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Nature Microbiology Feb 2017The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic...
The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.
Topics: Bacterial Proteins; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Endonucleases; Gene Expression; Gene Knockdown Techniques; Gene Silencing; Genetic Techniques; Mycobacterium; Mycobacterium tuberculosis; Operon; Streptococcus pyogenes; Streptococcus thermophilus; Transcription Initiation Site; Transcription, Genetic
PubMed: 28165460
DOI: 10.1038/nmicrobiol.2016.274 -
Proceedings of the National Academy of... Sep 2012Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria...
Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.
Topics: Bacterial Proteins; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; Endonucleases; RNA, Bacterial; Ribonucleoproteins; Streptococcus thermophilus
PubMed: 22949671
DOI: 10.1073/pnas.1208507109 -
Gastroenterology Jun 2013Changes in gut microbiota have been reported to alter signaling mechanisms, emotional behavior, and visceral nociceptive reflexes in rodents. However, alteration of the... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND & AIMS
Changes in gut microbiota have been reported to alter signaling mechanisms, emotional behavior, and visceral nociceptive reflexes in rodents. However, alteration of the intestinal microbiota with antibiotics or probiotics has not been shown to produce these changes in humans. We investigated whether consumption of a fermented milk product with probiotic (FMPP) for 4 weeks by healthy women altered brain intrinsic connectivity or responses to emotional attention tasks.
METHODS
Healthy women with no gastrointestinal or psychiatric symptoms were randomly assigned to groups given FMPP (n = 12), a nonfermented milk product (n = 11, controls), or no intervention (n = 13) twice daily for 4 weeks. The FMPP contained Bifidobacterium animalis subsp Lactis, Streptococcus thermophiles, Lactobacillus bulgaricus, and Lactococcus lactis subsp Lactis. Participants underwent functional magnetic resonance imaging before and after the intervention to measure brain response to an emotional faces attention task and resting brain activity. Multivariate and region of interest analyses were performed.
RESULTS
FMPP intake was associated with reduced task-related response of a distributed functional network (49% cross-block covariance; P = .004) containing affective, viscerosensory, and somatosensory cortices. Alterations in intrinsic activity of resting brain indicated that ingestion of FMPP was associated with changes in midbrain connectivity, which could explain the observed differences in activity during the task.
CONCLUSIONS
Four-week intake of an FMPP by healthy women affected activity of brain regions that control central processing of emotion and sensation.
Topics: Adolescent; Adult; Attention; Bifidobacterium; Brain; Cultured Milk Products; Female; Functional Neuroimaging; Humans; Lactobacillus; Lactococcus lactis; Magnetic Resonance Imaging; Middle Aged; Probiotics; Streptococcus thermophilus; Treatment Outcome; Young Adult
PubMed: 23474283
DOI: 10.1053/j.gastro.2013.02.043 -
Journal of Microbiology and... Oct 2018The growth of lactic acid bacteria (LAB) generates a high number of metabolites related to aromas and flavors in fermented dairy foods. These microbial proteases are... (Review)
Review
The growth of lactic acid bacteria (LAB) generates a high number of metabolites related to aromas and flavors in fermented dairy foods. These microbial proteases are involved in protein hydrolysis that produces necessary peptides for their growth and releases different molecules of interest, like bioactive peptides, during their activity. Each genus in particular has its own proteolytic system to hydrolyze the necessary proteins to meet its requirements. This review aims to highlight the differences between the proteolytic systems of and other lactic acid bacteria ( and ) since they are microorganisms that are frequently used in combination with other LAB in the elaboration of fermented dairy products. Based on genetic studies and in vitro and in vivo tests, the proteolytic system of has been divided into three parts: 1) a serine proteinase linked to the cellular wall that is activated in the absence of glutamine and methionine; 2) the transport of peptides and oligopeptides, which are integrated in both the Dpp system and the Ami system, respectively; according to this, it is worth mentioning that the Ami system is able to transport peptides with up to 23 amino acids while the Opp system of or transports chains with less than 13 amino acids; and finally, 3) peptide hydrolysis by intracellular peptidases, including a group of three exclusive of capable of releasing either aromatic amino acids or peptides with aromatic amino acids.
