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CRISPR-Cas is a form of adaptive sequence-specific immunity in microbes. This system offers unique opportunities for the study of coevolution between bacteria and their viral pathogens, bacteriophages. A full understanding of the coevolutionary dynamics of CRISPR-Cas requires knowing the magnitude of the cost of resisting infection. Here, using the gram-positive bacterium Streptococcus thermophilus and its associated virulent phage 2972, a well-established model system harbouring at least two type If functional CRISPR-Cas systems, we obtained different fitness measures based on growth assays in isolation or in pairwise competition. We measured the fitness cost associated with different components of this adaptive immune system: the cost of Cas protein expression, the constitutive cost of increasing immune memory through additional spacers, and the conditional costs of immunity during phage exposure. We found that Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins. Increasing immune memory by acquiring up to four phage-derived spacers was not associated with fitness costs. In addition, the activation of the CRLSPR-Cas system during phage exposure induces significant but small fitness costs. Together these results suggest that the costs of the CRISPR-Cas system arise mainly due to the maintenance of the defence system. We discuss the implications of these results for the evolution of CRISPR-Cas-mediated immunity.

Type III CRISPR-Cas systems in prokaryotes provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally active DNA and its transcripts by the Csm effector complex. The Cas10 subunit of the complex contains an HD nuclease domain that is responsible for DNA degradation and two Palm domains with elusive functions. In addition, Csm6, a ribonuclease that is not part of the complex, is also required to provide full immunity. We show here that target RNA binding by the Csm effector complex of Streptococcus thermophilus triggers Cas10 to synthesize cyclic oligoadenylates (cA n ; n = 2 to 6) by means of the Palm domains. Acting as signaling molecules, cyclic oligoadenylates bind Csm6 to activate its nonspecific RNA degradation. This cyclic oligoadenylate–based signaling pathway coordinates different components of CRISPR-Cas to prevent phage infection and propagation.

Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains. Bacterial immunity: versatile CRISPR/Cas CRISPR/Cas is a microbial immune system that is known to protect bacteria from viral infection. It is now shown that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA of both viral and plasmid origin. The system seems remarkably adapted to this end, and it is thought that CRISPR/Cas could be used to naturally generate safer and more robust bacteria that are resistant to the acquisition and spread of antibiotic resistance genes. CRISPR/Cas is a microbial immune system that is known to protect bacteria from virus infection. These authors show that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA.

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.

Streptococcus thermophilus is one of the most valuable homo-fermentative lactic acid bacteria, which, for a long time, has been widely used as a starter for the production of fermented dairy products. The key production characteristics of S. thermophilus, for example the production of extracellular polysaccharide, proteolytic enzymes and flavor substances as well as acidifying capacity etc., have an important effect on the quality of dairy products. The acidification capacity of the strains determines the manufacturing time and quality of dairy products. It depends on the sugar utilization ability of strains. The production of extracellular polysaccharide is beneficial for improving the texture of dairy products. Flavor substances increase the acceptability of dairy products. The proteolytic activity of the strain influences not only the absorption of the nitrogen source, but also the formation of flavor substances. Different strains have obvious differences in production characteristics via long-time evolution and adaptation to environment. Gaining new strains with novel and desirable characteristics is an important long-term goal for researchers and the fermenting industry. The understanding of the potential molecular mechanisms behind important characteristics of different strains will promote the screening and breeding of excellent strains. In this paper, key technological and functional properties of different S. thermophilus strains are discussed, including sugar metabolism, proteolytic system and amino acid metabolism, and polysaccharide and flavor substance biosynthesis. At the same time, diversity of genomes and plasmids of S. thermophilus are presented. Advances in research on key production characteristics and molecular levels of S. thermophilus will increase understanding of molecular mechanisms of different strains with different important characteristics, and improve the industrialization control level for fermented foods.

