RESUMO
ß-Aspartyl compounds, such as ß-aspartyl hydroxamate (serine racemase inhibitor), ß-aspartyl-l-lysine (moisture retention), and ß-aspartyl-l-tryptophan (immunomodulator) are physiologically active compounds. There is limited literature on the development of effective methods of production of ß-aspartyl compounds. In this study, we describe the biochemical characterization of asparagine synthetase (AS) from Streptococcus thermophilus NBRC 13957 (StAS) and the enzymatic synthesis of ß-aspartyl compounds using StAS. Recombinant StAS was expressed in Escherichia coli BL21(DE3) and it displayed activity towards hydroxylamine, methylamine, ethylamine, and ammonia, as acceptors of the ß-aspartyl moiety. StAS exhibited higher activity toward hydroxylamine and ethylamine as acceptor substrates compared with the enzymes from Lactobacillus delbrueckii NBRC 13953, Lactobacillus reuteri NBRC 15892, and E. coli. The coupling of the synthesis of ß-aspartyl compounds by StAS with an ATP-regeneration system using polyphosphate kinase from Deinococcus proteoliticus NBRC 101906 displayed an approximately 2.5-fold increase in the production of ß-aspartylhydroxamate from 1.06 mM to 2.53 mM after a 76-h reaction.
Assuntos
Aspartato-Amônia Ligase , Escherichia coli , Proteínas Recombinantes , Streptococcus thermophilus , Streptococcus thermophilus/enzimologia , Streptococcus thermophilus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Aspartato-Amônia Ligase/metabolismo , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Ácido Aspártico/metabolismo , Ácido Aspártico/biossíntese , Especificidade por Substrato , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/químicaRESUMO
To explore the roles of loops around active pocket in the reuteran type 4,6-α-glucanotransferase (StGtfB) from S. thermophilus, they were individually or simultaneously replaced with those of an isomalto/maltopolysaccharides type 4,6-α-glucanotransferase from L. reuteri. StGtfB with the replaced loops A1, A2 (A1A2) and A1, A2, B (A1A2B), respectively, showed 1.41- and 0.83-fold activities of StGtfB. Two mutants reduced crystallinity and increased starch disorder at 2, 4, and 8 U/g more than StGtfB and increased DP ≤ 5 short branches of starch by 38.01% at 2 U/g, much more than StGtfB by 4.24%. A1A2B modified starches had the lowest retrogradation over 14 days. A1A2 modified starches had the highest percentage of slowly digestible fractions, ranging from 40.32% to 43.34%. StGtfB and its mutants bind substrates by hydrogen bonding and van der Waals forces at their nonidentical amino acid residues, suggesting that loop replacement leads to a different conformation and changes activity and product structure.
Assuntos
Proteínas de Bactérias , Sistema da Enzima Desramificadora do Glicogênio , Streptococcus thermophilus , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Sistema da Enzima Desramificadora do Glicogênio/química , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/genética , Cinética , Amido/metabolismo , Amido/química , Streptococcus thermophilus/enzimologia , Streptococcus thermophilus/genética , Streptococcus thermophilus/química , Streptococcus thermophilus/metabolismo , Especificidade por SubstratoRESUMO
Streptococcus thermophilus is a common starter in yogurt production and plays an important role in the dairy industry. In this study, a galactose-positive (Gal+) mutant strain, IMAU20246Y, was produced using the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (NTG) from wild-type S. thermophilus IMAU20246, which is known to have good fermentation characteristics. The sugar content of milk fermented by either the mutant or the wild type was determined using HPLC; metabolism of lactose and galactose was significantly increased in the mutant strain. In addition, we used response surface methodology to optimize components of the basic M17 medium for survival ratio of the mutant strain. Under these optimal conditions, the viable counts of mutant S. thermophilus IMAU20246Y reached 4.15 × 108 cfu/mL and, following freeze-drying in the medium, retained cell viability of up to 67.42%. These results are conducive to production of a high-vitality starter culture and development of "low sugar, high sweetness" dairy products.
