Biochemical characterization of l-asparagine synthetase from Streptococcus thermophilus and its application in the enzymatic synthesis of ß-aspartyl compounds.

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.

Replacement of Loops at the Entrance of the Active Pocket of Streptococcus thermophilus 4,6-α-Glucanotransferase Changes Its Catalytic Activity and Product Specificity.

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.

A food-grade vector for Streptococcus thermophilus based on the α-complementation of ß-galactosidase.

Streptococcus thermophilus is a common yogurt starter that consumes lactose as its primary carbon source. The enzyme ß-galactosidase is essential for the lactose metabolism and the growth of this species. Streptococcus thermophilus appears to be a promising cell factory. Food-grade vectors have advantages in heterologous protein expression. This study aimed to determine whether the ß-galactosidase of S. thermophilus has the α-complementary characteristic and to develop a novel food-grade vector based on this phenomenon. The N-terminal 7 to 36 AA residues of the ß-galactosidase in S. thermophilus were deleted. The obtained mutant S. thermophilus Δα lost ß-galactosidase activity and growth ability in the lactose medium. Subsequently, plasmids expressing α-fragments with different lengths of 1 to 36 (Sα1), 1 to 53 (Sα2), and 1 to 88 (Sα3) AA were constructed and transformed into S. thermophilus Δα. Recombinant S. thermophilus Δα expressing Sα2 or Sα3 recovered the ability to grow in the lactose medium, and their ß-galactosidase activity accounted for 24.5% or 11.5% of the wild strain, respectively. These results indicated that the α-complementation system of ß-galactosidase existed in S. thermophilus. Based on the characteristic, a food-grade vector pSEα was constructed. Except for Sα2, vector pSEα expressed the α-donor derived from E. coli ß-galactosidase. This facilitated the construction of recombinant plasmids in E. coli DH5α and thus improved the transformation efficiency of S. thermophilus. Green fluorescent protein as a reporter protein could be highly expressed in S. thermophilus using this vector. As a result, pSEα is an efficient and safe vector for S. thermophilus with potential food applications.

Slowly digestible property of highly branched α-limit dextrins produced by 4,6-α-glucanotransferase from Streptococcus thermophilus evaluated in vitro and in vivo.

Starch molecules are first degraded to slowly digestible α-limit dextrins (α-LDx) and rapidly hydrolyzable linear malto-oligosaccharides (LMOs) by salivary and pancreatic α-amylases. In this study, we designed a slowly digestible highly branched α-LDx with maximized α-1,6 linkages using 4,6-α-glucanotransferase (4,6-αGT), which creates a short length of α-1,4 side chains with increasing branching points. The results showed that a short length of external chains mainly composed of 1-8 glucosyl units was newly synthesized in different amylose contents of corn starches, and the α-1,6 linkage ratio of branched α-LDx after the chromatographical purification was significantly increased from 4.6% to 22.1%. Both in vitro and in vivo studies confirmed that enzymatically modified α-LDx had improved slowly digestible properties and extended glycemic responses. Therefore, 4,6-αGT treatment enhanced the slowly digestible properties of highly branched α-LDx and promises usefulness as a functional ingredient to attenuate postprandial glucose homeostasis.

Three Distinct Proteases Are Responsible for Overall Cell Surface Proteolysis in Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely, the cell envelope protease PrtS and the housekeeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell surface protease, which shares significant homology with the SepM protease of Streptococcus mutans. Although all three proteases-PrtS, HtrA, and SepM-are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wild-type strain. IMPORTANCE Streptococcus thermophilus is a lactic acid bacterium used widely as a starter in the dairy industry. Due to its "generally recognized as safe" status and its weak cell surface proteolytic activity, it is also considered a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications.

Potato starch modified by Streptococcus thermophilus GtfB enzyme has low viscoelastic and slowly digestible properties.

Potato starch with high viscosity and digestibility cannot be added into some foods. To address this issue, a novel starch-acting enzyme 4,6-α-glucosyltransferase from Streptococcus thermophilus (StGtfB) was used. StGtfB decreased the iodine affinity and the molecular weight, but increased the degree of branching of starch at a mode quite different from glycogen 1,4-α-glucan branching enzyme (GBE). StGtfB at 5 U/g substrate mainly introduced DP 1-7 into amylose (AMY) or DP 1-12 branches into amylopectin (AMP), and increased the ratio of short- to long-branches from 0.32 to 2.22 or from 0.41 to 2.50. The DP 3 branch chain was the most abundant in both StGtfB-modified AMY and StGtfB-modified AMP. The DP < 6 branch chain contents in StGtfB-modified AMY were 42.68%, much higher than those of GBE-modified AMY. StGtfB significantly decreased viscoelasticity but still kept pseudoplasticity of starch. The modifications also slowed down the glucose generation rate of products at the mammalian mucosal α-glucosidase level. The slowly digestible fraction in potato starch increased from 34.29% to 53.22% using StGtfB of 5 U/g starch. This low viscoelastic and slowly digestible potato starch had great potential with respect to low and stable postprandial blood glucose.

Heterologous expression of cobalamin dependent class-III enzymes.

The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.

Streptococcus thermophilus Inhibits Colorectal Tumorigenesis Through Secreting ß-Galactosidase.

