Regulation of lactose, glucose and sucrose metabolisms in S. thermophilus.

Streptococcus thermophilus is a bacterium widely used in the production of yogurts and cheeses, where it efficiently ferments lactose, the saccharide naturally present in milk. It is also employed as a starter in dairy- or plant-based fermented foods that contain saccharides other than lactose (e.g., sucrose, glucose). However, little is known about how saccharide use is regulated, in particular when saccharides are mixed. Here, we determine the effect of the 5 sugars that S. thermophilus is able to use, at different concentration and when they are mixed on the promoter activities of the C-metabolism genes. Using a transcriptional fusion approach, we discovered that lactose and glucose modulated the activity of the lacS and scrA promoters in a concentration-dependent manner. When mixed with lactose, glucose also repressed the two promoter activities; when mixed with sucrose, lactose still repressed scrA promoter activity. We determined that catabolite control protein A (CcpA) played a key role in these dynamics. We also showed that promoter activity was linked with glycolytic flux, which varied depending on saccharide type and concentration. Overall, this study identified key mechanisms in carbohydrate metabolism - autoregulation and partial hierarchical control - and demonstrated that they are partly mediated by CcpA.

Microbial composition and viability of natural whey starters used in PDO Comté cheese-making.

Natural whey starters (NWS) are cultures with undefined multiple-strains species commonly used to speed up the fermentation process of cheeses. The aim of this study was to explore the diversity and the viability of Comté cheese NWS microbiota. Culture-dependent methods, i.e. plate counting and genotypic characterization, and culture-independent methods, i.e. qPCR, viability-qPCR, fluorescence microscopy and DNA metabarcoding, were combined to analyze thirty-six NWS collected in six Comté cheese factories at two seasons. Our results highlighted that NWS were dominated by Streptococcus thermophilus (ST) and thermophilic lactobacilli. These species showed a diversity of strains based on Rep-PCR. The dominance of Lactobacillus helveticus (LH) over Lactobacillus delbrueckii (LD) varied depending on the factory and the season. This highlighted two types of NWS: the type-ST/LD (LD > LH) and the type-ST/LH (LD < LH). The microbial composition varied depending on cheese factory. One factory was distinguished by its level of culturable microbial groups (ST, enterococci and yeast) and its fungi diversity. The approaches used to estimate the viability showed that most NWS cells were viable. Further investigations are needed to understand the microbial diversity of these NWS.

Comprehensive analysis of the CRISPR-Cas systems in Streptococcus thermophilus strains isolated from traditional yogurts.

Phage resistance is crucial for lactic acid bacteria in the dairy industry. However, identifying all phages affecting these bacteria is challenging. CRISPR-Cas systems offer a resistance mechanism developed by bacteria and archaea against phages and plasmids. In this study, 11 S. thermophilus strains from traditional yogurts underwent analysis using next-generation sequencing (NGS) and bioinformatics tools. Initial characterization involved molecular ribotyping. Bioinformatics analysis of the NGS raw data revealed that all 11 strains possessed at least one CRISPR type. A total of 21 CRISPR loci were identified, belonging to CRISPR types II-A, II-C, and III-A, including 13 Type II-A, 1 Type III-C, and 7 Type III-A CRISPR types. By analyzing spacer sequences in S. thermophilus bacterial genomes and matching them with phage/plasmid genomes, notable strains emerged. SY9 showed prominence with 132 phage matches and 30 plasmid matches, followed by SY12 with 35 phage matches and 25 plasmid matches, and SY18 with 49 phage matches and 13 plasmid matches. These findings indicate the potential of S. thermophilus strains in phage/plasmid resistance for selecting starter cultures, ultimately improving the quality and quantity of dairy products. Nevertheless, further research is required to validate these results and explore the practical applications of this approach.

Gene knock-out in Mycobacterium abscessus using Streptococcus thermophilus CRISPR/Cas.

The CRISPRi system using dCas9Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity.

Retention of the RNA ends provides the molecular memory for maintaining the activation of the Csm complex.

