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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 wildtype strain. [ABSTRACT FROM AUTHOR]

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 the 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 acids quantification, we used a multi-omics approach (peptidomics, proteomics and transcriptomics) to identify peptide 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 composition of the two yeast extracts differed qualitatively and quantitatively. Nevertheless, the two yeast extracts sustained similar 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 competencestate 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. [ABSTRACT FROM AUTHOR]

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 H2O2 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. [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.

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. [ABSTRACT FROM AUTHOR]