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The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely, the cell envelope protease PrtS and the housekeeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell surface protease, which shares significant homology with the SepM protease of Streptococcus mutans. Although all three proteases-PrtS, HtrA, and SepM-are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wild-type strain. IMPORTANCE Streptococcus thermophilus is a lactic acid bacterium used widely as a starter in the dairy industry. Due to its "generally recognized as safe" status and its weak cell surface proteolytic activity, it is also considered a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications.

Peptides present in growth media are essential for nitrogen nutrition and optimal growth of lactic acid bacteria. In addition, according to their amino acid composition, they can also directly or indirectly play regulatory roles and influence global metabolism. This is especially relevant during the propagation phase to produce high cell counts of active lactic acid bacteria used as starters in the dairy industry. In the present work, we aimed at investigating how the respective compositions of two different yeast extracts, with a specific focus on peptide content, influenced Streptococcus thermophilus metabolism during growth under pH-controlled conditions. In addition to free amino acid quantification, we used a multi-omics approach (peptidomics, proteomics, and transcriptomics) to identify peptides initially present in the two culture media and to follow S. thermophilus gene expression and bacterial protein production during growth. The free amino acid and peptide compositions of the two yeast extracts differed qualitatively and quantitatively. Nevertheless, the two yeast extracts sustained similar levels of growth of S. thermophilus and led to equivalent final biomasses. However, transcriptomics and proteomics showed differential gene expression and protein production in several S. thermophilus metabolic pathways, especially amino acid, citrate, urease, purine, and pyrimidine metabolisms. The probable role of the regulator CodY is discussed in this context. Moreover, we observed significant differences in the production of regulators and of a quorum sensing regulatory system. The possible roles of yeast extract peptides on the modulation of the quorum sensing system expression are evaluated. IMPORTANCE Improving the performance and industrial robustness of bacteria used in fermentations and food industry remains a challenge. We showed here that two Streptococcus thermophilus fermentations, performed with the same strain in media that differ only by their yeast extract compositions and, more especially, their peptide contents, led to similar growth kinetics and final biomasses, but several genes and proteins were differentially expressed/produced. In other words, subtle variations in peptide composition of the growth medium can finely tune the metabolism status of the starter. Our work, therefore, suggests that acting on growth medium components and especially on their peptide content, we could modulate bacterial metabolism and produce bacteria differently programmed for further purposes. This might have applications for preparing active starter cultures., (Copyright © 2020 American Society for Microbiology.)

In streptococci, intracellular quorum sensing pathways are based on quorum-sensing systems that are responsible for peptide secretion, maturation, and reimport. These peptides then interact with Rgg or ComR transcriptional regulators in the Rap, Rgg, NprR, PlcR, and PrgX (RRNPP) family, whose members are found in Gram-positive bacteria. Short hydrophobic peptides (SHP) interact with Rgg whereas ComS peptides interact with ComR regulators. To date, in Streptococcus thermophilus , peptide secretion, maturation, and extracellular fate have received little attention, even though this species has several (at least five) genes encoding Rgg regulators and one encoding a ComR regulator. We studied pheromone export in this species, focusing our attention on PptAB, which is an exporter of signaling peptides previously identified in Enterococcus faecalis , pathogenic streptococci and Staphylococcus aureus . In the S. thermophilus strain LMD-9, we showed that PptAB controlled three regulation systems, two SHP/Rgg systems (SHP/Rgg 1358 and SHP/Rgg 1299 ), and the ComS/ComR system, while using transcriptional fusions and that PptAB helped to produce and export at least three different mature SHPs (SHP 1358 , SHP 1299 , and SHP 279 ) peptides while using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using a deep sequencing approach (RNAseq), we showed that the exporter PptAB, the membrane protease Eep, and the oligopeptide importer Ami controlled the transcription of the genes that were located downstream from the five non-truncated rgg genes as well as few distal genes. This led us to propose that the five non-truncated shp / rgg loci were functional. Only three shp genes were expressed in our experimental condition. Thus, this transcriptome analysis also highlighted the complex interconnected network that exists between SHP/Rgg systems, where a few homologous signaling peptides likely interact with different regulators.

