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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]

Abstract: 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 &y& Elsevier]

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.

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/Rgg1358 and SHP/Rgg1299), and the ComS/ComR system, while using transcriptional fusions and that PptAB helped to produce and export at least three different mature SHPs (SHP1358, SHP1299, and SHP279) 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. [ABSTRACT FROM AUTHOR]