Streptococcus thermophilus growth in soya milk: Sucrose consumption, nitrogen metabolism, soya protein hydrolysis and role of the cell-wall protease PrtS.

Societal demand for plant-based foods is increasing. In this context, soya products fermented using lactic acid bacteria (LAB) are appealing because of their potential health and nutritional benefits. The thermophilic LAB Streptococcus thermophilus is an essential starter species in the dairy industry. However, while its physiology is well characterized, little is known about its general metabolic activity or its techno-functional properties when it is grown in soya milk. In this study, S. thermophilus LMD-9 growth, sugar production, and lactic acid production in soya milk versus cow's milk were measured. Additionally, the main metabolic pathways used by the bacterium when growing in soya milk were characterized using a proteomic approach. Streptococcus thermophilus LMD-9 growth decreased soya milk pH, from 7.5 to 4.9, in 5 h. During fermentation, acidification thus occurred in tandem with lactate production and increasing population size (final population: 1.0 × 109 CFU/ml). As growth proceeded, sucrose was consumed, and fructose was produced. The proteomic analysis (LC-MS/MS) of the strain's cytosolic and cell envelope-associated proteins revealed that proteins related to amino acid transport and nitrogen metabolism were the most common among the 328 proteins identified (63/328 = 19.2% of total proteins). The cell-wall protease PrtS was present, and an LMD-9 deletion mutant was constructed by interrupting the prtS gene (STER_RS04165 locus). Acidification levels, growth levels, and final population size were lower in the soya milk cultures when the ΔprtS strain versus the wild-type (wt) strain was used. The SDS-PAGE profile of the soluble proteins in the supernatant indicated that soya milk proteins were less hydrolyzed by the ΔprtS strain than by the wt strain. It was discovered that S. thermophilus can grow in soya milk by consuming sucrose, can hydrolyze soya proteins, and can produce acidification levels comparable to those in cow's milk. This study comprehensively examined the proteomics of S. thermophilus grown in soya milk and demonstrated that the cell-wall protease PrtS is involved in the LAB's growth in soya milk and in the proteolysis of soya proteins, which are two novel findings. These results clarify how S. thermophilus adapts to soya milk and can help inform efforts to develop new fermented plant-based foods with better-characterized biochemical and microbiological traits.

Characterization of Mucus-Related Properties of Streptococcus thermophilus: From Adhesion to Induction.

Mucus is a major component of the intestinal barrier involved both in the protection of the host and the fitness of commensals of the gut. Streptococcus thermophilus is consumed world-wide in fermented dairy products and is also recognized as a probiotic, as its consumption is associated with improved lactose digestion. We determined the overall effect of S. thermophilus on the mucus by evaluating its ability to adhere, degrade, modify, or induce the production of mucus and/or mucins. Adhesion was analyzed in vitro using two types of mucins (from pig or human biopsies) and mucus-producing intestinal HT29-MTX cells. The induction of mucus was characterized in two different rodent models, in which S. thermophilus is the unique bacterial species in the digestive tract or transited as a sub-dominant bacterium through a complex microbiota. S. thermophilus LMD-9 and LMG18311 strains did not grow in sugars used to form mucins as the sole carbon source and displayed weak binding to mucus/mucins relative to the highly adhesive TIL448 Lactococcus lactis. The presence of S. thermophilus as the unique bacteria in the digestive tract of gnotobiotic rats led to accumulation of lactate and increased the number of Alcian-Blue positive goblet cells and the amount of the mucus-inducer KLF4 transcription factor. Lactate significantly increased KLF4 protein levels in HT29-MTX cells. Introduction of S. thermophilusvia transit as a sub-dominant bacterium (103 CFU/g feces) in a complex endogenous microbiota resulted in a slight increase in lactate levels in the digestive tract, no induction of overall mucus production, and moderate induction of sulfated mucin production. We thus show that although S. thermophilus is a poor mucus-adhesive bacterium, it can promote mucus pathway at least in part by producing lactate in the digestive tract.

Characterization of Streptococcus thermophilus two-component systems: In silico analysis, functional analysis and expression of response regulator genes in pure or mixed culture with its yogurt partner, Lactobacillus delbrueckii subsp. bulgaricus.