The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus. Although single-stranded RNA is cleaved rapidly (within seconds), dual-color FCS experiments and single-molecule TIRF microscopy revealed that Csm remains bound to terminal RNA cleavage products with a half-life of over 1 hour while releasing the internal RNA fragments quickly. Using a continuous fluorescent DNA degradation assay, we observed that RNA-regulated single-stranded DNase activity decreases on a similar timescale. These findings suggest that after fast target RNA cleavage the terminal RNA cleavage products stay bound within the Csm complex, keeping the Cas10 subunit activated for DNA destruction. Additionally, we demonstrate that during Cas10 activation, the complex remains capable of RNA turnover, i.e. of ongoing degradation of target RNA.

Phenotypic, Technological, Safety, and Genomic Profiles of Gamma-Aminobutyric Acid-Producing Lactococcus lactis and Streptococcus thermophilus Strains Isolated from Cow's Milk.

Gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) can be used as starters in the development of GABA-enriched functional fermented foods. In this work, four GABA-producing strains each of Lactococcus lactis and Streptococcus thermophilus species were isolated from cow's milk, and their phenotypic, technological, and safety profiles determined. Genome analysis provided genetic support for the majority of the analyzed traits, namely, GABA production, growth in milk, and the absence of genes of concern. The operon harboring the glutamate decarboxylase gene (gadB) was chromosomally encoded in all strains and showed the same gene content and gene order as those reported, respectively, for L. lactis and S. thermophilus. In the latter species, the operon was flanked (as in most strains of this species) by complete or truncated copies of insertion sequences (IS), suggesting recent acquisition through horizontal gene transfer. The genomes of three L. lactis and two S. thermophilus strains showed a gene encoding a caseinolytic proteinase (PrtP in L. lactis and PrtS in S. thermophilus). Of these, all but one grew in milk, forming a coagulum of good appearance and an appealing acidic flavor and taste. They also produced GABA in milk supplemented with monosodium glutamate. Two L. lactis strains were identified as belonging to the biovar. diacetylactis, utilized citrate from milk, and produced significant amounts of acetoin. None of the strains showed any noticeable antibiotic resistance, nor did their genomes harbor transferable antibiotic resistance genes or genes involved in toxicity, virulence, or pathogenicity. Altogether these results suggest that all eight strains may be considered candidates for use as starters or components of mixed LAB cultures for the manufacture of GABA-enriched fermented dairy products.

Genetic diversity and phylogenetic relationships of Streptococcus thermophilus isolates from traditional Turkish yogurt: multilocus sequence typing (MLST).

Yogurt, a globally consumed fermented dairy product, is recognized for its taste and potential health benefits attributed to probiotic bacteria, particularly Streptococcus thermophilus. In this study, we employed Multilocus Sequence Typing (MLST) to investigate the genetic diversity and phylogenetic relationships of 13 S. thermophilus isolates from traditional Turkish yogurt samples. We also assessed potential correlations between genetic traits and geographic origins. The isolates were identified as S. thermophilus using VITEK® MALDI-TOF MS, ribotyping, and 16S rRNA analysis methods. MLST analysis revealed 13 different sequence types (STs), with seven new STs for Turkey. The most prevalent STs were ST/83 (n = 3), ST/135 (n = 2), and ST/134 (n = 2). eBURST analysis showed that these isolates mainly were singletons (n = 7) defined as sequence types (STs) that cannot be assigned to any group and differ at two or more alleles from every other ST in the sample. This information suggests that the isolates under study were genetically distinct from the other isolates in the dataset, highlighting their unique genetic profiles within the population. Genetic diversity analysis of ten housekeeping genes revealed polymorphism, with some genes showing higher allelic variation than others. Tajima's D values suggested that selection pressures differed among these genes, with some being more conserved, likely due to their vital functions. Phylogenetic analysis revealed distinct genetic diversity between Turkish isolates and European and Asian counterparts. These findings demonstrate the genetic diversity of S. thermophilus isolates in Turkish yogurt and highlight their unique evolutionary patterns. This research contributes to our understanding of local microbial diversity associated with yogurt production in Turkey and holds the potential for identifyic strains with enhanced functional attributes.

Repurposing endogenous type II CRISPR-Cas9 system for genome editing in Streptococcus thermophilus.