Lactobacillus delbrueckii subsp. bulgaricus ( L. bulgaricus ) and Streptococcus thermophilus ( S. thermophilus ) are commonly used starters in milk fermentation. Fermentation experiments revealed that L. bulgaricus-S. thermophilus interactions ( LbSt I ) substantially impact dairy product quality and production. Traditional biological humidity experiments are time-consuming and labor-intensive in screening interaction combinations, an artificial intelligence-based method for screening interactive starter combinations is necessary. However, in the current research on artificial intelligence based interaction prediction in the field of bioinformatics, most successful models adopt supervised learning methods, and there is a lack of research on interaction prediction with only a small number of labeled samples. Hence, this study aimed to develop a semi-supervised learning framework for predicting LbSt I using genomic data from 362 isolates (181 per species). The framework consisted of a two-part model: a co-clustering prediction model (based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) dataset) and a Laplacian regularized least squares prediction model (based on K-mer analysis and gene composition of all isolates datasets). To enhance accuracy, we integrated the separate outcomes produced by each component of the two-part model to generate the ultimate LbSt I prediction results, which were verified through milk fermentation experiments. Validation through milk fermentation experiments confirmed a high precision rate of 85% (17/20; validated with 20 randomly selected combinations of expected interacting isolates). Our data suggest that the biosynthetic pathways of cysteine, riboflavin, teichoic acid, and exopolysaccharides, as well as the ATP-binding cassette transport systems, contribute to the mutualistic relationship between these starter bacteria during milk fermentation. However, this finding requires further experimental verification. The presented model and data are valuable resources for academics and industry professionals interested in screening dairy starter cultures and understanding their interactions.

CRISPR-Cas and restriction-modification systems represent the core defense arsenal in Streptococcus thermophilus , but their effectiveness is compromised by phages encoding anti-CRISPR proteins (ACRs) and other counter-defense strategies. Here, we explore the defensome of 263 S. thermophilus strains to uncover other anti-phage systems. The defense landscape of S. thermophilus is enriched by 21 accessory defense systems, 13 of which have never been investigated in this species. Experimental validation of 17 systems with 14 phages reveals a range of anti-phage activities, highlighting both broad and narrow specificities across the five viral genera infecting S. thermophilus . Synergies are observed when combining CRISPR immunity with accessory systems. We also assess the fitness cost associated with the chromosomal integration of these systems in their native context and find no impact under laboratory or industrial conditions. These findings underscore the potential of these accessory defense systems to enhance the resistance of S. thermophilus , particularly against ACR-encoding phages. A study by Leprince et al. shows the effectiveness of Streptococcus thermophilus defense systems against dairy phages, their synergy with CRISPR-Cas, and the absence of fitness cost under lab or industrial conditions when they are chromosomally integrated.

RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications.

This study investigated the probiotic properties and antibiofilm potential of Streptococcus thermophilus strains obtained from homemade and commercial dahi. The S. thermophilus strain from homemade dahi had greater acid tolerance than the commercial strain, indicating a greater capacity to live in the acidic environments of the stomach. The commercial strain had increased survivability in bile salts and was more hydrophobic than the homemade strain. These findings suggest improved adaptability and increased colonization in the gut. The genomes of both strains included genes associated with probiotic characteristics implying that the two strains may provide unique probiotic advantages. These findings highlight the importance of cell-free supernatants (CFS) of these strains in reducing biofilm formation of pathogenic bacteria. Gas chromatography-mass spectrometry demonstrated that 2, 4-di-tert-butylphenol was a shared metabolite in the CFSs of both strains; however, 2-butanol was found only in the CFS of the homemade dahi strain. In-silico investigations revealed that compounds have drug-like characteristics, suggesting that they could be used for treating biofilm-associated diseases. This study highlights the health advantages of probiotics found in traditional dahi, but it also provides a way to develop natural antibacterial medicines.

Clustered regularly interspaced short palindromic repeats (CRISPR)‐encoded immunity in Type I systems relies on the Cascade (CRISPR‐associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4‐Cas (CRISPR‐associated) system (St‐CRISPR4‐Cas), we isolated an effector complex (St‐Cascade) containing 61‐nucleotide CRISPR RNA (crRNA). We show that St‐Cascade, guided by crRNA, binds in vitro to a matching proto‐spacer if a proto‐spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (−1 position) of the proto‐spacer. In the presence of a correct PAM, St‐Cascade binding to the target DNA generates an R‐loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R‐loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St‐CRISPR4‐Cas and other Type I systems. Bacterial CRISPR systems confer immunity by degrading invading DNA species. Reconstituting the Type I CRISPR Cascade complex from S. thermophilus demonstrates the molecular basis for recognition and cleavage of DNA by the nuclease Cas3.