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Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP 1358 ) and a transcriptional regulator (Rgg 1358 ) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP 1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus , whose myriad applications include the delivery of therapeutic peptides or proteins.

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.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30-220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of "elementary bodies" and "not-cell bound antigens"., Competing Interests: The authors have declared that no competing interests exist.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the "xyl-doc" cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics., Competing Interests: We have the following interests: Victor Sabarly and Marc Dubois are employed by Omics Services. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism.

Objective: No Crohn's disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls., Design: We first developed and validated a workflow-including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS-to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions., Results: Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins., Conclusions: This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

We described a quorum-sensing mechanism in the streptococci genus involving a short hydrophobic peptide (SHP), which acts as a pheromone, and a transcriptional regulator belonging to the Rgg family. The shp/rgg genes, found in nearly all streptococcal genomes and in several copies in some, have been classified into three groups. We used a genetic approach to evaluate the functionality of the SHP/Rgg quorum-sensing mechanism, encoded by three selected shp/rgg loci, in pathogenic and non-pathogenic streptococci. We characterized the mature form of each SHP pheromone by mass-spectrometry. We produced synthetic peptides corresponding to these mature forms, and used them to study functional complementation and cross-talk between these different SHP/Rgg systems. We demonstrate that a SHP pheromone of one system can influence the activity of a different system. Interestingly, this does not seem to be dependent on the SHP/Rgg group and cross-talk between pathogenic and non-pathogenic streptococci is observed.

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.

Phosphorylation is the most common and widely studied post-translational protein modification in bacteria. It plays an important role in all kinds of cellular processes and controls key regulatory mechanisms, including virulence in certain pathogens. To gain insight into the role of protein phosphorylation in the pathogen Listeria monocytogenes, the serine (Ser), threonine (Thr) and tyrosine (Tyr) phosphoproteome of this bacterium was determined. We used the "gel free" proteomic approach with high accuracy mass spectrometry after enrichment of phosphopeptides. A total of 143 sites of phosphorylation were clearly identified, on 155 unique peptides of 112 phosphoproteins. The Ser/Thr/Tyr phosphorylation site distribution was 93:43:7. All identified phosphopeptides are monophosphorylated, except one and many identified phosphoproteins are related to virulence, translation, phosphoenolpyruvate:sugar phosphotransferase system, glycolysis and stress response. A description of these phosphoproteins is provided together with a comparison of the phosphosites in the L. monocytogenes proteins and in their homologues of other bacteria for which the phosphoproteome has been determined. Compared with the previous studies, we noticed a more extended conservation of the phosphorylation sites in glycolytic enzymes as well as ribosomal proteins., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In prokaryotes, transcription results from the activity of a 400 kDa RNA polymerase (RNAP) protein complex composed of at least five subunits (2α, β, β', ω). To ensure adequate responses to changing environmental cues, RNAP activity is tightly controlled by means of interacting regulatory proteins. Here, we report the affinity-purification of the Bacillus subtilis RNAP complexes from cells in different growth states and stress conditions, and the quantitative assessment by mass spectrometry of the dynamic changes in the composition of the RNAP complex. The stoichiometry of RNA polymerase was determined by a comparison of two mass spectrometry-based quantification methods: a label-based and a label-free method. The validated label-free method was then used to quantify the proteins associated with RNAP. The levels of sigma factors bound to RNAP varied during growth and exposure to stress. Elongation factors, helicases such as HelD and PcrA, and novel unknown proteins were also associated with RNAP complexes. The content in 6S RNAs of purified RNAP complexes increased at the onset of the stationary phase. These quantitative variations in the protein and RNA composition of the RNAP complexes well correlate with the known physiology of B. subtilis cells under different conditions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We characterized the insoluble proteome of Lactococcus lactis using 1D electrophoresis-LC-MS/MS and identified 313 proteins with at least two different peptides. The identified proteins include 89 proteins having a predicted signal peptide and 25 predicted to be membrane-located. In addition, 67 proteins had alkaline isoelectric point values. Using spectra and peptide counts, we compared protein abundances in two different conditions: growth in rich medium, and after transit in the mouse digestive tract. We identified the large mechanosensitive channel and a putative cation transporter as membrane markers of bacterial adaptation to the digestive tract.

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.

Lactococcus lactis is a model of food-grade lactic acid bacterium, which can durably colonize the digestive tract of germ-free mice. To study in vivo the bacterial adaptation to a novel nutritional resource brought by alimentation, the lactose-catabolizing strain IL2661 of L. lactis was established in monoxeny in mice. Half of the mice then received a lactose-rich diet. The mouse has no efficient intestinal lactase and is well adapted to a follow-up of the metabolic activity of microbial origin. The analysis of lactose and lactate in the feces suggested that L. lactis was able to use lactose in vivo. We developed a proteomic approach to evaluate in deeper details the metabolic response of the bacterium. We observed that L. lactis switched its metabolism to use the novel carbon source and reduced the level of proteins involved in an alternative mode of ATP production. In parallel, we also found that the amount of proteins involved in transcriptional regulation, transport and catabolism decreased in the presence of lactose. The proteome analysis informed us about the resources used by the bacteria in absence of lactose. In competition experiments, we found that the metabolic adaptation gives a strong ecological advantage to the bacteria able to efficiently utilize lactose.

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.