Streptococcus thermophilus has been extensively used in industrial milk fermentation. However, lack of efficient genetic manipulation approaches greatly hampered the industrial application of this species. Here, we repurposed the endogenous CRISPR1 and CRISPR3 systems, both belong to type II-A CRISPR-Cas9, by delivering a self-targeting CRISPR array with DNA repair template into S. thermophilus LMD-9. We achieved 785-bp deletion in lacZ gene by repurposing CRISPR1 and CRISPR3 systems with efficiencies of 35% and 59%, respectively, when 1-kb DNA repair template was provided. While providing with 1.5-kb repair template, the editing efficiency for deletion in lacZ gene reached 90% using CRISPR3 systems. Diverse editing outcomes encompassing a stop code insertion and single nucleotide variation within lacZ, as well as a 234-bp DNA fragment insertion upstream of ster_0903, were generated with high efficiencies of 75%-100% using the CRISPR3 system. Harnessing the customized endogenous CRISPR3 system to target six genes of eps gene cluster, we obtained six single-gene knockout mutants with efficiencies of 29%-80%, and proved that the epsA, epsE, and epsG were the key genes affecting exopolysaccharides biosynthesis in S. thermophilus LMD-9. Altogether, repurposing the native type II-A CRISPR-Cas9 can be served as a toolkit for precise genome engineering in S. thermophilus for biotechnological applications.

Genome-scale reconstruction of the metabolic network in Streptococcus thermophilus S-3 and assess urea metabolism.

BACKGROUND: Streptococcus thermophilus is an important strain widely used in dairy fermentation, with distinct urea metabolism characteristics compared to other lactic acid bacteria. The conversion of urea by S. thermophilus has been shown to affect the flavor and acidification characteristics of milk. Additionally, urea metabolism has been found to significantly increase the number of cells and reduce cell damage under acidic pH conditions, resulting in higher activity. However, the physiological role of urea metabolism in S. thermophilus has not been fully evaluated. A deep understanding of this metabolic feature is of great significance for its production and application. Genome-scale metabolic network models (GEMs) are effective tools for investigating the metabolic network of organisms using computational biology methods. Constructing an organism-specific GEM can assist us in comprehending its characteristic metabolism at a systemic level. RESULTS: In the present study, we reconstructed a high-quality GEM of S. thermophilus S-3 (iCH492), which contains 492 genes, 608 metabolites and 642 reactions. Growth phenotyping experiments were employed to validate the model both qualitatively and quantitatively, yielding satisfactory predictive accuracy (95.83%), sensitivity (93.33%) and specificity (100%). Subsequently, a systematic evaluation of urea metabolism in S. thermophilus was performed using iCH492. The results showed that urea metabolism reduces intracellular hydrogen ions and creates membrane potential by producing and transporting ammonium ions. This activation of glycolytic fluxes and ATP synthase produces more ATP for biomass synthesis. The regulation of fluxes of reactions involving NAD(P)H by urea metabolism improves redox balance. CONCLUSION: Model iCH492 represents the most comprehensive knowledge-base of S. thermophilus to date, serving as a potent tool. The evaluation of urea metabolism led to novel insights regarding the role of urease. © 2023 Society of Chemical Industry.

Safety assessment, whole genome sequence, and metabolome analysis of Streptococcus thermophilus CICC 20372 for bone cement fermentation.

Bone is a kind of meat processing by-product with high nutritional value but low in calorie, which is a typical food in China and parts of East Asian countries. Microbial fermentation by lactic acid bacteria showed remarkable advantages to increase the absorption of nutrients from bone cement by human body. Streptococcus thermophilus CICC 20372 is proven to be a good starter for bone cement fermentation. No genes encoding virulence traits or virulence factors were found in the genome of S. thermophilus CICC 20372 by a thorough genomic analysis. Its notable absence of antibiotic resistance further solidifies the safety. Furthermore, the genomic analysis identified four types of gene clusters responsible for the synthesis of antimicrobial metabolites. A comparative metabolomic analysis was performed by cultivating the strain in bone cement at 37 °C for 72 h, with the culture in de Man, Rogosa, and Sharpe (MRS) medium as control. Metabolome analysis results highlighted the upregulation of pathways involved in 2-oxocarboxylic acid metabolism, ATP-binding cassette (ABC) transporters, amino acid synthesis, and nucleotide metabolism during bone cement fermentation. S. thermophilus CICC 20372 produces several metabolites with health-promoting function during bone cement fermentation, including indole-3-lactic acid, which is demonstrated ameliorative effects on intestinal inflammation, tumor growth, and gut dysbiosis. In addition, lots of nucleotide and organic acids were accumulated at higher levels, which enriched the fermented bone cement with a variety of nutrients. Collectively, these features endow S. thermophilus CICC 20372 a great potential strain for bone food processing.