In streptococci, ComX is the alternative sigma factor controlling the transcription of the genes encoding the genetic transformation machinery. In Streptococcus thermophilus, comX transcription is controlled by a complex consisting of a transcriptional regulator of the Rgg family, ComR, and a signaling peptide, ComS, which controls ComR activity. Following its initial production, ComS is processed, secreted, and imported back into the cell by the Ami oligopeptide transporter. We characterized these steps and the partners interacting with ComS during its extracellular circuit in more detail. We identified the mature form of ComS and demonstrated the involvement of the membrane protease Eep in ComS processing. We found that ComS was secreted but probably not released into the extracellular medium. Natural competence was first discovered in a chemically defined medium without peptides. We show here that the presence of a high concentration of nutritional peptides in the medium prevents the triggering of competence. In milk, the ecological niche of S. thermophilus, competence was found to be functional, suggesting that the concentration of nutritional peptides was too low to interfere with ComR activation. The kinetics of expression of the comS, comR, and comX genes and of a late competence gene, dprA, in cultures inoculated at different initial densities revealed that the activation mechanism of ComR by ComS is more a timing device than a quorum-sensing mechanism sensu stricto. We concluded that the ComS extracellular circuit facilitates tight control over the triggering of competence in S. thermophilus.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography-tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

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.

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.

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.

Streptococcus thermophilus is a major dairy starter used for the manufacture of yoghurt and cheese. The access to three genome sequences, comparative genomics and multilocus sequencing analyses suggests that this species recently emerged and is still undergoing a process of regressive evolution towards a specialised bacterium for growth in milk. Notably, S. thermophilus has maintained a well-developed nitrogen metabolism whereas its sugar catabolism has been subjected to a high level of degeneracy due to a paucity of carbon sources in milk. Furthermore, while pathogenic streptococci are recognised for a high capacity to expose proteins at their cell surface in order to achieve cell adhesion or to escape the host immune system, S. thermophilus has nearly lost this unique feature as well as many virulence-related functions. Although gene decay is obvious in S. thermophilus genome evolution, numerous small genomic islands, which were probably acquired by horizontal gene transfer, comprise important industrial phenotypic traits such as polysaccharide biosynthesis, bacteriocin production, restriction-modification systems or oxygen tolerance.

Protein phosphorylation especially on serine/threonine/tyrosine residues are frequent in many bacteria. This post-translational modification has been associated with pathogenicity and virulence in various species. However, only few data have been produced so far on generally recognized as safe bacteria used in food fermentations. A family of kinases known as Hanks-type kinases is suspected to be responsible for, at least, a part of these phosphorylations in eukaryotes as in bacteria. The objective of our work was to establish the first phosphoproteome of Streptococcus thermophilus , a lactic acid bacterium widely used in dairy fermentations in order to identified the proteins and pathways tagged by Ser/Thr/Tyr phosphorylations. In addition, we have evaluated the role in this process of the only Hanks-type kinase encoded in the S. thermophilus genome. We have constructed a mutant defective for the Hanks type kinase in S. thermophilus and established the proteomes and phosphoproteomes of the wild type and the mutant strains. To do that, we have enriched our samples in phosphopeptides with titane beads and used dimethyl tags to compare phosphopeptide abundances. Peptides and phosphopeptides were analyzed on a last generation LC-MS/MS system. We have identified and quantified 891 proteins representing half of the theoretical proteome. Among these proteins, 106 contained phosphorylated peptides. Various functional groups of proteins (amino acid, carbon and nucleotide metabolism, translation, cell cycle, stress response, ...) were found phosphorylated. The phosphoproteome was only weakly reduced in the Hanks-type kinase mutant indicating that this enzyme is only one of the players in the phosphorylation process. The proteins that are modified by the Hanks-type kinase mainly belong to the divisome. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus , an extensively used lactic starter, is generally produced in yeast extract-based media containing a complex mixture of peptides whose exact composition remains elusive. In this work, we aimed at investigating the peptide content of a commercial yeast extract (YE) and identifying dynamics of peptide utilization during the growth of the industrial S. thermophilus N4L strain, cultivated in 1 l bioreactors under pH-regulation. To reach that goal, we set up a complete analytical workflow based on mass spectrometry (peptidomics). About 4,600 different oligopeptides ranging from 6 to more than 30 amino acids in length were identified during the time-course of the experiment. Due to the low spectral abundance of individual peptides, we performed a clustering approach to decipher the rules of peptide utilization during fermentation. The physicochemical characteristics of consumed peptides perfectly matched the known affinities of the oligopeptide transport system of S. thermophilus. Moreover, by analyzing such a large number of peptides, we were able to establish that peptide net charge is the major factor for oligopeptide transport in S. thermophilus N4L. [ABSTRACT FROM AUTHOR]

Describes the growth phases of Streptococcus thermophilus in milk. Relationship between proteolysis and growth of S. thermophilus; Ability to produce a functional cell wall proteinase; Increase in the concentrations of free amino acids.