Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca 2؉ ions. Its activity was optimal at 37°C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.Lactic acid bacteria (LAB) are widely used as starters in fermented milk products due to their properties of milk acidification and flavor development. For these applications, their capacity to grow fast in milk is of major importance. LAB are fastidious microorganisms and require an exogenous source of amino acids or peptides for optimal growth. As milk is poor in these low-molecular-weight compounds, their growth largely depends on their proteolytic system to achieve hydrolysis of caseins (65). The cell envelope proteinase (CEP) is the key enzyme of this process since it is the only enzyme capable of initiating the breakdown of caseins into oligopeptides. The latter are then transported into the bacteria and further degraded by a complex set of intracellular peptidases (12).The cell envelope proteinases of lactococci, and to a lesser extent those of lactobacilli, have been the subject of intensive biochemical and genetic investigation (for a review, see reference 37). Lactococcal proteinase PrtP is synthesized as an inactive preproenzyme and maturated via an autoproteolytic process involving a chaperone lipoprotein PrtM, and it is anchored to the cell wall. The hydrolysis specificity of CEPs determined on caseins or casein peptides varies among strains, and several classifications have been proposed (5,18,21,22,24,40,66). The differences observed in substrate specificity are only due...

Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yoghurt manufacture, where it is associated with Lactobacillus delbrueckii subsp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG 18311 in response to the presence of L. delbrueckii subsp. bulgaricus ATCC 11842 during growth in milk at two growth stages. Seventy-seven different genes or proteins (4.1% of total coding sequences), implicated mainly in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in coculture. One of the most unpredicted results was a significant decrease of most of the transcripts and enzymes involved in purine biosynthesis. Interestingly, the expression of nearly all genes potentially encoding iron transporters of S. thermophilus decreased, whereas that of iron-chelating dpr as well as that of the fur (perR) regulator genes increased, suggesting a reduction in the intracellular iron concentration, probably in response to H 2 O 2 production by L. bulgaricus. The present study reveals undocumented nutritional exchanges and regulatory relationships between the two yoghurt bacteria, which provide new molecular clues for the understanding of their associative behavior.

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 PrtBnegative 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.

We investigated the adaptation to milk of Streptococcus thermophilus LMG18311 using a proteomic approach. Two-dimensional electrophoresis of cytosolic proteins were performed after growth in M17 medium or in milk. A major modification of the proteome concerned proteins involved in the supply of amino acids, like the peptidase PepX, and several enzymes involved in amino acid biosynthesis. In parallel, we observed the upregulation of the synthesis of seven enzymes directly involved in the synthesis of purines, as well as formyl-tetrahydrofolate (THF) synthetase and serine hydroxy-methyl transferase, two enzymes responsible for the synthesis of compounds (THF and glycine, respectively) feeding the purine biosynthetic pathway. The analysis also revealed a massive increase in the synthesis of pyruvate formate-lyase (PFL), the enzyme which converts pyruvate into acetyl coenzyme A and formate. PFL has been essentially studied for its role in mixed-acid product formation in lactic acid bacteria during anaerobic fermentation. However, formate is an important methyl group donor for anabolic pathway through the formation of folate derivates. We hypothesized that PFL was involved in purine biosynthesis during growth in milk. We showed that PFL expression was regulated at the transcriptional level and that pfl transcription occurred during the exponential growth phase in milk. The complementation of milk with formate or purine bases was shown to reduce pfl expression, to suppress PFL synthesis, and to stimulate growth of S. thermophilus. These results show a novel regulatory mechanism controlling the synthesis of PFL and suggest an unrecognized physiological role for PFL as a formate supplier for anabolic purposes.The thermophilic bacteria Streptococcus thermophilus is one of the most widely used lactic acid bacteria (LAB) in the dairy fermentation industry for yoghurt and cheese production. In the industrial implementation of S. thermophilus, fast-growing capacity is crucial to enable rapid and intense acidification of milk. Identification of functions activated during growth in milk should help to understand the molecular adaptation of this bacterium to milk and provide the basis for targeted strain selection.In contrast with other LAB, the only environment from which S. thermophilus has been isolated is milk (38). In line with this restricted ecological niche, lactose, not glucose, is the preferred carbon source for S. thermophilus (31). The capacity to ferment lactose, the main sugar of milk, into lactic acid is essential for growth in milk and depends on a non-phosphotransferase system lactose permease (LacS) and a beta-galactosidase (LacZ) (40). Concerning the capacity of S. thermophilus to fulfill its amino acid needs during growth in milk, the species displays only a few amino acid auxotrophies compared to the model LAB, L. lactis (29,33). The growth of Lactococcus lactis in milk depends largely on the activity of a cell-wallbound proteinase (PrtP) that is responsible for casein hydrolysis, the main source of amino a...

The thermophilic lactic acid bacterium Streptococcus thermophilus is widely and traditionally used in the dairy industry. Despite the vast level of consumption of S. thermophilus through yogurt or probiotic functional food, very few data are available about its physiology in the gastrointestinal tract (GIT). The objective of the present work was to explore both the metabolic activity and host response of S. thermophilus in vivo. Our study profiles the protein expression of S. thermophilus after its adaptation to the GIT of gnotobiotic rats and describes the impact of S. thermophilus colonization on the colonic epithelium. S. thermophilus colonized progressively the GIT of germ-free rats to reach a stable population in 30 days (10 8 cfu/g of feces). This progressive colonization suggested that S. thermophilus undergoes an adaptation process within GIT. Indeed, we showed that the main response of S. thermophilus in the rat's GIT was the massive induction of the glycolysis pathway, leading to formation of lactate in the cecum. At the level of the colonic epithelium, the abundance of monocarboxylic acid transporter mRNAs (SLC16A1 and SLC5A8) and a protein involved in the cell cycle arrest (p27 kip1 ) increased in the presence of S. thermophilus compared with germ-free rats. Based on different mono-associated rats harboring two different strains of S. thermophilus (LMD-9 or LMG18311) or weak lactate-producing commensal bacteria (Bacteroides thetaiotaomicron and Ruminococcus gnavus), we propose that lactate could be a signal produced by S. thermophilus and modulating the colon epithelium.Streptoccocus thermophilus belongs to the group of the thermophilic lactic acid bacteria and is traditionally and widely used as a starter in manufacturing dairy products (Emmental, Gruyere, Parmigiano, mozarella, yogurt, etc.). Yogurt, which results from the fermentation of milk by S. thermophilus and Lactobacillus delbrueckii sp. bulgaricus (L. bulgaricus), fulfills the current specifications required to be recognized as a probiotic product (1). The health beneficial effect of yogurt consumption is linked to the metabolic properties of S. thermophilus and L. bulgaricus. As such, it improves lactose digestion in the gastrointestinal tract (GIT) 3 through their lactose-hydrolyzing activity present in yogurt and in the GIT, thus reducing symptoms of lactose intolerance (2, 3).Yogurt cultures have been shown to induce other health benefits, such as reduction of diarrhea or allergic disorders as well as modulation of the immune system (1, 4). S. thermophilus is also present at high concentration in VSL#3, a probiotic mixture of eight different bacterial strains that possesses beneficial effects in several intestinal conditions (5, 6).Recent data indicate that strains related to S. thermophilus LMD-9 are among the 57 bacteria species found in the intestinal microbiota of 90% of 124 European individuals (7). In comparison with the overall human intestinal microbiota, S. thermophilus is a numerically nondominant species with variable levels...