Topics: Amino Acid Transport Systems; Amino Acids; Cultured Milk Products; Lactobacillales; Peptide Hydrolases; Proteolysis; Streptococcus thermophilus; Substrate Specificity
PubMed: 30196594
DOI: 10.4014/jmb.1807.07017 -
FEMS Microbiology Reviews Jul 2023Bacteriophages (or phages) represent a persistent threat to the success and reliability of food fermentation processes. Recent reports of phages that infect... (Review)
Review
Bacteriophages (or phages) represent a persistent threat to the success and reliability of food fermentation processes. Recent reports of phages that infect Streptococcus thermophilus have highlighted the diversification of phages of this species. Phages of S. thermophilus typically exhibit a narrow range, a feature that is suggestive of diverse receptor moieties being presented on the cell surface of the host. Cell wall polysaccharides, including rhamnose-glucose polysaccharides and exopolysaccharides have been implicated as being involved in the initial interactions with several phages of this species. Following internalization of the phage genome, the host presents several defences, including CRISPR-Cas and restriction and modification systems to limit phage proliferation. This review provides a current and holistic view of the interactions of phages and their S. thermophilus host cells and how this has influenced the diversity and evolution of both entities.
Topics: Bacteriophages; Streptococcus thermophilus; Streptococcus Phages; Reproducibility of Results; Polysaccharides
PubMed: 37339909
DOI: 10.1093/femsre/fuad032 -
Frontiers in Microbiology 2018CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) loci, which provide a specific immunity against exogenous elements, are... (Review)
Review
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) loci, which provide a specific immunity against exogenous elements, are hypervariable among distinct prokaryotes. Based on previous researches, this review focuses on concluding systematical genome editing protocols in Firstly, its protocols and optimized conditions in gene editing are introduced. What's more, classification and diversity analyses of CRISPR-Cas benefit the further understanding of evolution relationship among Ability of its foreign segment integration and spacer source analyses also indicate a new direction of phage resistance. Above all, all of these point out its potential to be regarded as another model system other than type II CRISPR-Cas in .
PubMed: 29515542
DOI: 10.3389/fmicb.2018.00257 -
Archives of Razi Institute Dec 2022Biopolymers, particularly exopolysaccharides produced by microorganisms such as bacteria, yeasts, and algae, have gained popularity in recent years due to their...
Biopolymers, particularly exopolysaccharides produced by microorganisms such as bacteria, yeasts, and algae, have gained popularity in recent years due to their physical, chemical, and functional properties that are widely useful in food, industrial, cosmetic, and pharmaceutical systems. Hyaluronic acid is one type of these polysaccharide. This study investigated the optimal conditions for producing hyaluronic acid from the bacterial strain. The isolated were cultured on MRS broth, Skim milk, and M17 broth with an addition of 1% lactose. The diagnosed bacterial strains were grown in 100 ml of culture media, placed in volumetric flasks of 250 ml capacity, and incubated at 42˚C for 24 hours, pH 6.8, inoculum volume 1%, and a vibrating incubator at 150 rpm. After the end of the fermentation period, the isolation and purification of HA have performed accordingly: proteins were removed using 1% trichloroacetic acid (TCA), and HA in the supernatant was collected by isopropanol precipitation. The collected HA was dialyzed against ultrapure water and lyophilized. The amount of acid produced was estimated. The results show that the best production of hyaluronic acid was from the bacterial strain grown on the alternative medium containing whey at a ratio of 450 ml/L and 7.5 g/L yeast extract at 40 ˚C, with a 3% of inoculum volume and 102×10 colony-forming units/ml of bacterial cells, in pH 6.8 and agitation speed of 150 rpm for 18 h, which had the most significant effect on the fermentation process and gave the highest value of HA production of 0.598 g/L and biomass of 6.08 g/L. These results showed the best production method for HA to achieve maximal production yelled.
Topics: Animals; Streptococcus thermophilus; Hyaluronic Acid; Milk; Lactose; Fermentation
PubMed: 37274889
DOI: 10.22092/ARI.2022.358612.2262 -
Applied and Environmental Microbiology Jun 2020is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes...
is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other phages. In fact, these two novel phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment. Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species These two related viruses represent a fifth group of phages, as they are significantly distinct from other known phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.