Assuntos
Fermentação , Galactose , Streptococcus thermophilus , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Galactose/metabolismo , Lactose/metabolismo , Animais , Leite , Iogurte/microbiologia , MutaçãoRESUMO
Our study aims to investigate the impact of probiotic consumption during pregnancy on gut microbiota functional diversity in healthy pregnant women. Thirty-two pregnant women were randomly assigned to two groups. The probiotic group (PG) consisted of pregnant women who consumed triple viable Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, and Streptococcus thermophilus tablets from the 32nd week of pregnancy until delivery. The functional profiles of the gut microbiota were predicted through high-throughput 16S rRNA sequencing results using PICRUSt software and referencing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In the gut microbiota of the PG, the genera Blautia and Ruminococcus, as well as the species Subdoligranulum, showed significantly higher relative abundances compared to the control group (CG) (P < 0.05). At Level 1 of the KEGG signaling pathways, there was a significant reduction in the functional genes of the gut microbiota involved in Organismal Systems in the PG (P < 0.05). In Level 2 of the KEGG signaling pathways, there was a significant reduction in the functional genes of the gut microbiota involved in Infectious Disease in the PG (P < 0.05). In Level 3 of the KEGG signaling pathways, the PG exhibited a significant increase in the functional genes of the gut microbiota involved in ABC transporters, Oxidative phosphorylation, Folate biosynthesis, and Biotin metabolism (P < 0.05). The CG showed a significant increase in the functional genes related to Cysteine and methionine metabolism, Vitamin B6 metabolism, Tuberculosis, and Vibrio cholerae pathogenic cycle (P < 0.05). In conclusion, our findings suggest that probiotic supplementation during pregnancy has a significant impact on functional metabolism in healthy pregnant women. IMPORTANCE: Probiotics are considered beneficial to human health. There is limited understanding of how probiotic consumption during pregnancy affects the functional diversity of the gut microbiota. The aim of our study is to investigate the impact of probiotic consumption during pregnancy on the functional diversity of the gut microbiota. Our findings suggest that probiotic supplementation during pregnancy has a significant impact on functional metabolism. This could potentially open up new avenues for preventing various pregnancy-related complications. This also provides new insights into the effects of probiotic consumption during pregnancy on the gut microbiota and offers a convenient method for exploring the potential mechanisms underlying the impact of probiotics on the gut microbiota of pregnant women.
Assuntos
Microbioma Gastrointestinal , Probióticos , RNA Ribossômico 16S , Humanos , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Probióticos/administração & dosagem , Gravidez , Adulto , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Bactérias/efeitos dos fármacos , Fezes/microbiologia , Streptococcus thermophilus/genética , Bifidobacterium longum , Adulto Jovem , Lactobacillus delbrueckii/genéticaRESUMO
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.
Assuntos
Bacteriófagos , Streptococcus thermophilus , Streptococcus thermophilus/genética , Sistemas CRISPR-Cas , Iogurte , Bacteriófagos/genética , Plasmídeos/genéticaRESUMO
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.
Assuntos
Glucose , Lactose , Lactose/metabolismo , Glucose/metabolismo , Metabolismo dos Carboidratos , Glicólise , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Sacarose/metabolismoRESUMO
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.
Assuntos
Queijo , Lactobacillus delbrueckii , Lactobacillus helveticus , Soro do Leite , Queijo/microbiologia , Microbiologia de Alimentos , Proteínas do Soro do Leite/análise , Streptococcus thermophilus/genéticaRESUMO
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.
Assuntos
Mycobacterium abscessus , Mycobacterium tuberculosis , Mycobacterium abscessus/genética , Sistemas CRISPR-Cas , Streptococcus thermophilus/genética , Mycobacterium tuberculosis/genéticaRESUMO
The synergistic fermentation of milk by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus is one of the key factors that determines the quality of yogurt. In this study, the mechanism whereby yogurt flavor compounds are produced by a mixture of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B were investigated by examining the flavor production, growth, and gene transcription of these strains. The results showed that yogurt produced by a 10:1 mixture of the aforementioned strains had the highest abundance of acetoin, whereas yogurt produced by a 1:1 mixture had the highest abundance of diacetyl and acetaldehyde. In addition, the growth of S. thermophilus SIT-20.S was enhanced in the 10:1 mixture. Transcriptomic analysis revealed differentially expressed genes in the flavor-compound-related pathways of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B in yogurts produced by 10:1 and 1:1 mixtures compared with those produced by either strain alone. Mixed fermentations regulated the expression of genes related to glycolysis, resulting in an increase of pyruvate, which is an important precursor for diacetyl and acetoin synthesis. The gene encoding the acetoin reductase (SIT-20S_orf01454) was decreased in S. thermophilus SIT-20.S, which ensured the accumulation of acetoin. In addition, the gene encoding the acetaldehyde dehydrogenase (SIT-20S_orf00949) was upregulated in S. thermophilus SIT-20.S, and the expression of alcohol dehydrogenase (SIT-20S_orf01479; SIT-17B_orf00943) was downregulated in both strains, maintaining the abundance of acetaldehyde. In addition, the gene encoding the NADH oxidase (SIT-17B_orf00860) in L. delbrueckii ssp. bulgaricus SIT-17.B were upregulated, which promoted the accumulation of diacetyl and acetoin. Overall, we characterized the mechanism by which S. thermophilus and L. delbrueckii ssp. bulgaricus synergistically generated yogurt flavor compounds during their production of yogurt and highlighted the importance of appropriate proportions of fermentation starters for improving the flavor of yogurts.
