Streptococcus Thermophilus In Milk Research Articles

Role of the Sortase A in the Release of Cell-Wall Proteinase PrtS in the Growth Medium of Streptococcus thermophilus 4F44

Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.

Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions

The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

Three Oligopeptide-binding Proteins Are Involved in the Oligopeptide Transport of Streptococcus thermophilus

The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.

La biosynthèse des acides aminés à chaîne branchée et des purines : deux voies essentielles pour une croissance optimale de Streptococcus thermophilus dans le lait

Branched-chain amino acids and purine biosynthesis: two pathways essential for optimal growth of Streptococcus thermophilus in milk. Slowing down and delays in Streptococcus thermophilusgrowth in milk have negative effects for dairy technology. Our objectives are to under- stand the reasons for those delays, and to identify some of the functions essential for optimal growth of S. thermophilusin milk. To reach this goal, a random insertional mutagenesis was performed on a S. thermophilus strain chosen for its ability to grow rapidly in milk. We obtained 14 different mutants, affected in their capacity to grow quickly in milk. Four of them were further character- ized: two were mutated in the branched-chain amino acids biosynthesis operon and two others were mutated in the purine nucleotides biosynthesis operon. Consequently, branched-chain amino acids and purine biosynthesis pathways are functional in S. thermophilus. Our results suggested that the amount of both branched-chain amino acids and purine nucleotides, present in milk, and directly consumable is not sufficient for an optimal growth. Moreover, we identified the branched-chain amino acids and purine biosynthesis pathways as essential functions for optimal growth of S. thermophilusin milk. milk / Streptococcus thermophilus / growth / branched chain amino acid / purine

Branched-chain amino acid biosynthesis is essential for optimal growth of Streptococcus thermophilus in milk.

Lactic acid bacteria are nutritionally demanding bacteria which need, among other things, amino acids for optimal growth. We identified the branched-chain amino acid (BCAA) biosynthesis pathway as an essential pathway for optimal growth of Streptococcus thermophilus in milk. Through random insertional mutagenesis, we isolated and characterized two mutants for which growth in milk is affected as a consequence of ilvB and ilvC gene interruptions. This situation demonstrates that the BCAA biosynthesis pathway is active in S. thermophilus. BCAA biosynthesis is necessary but not sufficient for optimal growth of S. thermophilus and is subject to retro-inhibition processes. The specificity of the BCAA biosynthesis pathway in S. thermophilus lies in the independent transcription of the ilvC gene encoding a keto acid reductoisomerase acting on acetolactate at the junction of the BCAA and acetoin biosynthesis pathways. The possible advantages for S. thermophilus of keeping this biosynthesis pathway active could be linked either to adaptation of the organism to milk, which is different than that of other dairy bacteria, or to the role of the pathway in maintaining the internal pH.

Effect of the addition of extracts of thermophilic lactobacilli on acid production by Streptococcus thermophilus in milk.

Soluble extracts of 20 strains of thermophilic lactobacilli (Lactobacillus helveticus, L. lactis and L. bulgaricus) were prepared and added to milk for the culture of 10 strains of Streptococcus thermophilus. Acid production was stimulated in 64.5% of cases for 9 of these 10 strains. The L. helveticus extracts were the most stimulatory, but the same extracts did not always strongly stimulate each strain of Str. thermophilus. The stimulatory effects observed varied with the volume of extract and the strain of Str. thermophilus. The exception was Str. thermophilus 385, which was never stimulated. The stimulatory effects observed were due to aminopeptidases present in the lactobacillus extracts and were not related to a general caseinolytic activity. The possible addition of such extracts to milk for cooked hard cheese is discussed.

Isolation and amino acid composition of casein peptides stimulating growth if Streptococcus thermophilus

In order to ascertain the physiological role of the peptides which enhanced the growth of Streptococcus thermophilus in milk, fourteen stimulating peptides were isolated and purified and their qualitative amino‐acid composition was determined. The purification process was the following: after hydrolysis at pH 7.5 of whole casein by the neutral protease from Micrococcus caseolyticus the stimulating peptides were fractionated by gel chromatography (Bio‐Gel P6), by column chromatography on cation‐exchange resins AG SOW X2 (0.05 M pyridine‐formate buffer at pH 3.10 to 1.5M pyridine‐acetate buffer at pH 5.10). Further purification was made by column chromatography on anion‐exchange resins AG1 X2 (0.1 M pyridine‐acetate buffer of pH 7.5 to 6 M acetic acid with a concave gradient system) and by paper chromatography (solvent: butanol–acetic acid–water, 4:1:5, v/v/v) and high tension electrophoresis (30 min; 60 V/cm).Gel filtration of enzymic digest of the whole casein led to six peptide fractions. Three fractions were stimulatory and exhibited a mean molecular weight of (B) 2500, (C) 1500, (D) 1000. Fraction C was the most stimulatory. Three other fractions were inhibitory and contained the highest molecular‐weight peptides (about 5000) and peptides with high contents of aromatic residues. Ten and twenty peptidic fractions were isolated respectively from Fractions B and D by chromatography on ion‐exchange resins; but the peptides obtained after further purification were no more stimulatory. Eleven stimulatory and two inhibitory fractions were separated from Fraction C by column chromatography on cation‐exchange resins. Purified stimulatory peptides were obtained from two fractions (C1 and C42) only, after anion‐exchange resins (three peptides from Fraction C42, eleven peptides from Fraction C1).These fourteen purified stimulatory peptides display a very variable qualitative amino‐acid composition. The polar part (Lys, Glx, Asx) was as important as the hydrophobic part (Pro, Leu, Val, Ala, Gly). The number of distinct ammo‐acid residues from one peptide was also quite variable: fourteen to four residues. Only lysyl residue was present in all peptides and tryptophan was not present. Three peptides that contained cysteine were issued from ϰ‐casein [18]; two peptides (Cla2α and C1C3) could be located respectively to Ala26… Gln52 and Ile81…Arg119 regions from bovine αs1B casein.A complete mixture of amino acids exerted growth stimulation. The set of the ammo acids present in a stimulatory peptide was always stimulatory.These data substantiate the following conclusions: stimulatory peptides acted as amino‐acid source; the amino acids have to be supplied in specific proportions in order not to disturb the cellular amino acids pool and the regulation of amino acid synthesis by Str. thermophilus; (b) the requirements in the peptide‐length residues and lysyl residue could be explained by a control system of peptide intake, for example, by a peptide‐transport system that would differ from that described in Escherichia coli.