Topics: Microscopy, Electron, Transmission; Sequence Analysis, DNA; Siphoviridae; Streptococcus Phages; Streptococcus thermophilus
PubMed: 32303549
DOI: 10.1128/AEM.00227-20 -
Journal of Dairy Science Jan 2020The ability to use lactose is critical for the application of Streptococcus thermophilus in fermented dairy products. Most studies have evaluated the use of lactose of...
The ability to use lactose is critical for the application of Streptococcus thermophilus in fermented dairy products. Most studies have evaluated the use of lactose of S. thermophilus by measuring lactose utilization, but its correlation with β-galactosidase and urease has rarely been investigated. In this study, 10 strains of S. thermophilus isolated from fermented yak milk exhibited a diversity of β-galactosidase and urease activities, growth, and acid production in de Man, Rogosa, and Sharpe-lactose. Among the strains, 15G5 possessed the highest β-galactosidase activity and showed the highest cell growth, lactic acid production, and titratable acidity during fermentation. In contrast, 7G10, with the weakest β-galactosidase activity, produced the lowest lactic acid content and change in titratable acidity. Further investigation indicated that β-galactosidase activity of S. thermophilus showed significant positive correlations with the growth of cell densities, the production of lactic acid, and titratable acidity, and urease activity of S. thermophilus showed a significant correlation with the use of lactose and the production of lactic acid and acetaldehyde. These findings suggest that the differences of β-galactosidase and urease activities are essential for the performance in the lactose metabolism, growth, and acid production of S. thermophilus, providing new insights into strain selection and application.
Topics: Animals; Carbohydrate Metabolism; Fermentation; Lactic Acid; Lactose; Milk; Streptococcus thermophilus; Urease; beta-Galactosidase
PubMed: 31704010
DOI: 10.3168/jds.2019-17009 -
Microbiology Spectrum Apr 2022Lactic acid bacteria such as Streptococcus thermophilus are known to produce extracellular polysaccharide (EPS) in fermented foods that enhance the creaminess and...
Lactic acid bacteria such as Streptococcus thermophilus are known to produce extracellular polysaccharide (EPS) in fermented foods that enhance the creaminess and mouthfeel of the product, such as yogurt. Strains producing larger amounts of EPS are highly sought-after, and therefore, robust and accurate quantification methodologies are important. This study found that two commonly used methodologies significantly underestimated the amount of EPS produced as measured using a milk matrix. To this end, a proteolytic step was implemented prior to EPS extraction (Method C). An initial proteolytic step using xanthan gum-spiked milk significantly increased recovery yield to 64%, compared to 27.8% for Method A and 34.3% for Method B. Method C showed no improvement when assessed using a chemically defined medium. Method C was further validated using three strains of S. thermophilus with varying EPS-production capabilities (ST, ST, ST). Overall, Method C demonstrated significant improvements in the EPS extraction yield for all three S. thermophilus strains in fermented milk. On average, Method C improved isolation yield by ∼3- to 6-fold compared with Method A and by ∼2- to 3-fold compared with method B. There were no significant differences between samples when they were grown in a chemically defined medium, highlighting the importance of a proteolytic step specifically for fermented milk samples. In commercial applications, accurate quantification of EPS-production is an important aspect when finding new strains. Extracellular polysaccharide (EPS) production by milk-fermenting microorganisms is a highly sought-after trait in improving the perceived thickness, creaminess, and mouthfeel of yogurt. Streptococcus thermophilus are commonly isolated and their EPS production is quantified in the search for higher-producing strains. In this study, we demonstrated that two commonly used methods for isolating EPS from milk samples significantly underestimated the true amount of EPS present. We demonstrated that the addition of a proteolytic step prior to EPS extraction isolated over 2-fold more EPS than identical samples processed using the traditional protocols. We further validated this method in fermented milk samples from three strains of S. thermophilus that included a low-, mid-, and high-EPS producing strain. Again, we showed significant improvements in EPS isolation using a proteolytic step. In the search for new S. thermophilus strains with enhanced EPS production, accurate quantification in an optimal medium is essential.
Topics: Animals; Dietary Carbohydrates; Fermentation; Milk; Polysaccharides, Bacterial; Streptococcus thermophilus; Yogurt
PubMed: 35343770
DOI: 10.1128/spectrum.02280-21