Assuntos
Fermentação , Iogurte , Animais , Aromatizantes , Acetoína/metabolismo , Lactobacillus delbrueckii/genética , Lactobacillus delbrueckii/metabolismo , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Leite/química , Transcriptoma , Paladar , Diacetil/metabolismoRESUMO
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.
Assuntos
Streptococcus thermophilus , Iogurte , Tipagem de Sequências Multilocus/métodos , Streptococcus thermophilus/genética , Filogenia , RNA Ribossômico 16S/genética , Turquia , Polimorfismo Genético , Variação GenéticaRESUMO
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.
Assuntos
Queijo , Lactobacillales , Lactococcus lactis , Animais , Leite/microbiologia , Lactococcus lactis/genética , Streptococcus thermophilus/genética , Ácido gama-Aminobutírico , Genômica , Fermentação , Queijo/microbiologiaRESUMO
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.
Assuntos
Streptococcus thermophilus , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , RNA/metabolismo , RNA/química , Proteínas Associadas a CRISPR/metabolismo , DNA/metabolismo , DNA/química , DNA/genética , Cinética , Clivagem do RNA , Hidrólise , Imagem Individual de Molécula , Ligação ProteicaRESUMO
The health benefits conferred by probiotics is specific to individual probiotic strains, highlighting the importance of identifying specific strains for research and production purposes. Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 are exceedingly valuable for commercial use with an excellent mixed-culture fermentation. To differentiate these 2 strains from other S. thermophilus and L. delbrueckii ssp. bulgaricus, a specific, sensitive, accurate, rapid, convenient, and cost-effective method is required. In this study, we conducted a pan-genome analysis of S. thermophilus and L. delbrueckii ssp. bulgaricus to identify species-specific core genes, along with strain-specific SNPs. These genes were used to develop suitable PCR primers, and the conformity of sequence length and unique SNPs was confirmed by sequencing for qualitative identification at the strain level. The results demonstrated that SNPs analysis of PCR products derived from these primers could distinguish CICC 6038 and CICC 6047 accurately and reproducibly from the other strains of S. thermophilus and L. delbrueckii ssp. bulgaricus, respectively. The strain-specific PCR method based on SNPs herein is universally applicable for probiotics identification. It offers valuable insights into identifying probiotics at the strain level that is fit-for-purpose in quality control and compliance assessment of commercial dairy products.
Assuntos
Lactobacillus delbrueckii , Polimorfismo de Nucleotídeo Único , Streptococcus thermophilus , Streptococcus thermophilus/genética , Lactobacillus delbrueckii/genética , Probióticos , Genômica , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Redes e Vias Metabólicas , Streptococcus thermophilus , Animais , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Fermentação , Leite/química , Ureia/metabolismo , Trifosfato de Adenosina/análiseRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Streptococcus thermophilus/genética , DNARESUMO
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.
Assuntos
Cimentos Ósseos , Streptococcus thermophilus , Humanos , Fermentação , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Cimentos Ósseos/metabolismo , Metaboloma , Nucleotídeos/metabolismoRESUMO
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.
Assuntos
Queijo , Lactococcus lactis , Animais , Acetoína/metabolismo , Diacetil/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Streptococcus thermophilus/genética , Fermentação , Leite , Microbiologia de AlimentosRESUMO
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.
Assuntos
Leite Humano , Probióticos , Feminino , Humanos , Animais , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Leite/microbiologia , Genômica , Probióticos/metabolismoRESUMO
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.
Assuntos
Bacteriófagos , Fagos de Streptococcus , Humanos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Fagos de Streptococcus/genética , Sistemas CRISPR-Cas/genética , Edição de GenesRESUMO
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.