Microbial interactions shape cheese flavour formation.

Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α-ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus, highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile.

Dairy phages escape CRISPR defence of Streptococcus thermophilus via the anti-CRISPR AcrIIA3.

Bacterial community collapse due to phage infection is a major risk in cheese making processes. As virulent phages are ubiquitous and diverse in milk fermentation factories, the use of phage-resistant lactic acid bacteria (LAB) is essential to obtain high-quality fermented dairy products. The LAB species Streptococcus thermophilus contains two type II-A CRISPR-Cas systems (CRISPR1 and CRISPR3) that can effectively protect against phage infection. However, virulent streptococcal phages carrying anti-CRISPR proteins (ACR) that block the activity of CRISPR-Cas systems have emerged in yogurt and cheese environments. For example, phages carrying AcrIIA5 can impede both CRISPR1 and CRISPR3 systems, while AcrIIA6 stops only CRISPR1. Here, we explore the activity and diversity of a third streptococcal phage anti-CRISPR protein, namely AcrIIA3. We were able to demonstrate that AcrIIA3 is efficiently active against the CRISPR3-Cas system of S. thermophilus. We used AlphaFold2 to infer the structure of AcrIIA3 and we predicted that this new family of functional ACR in virulent streptococcal phages has a new α-helical fold, with no previously identified structural homologs. Because ACR proteins are being explored as modulators in genome editing applications, we also tested AcrIIA3 against SpCas9. We found that AcrIIA3 could block SpCas9 in bacteria but not in human cells. Understanding the diversity and functioning of anti-defence mechanisms will be of importance in the design of long-term stable starter cultures.

Genomic and phenotypic-based safety assessment and probiotic properties of Streptococcus thermophilus FUA329, a urolithin A-producing bacterium of human milk origin.

Streptococcus thermophilus FUA329, a urolithin A-producing bacterium, is isolated from human breast milk. The complete genome sequence of FUA329 did not contain any plasmids and at least 20 proteins were related to extreme environment resistance. Phenotypic assay results demonstrated that FUA329 was susceptible to 12 kinds of antibiotics and did not exhibit any hemolytic or nitrate reductase activity. Three free radical scavenging assays revealed that FUA329 have high antioxidant capability. FUA329 exhibited a cell surface hydrophobicity of 52.58 ± 1.17% and an auto-aggregation rate of 18.69 ± 2.48%. Moreover, FUA329 demonstrated a survival rate of over 60% in strong acid and bile salt environments, indicating that FUA329 may be stable colonization in the gastrointestinal tract. Additionally, we firstly found 3 potential proteins and 11 potential genes of transforming ellagic acid to urolithins in FUA329 genome. The above results indicate that FUA329 has credible safety and probiotic properties, as well as the potential to be developed as a new generation of urolithin A-producing probiotics.

Development of a Tetracycline-Inducible System for Conditional Gene Expression in Lactococcus lactis and Streptococcus thermophilus.

Inducible gene expression systems are invaluable tools for the functional characterization of genes and in the construction of protein overexpression hosts. Controllable expression is especially important for the study of essential and toxic genes or genes where the level of expression tightly influences their cellular effect. Here, we implemented the well-characterized tetracycline-inducible expression system in two industrially important lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus. Using a fluorescent reporter gene, we show that optimization of the repression level is necessary for efficient induction using anhydrotetracycline in both organisms. Random mutagenesis in the ribosome binding site of the tetracycline repressor TetR in Lactococcus lactis indicated that altering the expression levels of TetR was necessary for efficient inducible expression of the reporter gene. Through this approach, we achieved plasmid-based, inducer-responsive, and tight gene expression in Lactococcus lactis. We then verified the functionality of the optimized inducible expression system in Streptococcus thermophilus following its chromosomal integration using a markerless mutagenesis approach and a novel DNA fragment assembly tool presented herein. This inducible expression system holds several advantages over other described systems in lactic acid bacteria, although more efficient techniques for genetic engineering are still needed to realize these advantages in industrially relevant species, such as S. thermophilus. Our work expands the molecular toolbox of these bacteria, which can accelerate future physiological studies. IMPORTANCE Lactococcus lactis and Streptococcus thermophilus are two industrially important lactic acid bacteria globally used in dairy fermentations and, therefore, are of considerable commercial interest to the food industry. Moreover, due to their general history of safe usage, these microorganisms are increasingly being explored as hosts for the production of heterologous proteins and various chemicals. Development of molecular tools in the form of inducible expression systems and mutagenesis techniques facilitates their in-depth physiological characterization as well as their exploitation in biotechnological applications.

Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.

Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.

Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus in Physiological Conditions Mimicking the Human Digestive Ecosystem.

Metagenome analyses of the human microbiome suggest that horizontal gene transfer (HGT) is frequent in these rich and complex microbial communities. However, so far, only a few HGT studies have been conducted in vivo. In this work, three different systems mimicking the physiological conditions encountered in the human digestive tract were tested, including (i) the TNO gastro-Intestinal tract Model 1 (TIM-1) system (for the upper part of the intestine), (ii) the ARtificial COLon (ARCOL) system (to mimic the colon), and (iii) a mouse model. To increase the likelihood of transfer by conjugation of the integrative and conjugative element studied in the artificial digestive systems, bacteria were entrapped in alginate, agar, and chitosan beads before being placed in the different gut compartments. The number of transconjugants detected decreased, while the complexity of the ecosystem increased (many clones in TIM-1 but only one clone in ARCOL). No clone was obtained in a natural digestive environment (germfree mouse model). In the human gut, the richness and diversity of the bacterial community would offer more opportunities for HGT events to occur. In addition, several factors (SOS-inducing agents, microbiota-derived factors) that potentially increase in vivo HGT efficiency were not tested here. Even if HGT events are rare, expansion of the transconjugant clones can happen if ecological success is fostered by selecting conditions or by events that destabilize the microbial community. IMPORTANCE The human gut microbiota plays a key role in maintaining normal host physiology and health, but its homeostasis is fragile. During their transit in the gastrointestinal tract, bacteria conveyed by food can exchange genes with resident bacteria. New traits acquired by HGT (e.g., new catabolic properties, bacteriocins, antibiotic resistance) can impact the gut microbial composition and metabolic potential. We showed here that TIM-1, a system mimicking the upper digestive tract, is a useful tool to evaluate HGT events in conditions closer to the physiological ones. Another important fact pointed out in this work is that Enterococcus faecalis is a good candidate for foreign gene acquisition. Due to its high ability to colonize the gut and acquire mobile genetic elements, this commensal bacterium could serve as an intermediate for HGT in the human gut.

The Ability of Streptococcus thermophilus BT01 to Modulate Urease Activity in Healthy Subjects' Fecal Samples Depends on the Biomass Production Process.

SCOPE: This study evaluates how manufacturing conditions of probiotic biomass production, using two different cryoprotectants, Cryo-A and Cryo-B, can affect Streptococcus thermophilus BT01 in vivo gastrointestinal tract survival and its ability to modulate the level of urease activity in fecal samples of healthy subjects. METHODS AND RESULTS: A randomized controlled cross-over study is carried out on 20 adult healthy subjects to evaluate total and viable loads, persistence of S. thermophilus BT01, and urease activity in fecal samples. Strain-specific quantification by using developed culture-based method and molecular qPCR tool allows to quantify viable S. thermophilus BT01 strain in 90% of the subjects. The quantification of both total DNA and recovered viable S. thermophilus BT01 in fecal samples does not reveal significant differences between Cryo-A or Cryo-B treated biomass. However, the administration of S. thermophilus BT01 produced with Cryo-A results in a decreased urease activity in fecal samples compared to Cryo-B protected cells. CONCLUSION: This study i) highlights how the manufacturing conditions can play a role in influencing the probiotic functionality in vivo and ii) represents the first evidence that links S. thermophilus to a specific probiotic mechanism, the reduction of urease activity in fecal samples.

Bipartite rgp Locus Diversity in Streptococcus thermophilus Corresponds to Backbone and Side Chain Differences of Its Rhamnose-Containing Cell Wall Polysaccharide.