The lactic acid bacterium Streptococcus thermophilus (S. thermophilus) is widely used in the dairy industry. As a food bacterium, it has to cope with changing environments such as milk, yogurt, as well as the digestive tract, after the product has been ingested. In bacteria, two-component systems (TCS) are one of the most prevalent mechanisms to sense and respond appropriately to a wide range of signals. They are typically composed of a sensor kinase (HK) that detects a stimulus and a response regulator (RR) which acts as a transcriptional regulator. Our objective was to make an inventory of the TCS present in S. thermophilus LMD-9 and investigate the contribution of each TCS to LMD-9 growth in milk. For that purpose, we performed in silico, transcriptomic as well as functional analysis. The LMD-9 genome presented 6 complete TCS with both HK and RR (TCS 2, 4, 5, 6, 7, and 9) and 2 orphan RRs (RR01 and 08) with truncated HK. Our in silico analysis revealed that for 5 TCS out of the 8, orthologs with known functions were found in other bacterial species whereas for TCS02, 4 and 6 the function of the orthologs are unidentified. Transcriptomic studies (using quantitative PCR) revealed that all S. thermophilus LMD-9 response regulator genes were expressed in milk; they were expressed at different levels and with different profiles during growth. In mixed culture with Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), the S. thermophilus partner in yogurt, the expression of four S. thermophilus LMD-9 response regulator increased; two of them, rr02 and rr09, increased by a factor of 6. These results indicate that the presence of L. bulgaricus induces regulatory changes in S. thermophilus. We also demonstrated that a response regulator (rr02) can exert its regulatory function on its target genes even when expressed at very low levels. We showed that RR05-an ortholog of Bacillus subtilis YycF or Staphylococcus aureus WalR-was essential for the growth of S. thermophilus. For the 7 other RRs, the absence of a single response regulator gene was insufficient to notably impact the growth of LMD-9 in milk, with or without supplementation with purines, formate, or stress agents (lactate, H2O2). We demonstrated here that the 8 response regulators of LMD-9 are expressed--and thus potentially active--during growth in milk and suggested that the response regulators have possibly overlapping regulons and/or functions not essential under the conditions tested.

Impact of the metabolic activity of Streptococcus thermophilus on the colon epithelium of gnotobiotic rats.

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.

Carbohydrate metabolism is essential for the colonization of Streptococcus thermophilus in the digestive tract of gnotobiotic rats.

Streptococcus thermophilus is the archetype of lactose-adapted bacterium and so far, its sugar metabolism has been mainly investigated in vitro. The objective of this work was to study the impact of lactose and lactose permease on S. thermophilus physiology in the gastrointestinal tract (GIT) of gnotobiotic rats. We used rats mono-associated with LMD-9 strain and receiving 4.5% lactose. This model allowed the analysis of colonization curves of LMD-9, its metabolic profile, its production of lactate and its interaction with the colon epithelium. Lactose induced a rapid and high level of S. thermophilus in the GIT, where its activity led to 49 mM of intra-luminal L-lactate that was related to the induction of mono-carboxylic transporter mRNAs (SLC16A1 and SLC5A8) and p27(Kip1) cell cycle arrest protein in epithelial cells. In the presence of a continuous lactose supply, S. thermophilus recruited proteins involved in glycolysis and induced the metabolism of alternative sugars as sucrose, galactose, and glycogen. Moreover, inactivation of the lactose transporter, LacS, delayed S. thermophilus colonization. Our results show i/that lactose constitutes a limiting factor for colonization of S. thermophilus, ii/that activation of enzymes involved in carbohydrate metabolism constitutes the metabolic signature of S. thermophilus in the GIT, iii/that the production of lactate settles the dialogue with colon epithelium. We propose a metabolic model of management of carbohydrate resources by S. thermophilus in the GIT. Our results are in accord with the rationale that nutritional allegation via consumption of yogurt alleviates the symptoms of lactose intolerance.

Postgenomic analysis of streptococcus thermophilus cocultivated in milk with Lactobacillus delbrueckii subsp. bulgaricus: involvement of nitrogen, purine, and iron metabolism.

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.

Physiology of Streptococcus thermophilus during the late stage of milk fermentation with special regard to sulfur amino-acid metabolism.

Streptococcus thermophilus is a thermophilic lactic acid bacterium widely used as starter in the manufacture of dairy products in particular in yoghurt manufacture in combination with Lactobacillus delbrueckii ssp. bulgaricus. However, in spite of its massive use, the physiological state of S. thermophilus in milk has hardly been investigated. We established the first map of the cytosolic proteome of S. thermophilus LMG18311 grown in milk. It comprises 203 identified proteins corresponding to 32% of theoretical proteome. In addition, using proteomic and transcriptomic approaches, we analyzed the physiology of LMG18311 during its late stage of growth in milk (between 2h30 and 5h30). It revealed the up-regulation of (i) peptides and AA transporters and of specific AA biosynthetic pathways notably for sulfur AA and (ii) genes and proteins involved in the metabolism of various sugars. These two effects were also observed in LMG18311 grown in milk in coculture with L. bulgaricus although the effect on sugar metabolism was less pronounced. It suggests that the stimulatory effect of Lactobacillus on the Streptococcus growth is more complex than AA or peptides supply.

Proteome analysis of Streptococcus thermophilus grown in milk reveals pyruvate formate-lyase as the major upregulated protein.

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.