strains 1159 and 1038, respectively. We thank M. Nardi, for her helpful suggestions concerning mutant construction, and M. Diard for his technical assistance.

 
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Received 4 April 2002; revised 20 June 2002; accepted 15 July 2002.
 

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