The rhamnose-glucose polysaccharide (Rgp) of Streptococcus thermophilus represents a major cell wall component, and the gene cluster responsible for its biosynthesis (termed rgp) has recently been identified. Significant genetic diversity among these loci has previously been reported, with five distinct rgp genotypes identified (designated rgp1 through -5). In the present study, two additional genotypes were identified (designated rgp6 and rgp7) through comparative analysis of the rgp loci of 78 Streptococcus thermophilus genomes. The rgp locus of a given S. thermophilus strain encoded the biosynthetic machinery for a rhamnan-rich backbone and a variable side chain component, the latter being associated with the highly specific interactions with many bacteriophages that infect this species. The chemical structure of the Rgp from three S. thermophilus strains, representing the rgp2, -3, and -4 genotypes, was elucidated, and based on bioinformatic and biochemical analyses we propose a model for Rgp biosynthesis in dairy streptococci. Furthermore, we exploited the genetic diversity within the S. thermophilus bipartite rgp locus to develop a two-step multiplex PCR system to classify strains based on gene content associated with the biosynthesis of the variable side chain structure as well as the rhamnan backbone. IMPORTANCE Streptococcus thermophilus is present and applied in industrial and artisanal dairy fermentations for the production of various cheeses and yogurt. During these fermentations, S. thermophilus is vulnerable to phage predation, and recent studies have identified the rhamnose-glucose polymer (Rgp) as the definitive receptor for at least one problematic phage species. Detailed analysis of S. thermophilus rgp loci has revealed an unprecedented level of genetic diversity, particularly within the glycosyltransferase-encoding gene content of a given locus. Our study shows that this genetic diversity reflects the biochemical structure(s) of S. thermophilus Rgp. As such, we harnessed the genetic diversity of S. thermophilus rgp loci to develop a two-step multiplex PCR method for the classification of strain collections and, ultimately, the formation of phage-robust rational starter sets.

Elucidation of the initial bacterial community of Ezine PDO cheese using next-generation sequencing.

This study aims to reveal initial bacterial consortia of Ezine PDO cheeses comprehensively by following a metagenomic approach. A total of 8 artisanal Ezine cheese samples were collected from the Bayramiç and Ezine districts of Çanakkale province of Turkey. Ezine cheese was found to contain Firmicutes, Bacteroidetes, and Proteobacteria phyla dominantly. Streptococcus, Lactococcus, and Lactobacillus genera dominated the microbiota with relative abundances of 4.47-56.07%, 7.33-20.34%, and 1.21-25.12%, respectively, followed by Bacteroides and Prevotella genera. Excluding two cheese samples obtained from the Ezine district, the most dominant species was Streptococcus thermophilus (8.24-54.34%). It was also found in greater proportions in the cheeses of the Bayramiç district. Unexpectedly, Lactobacillus graminis (11.50-23.63%) was the most abundant species in two samples collected from the Ezine district. However, lower bacterial diversity was determined in the samples collected from the Bayramiç district. The lowest species richness was 129 OTU-species in the cheeses from the Bayramiç district while the highest species richness was 267 OTU-species in cheeses from the Ezine district. In addition, the Simpson index was the highest in cheeses from the Ezine district, where different species were evenly distributed. Permutational multivariate analysis of variance tests also confirmed that the differences in the structure of bacterial consortia in cheeses from two different districts were statistically significant. This study will provide pioneer data for further investigations on the role of complex bacterial composition in maintaining and improving the quality and safety of Ezine cheese.

Enhanced Stability and Function of Probiotic Streptococcus thermophilus with Self-Encapsulation by Increasing the Biosynthesis of Hyaluronan.

The harsh conditions of the gastrointestinal tract limit the potential health benefits of oral probiotics. It is promising that oral bioavailability is improved by strengthening the self-protection of probiotics. Here, we report the encapsulation of a probiotic strain by endogenous production of hyaluronan to enhance the effects of oral administration of the strain. The traditional probiotic Streptococcus thermophilus was engineered to produce hyaluronan shells by using traceless genetic modifications and clustered regularly interspaced short palindromic repeat interference. After oral delivery to mice in the form of fermented milk, hyaluronan-coated S. thermophilus (204.45 mg/L hyaluronan in the milk) exhibited greater survival and longer colonization time in the gut than the wild-type strain. In particular, the engineered probiotic strain could also produce hyaluronan after intestinal colonization. Importantly, S. thermophilus self-encapsulated with hyaluronan increased the number of goblet cells, mucus production, and abundance of the microorganisms related to the biosynthesis of short-chain fatty acids, resulting in the enhancement of the intestinal barrier. The coating formed by endogenous hyaluronan provides an ideal reference for the effective oral administration of probiotics.