BACKGROUND & AIMS: Streptococcus thermophilus was identified to be depleted in patients with colorectal cancer (CRC) by shotgun metagenomic sequencing of 526 multicohort fecal samples. Here, we aim to investigate whether this bacterium could act as a prophylactic for CRC prevention. METHODS: The antitumor effects of S thermophilus were assessed in cultured colonic epithelial cells and in 2 murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S thermophilus was identified by mass spectrometry and followed by ß-galactosidase activity assay. The mutant strain of S thermophilus was constructed by homologous recombination. The effect of S thermophilus on the gut microbiota composition was assessed by shotgun metagenomic sequencing. RESULTS: Oral gavage of S thermophilus significantly reduced tumor formation in both Apcmin/+ and azoxymethane-injected mice. Coincubation with S thermophilus or its conditioned medium decreased the proliferation of cultured CRC cells. ß-Galactosidase was identified as the critical protein produced by S thermophilus by mass spectrometry screening and ß-galactosidase activity assay. ß-Galactosidase secreted by S thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S thermophilus without functional ß-galactosidase lost its tumor-suppressive effect. Also, S thermophilus increased the gut abundance of known probiotics, including Bifidobacterium and Lactobacillus via ß-galactosidase. ß-Galactosidase-dependent production of galactose interfered with energy homeostasis to activate oxidative phosphorylation and downregulate the Hippo pathway kinases, which partially mediated the anticancer effects of S thermophilus. CONCLUSION: S thermophilus is a novel prophylactic for CRC prevention in mice. The tumor-suppressive effect of S thermophilus is mediated at least by the secretion of ß-galactosidase.

Streptococcus thermophilus growth in soya milk: Sucrose consumption, nitrogen metabolism, soya protein hydrolysis and role of the cell-wall protease PrtS.

Societal demand for plant-based foods is increasing. In this context, soya products fermented using lactic acid bacteria (LAB) are appealing because of their potential health and nutritional benefits. The thermophilic LAB Streptococcus thermophilus is an essential starter species in the dairy industry. However, while its physiology is well characterized, little is known about its general metabolic activity or its techno-functional properties when it is grown in soya milk. In this study, S. thermophilus LMD-9 growth, sugar production, and lactic acid production in soya milk versus cow's milk were measured. Additionally, the main metabolic pathways used by the bacterium when growing in soya milk were characterized using a proteomic approach. Streptococcus thermophilus LMD-9 growth decreased soya milk pH, from 7.5 to 4.9, in 5 h. During fermentation, acidification thus occurred in tandem with lactate production and increasing population size (final population: 1.0 × 109 CFU/ml). As growth proceeded, sucrose was consumed, and fructose was produced. The proteomic analysis (LC-MS/MS) of the strain's cytosolic and cell envelope-associated proteins revealed that proteins related to amino acid transport and nitrogen metabolism were the most common among the 328 proteins identified (63/328 = 19.2% of total proteins). The cell-wall protease PrtS was present, and an LMD-9 deletion mutant was constructed by interrupting the prtS gene (STER_RS04165 locus). Acidification levels, growth levels, and final population size were lower in the soya milk cultures when the ΔprtS strain versus the wild-type (wt) strain was used. The SDS-PAGE profile of the soluble proteins in the supernatant indicated that soya milk proteins were less hydrolyzed by the ΔprtS strain than by the wt strain. It was discovered that S. thermophilus can grow in soya milk by consuming sucrose, can hydrolyze soya proteins, and can produce acidification levels comparable to those in cow's milk. This study comprehensively examined the proteomics of S. thermophilus grown in soya milk and demonstrated that the cell-wall protease PrtS is involved in the LAB's growth in soya milk and in the proteolysis of soya proteins, which are two novel findings. These results clarify how S. thermophilus adapts to soya milk and can help inform efforts to develop new fermented plant-based foods with better-characterized biochemical and microbiological traits.

Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9.

The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR-Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference-mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.

Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish.

RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization in Tg(myl7:GFP) and Tg(fli1:EGFP) fish that express EGFP zygotically. StCsm targeted against the endogenous tdgf1 elicited the characteristic one-eyed phenotype with greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm-mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown without significant off-target effects.

Identification and characterization of a moonlighting protein-enolase for surface display in Streptococcus thermophilus.

BACKGROUND: Streptococcus thermophilus is an important food starter and receiving more attention to serve as cell factories for production of high-valued metabolites. However, the low yields of intracellular or extracellular expression of biotechnological and biomedical proteins limit its practical applications. RESULTS: Here, an enolase EnoM was identified from S. thermophilus CGMCC7.179 with about 94% identities to the surface-located enolases from other Streptococcus spp. strains. The EnoM was used as an anchor to achieve surface display in S. thermophilus using GFP as a reporter. After respectively mixing the GFP-EnoM fusion protein or GFP with S. thermophilus cells in vitro, the relative fluorescence units (RFU) of the S. thermophilus cells with GFP-EnoM was 80-folds higher than that with purified GFP. The sharp decrease in the RFU of sodium dodecyl sulfate (SDS) pretreated cells compared to those of non-pretreated cells demonstrated that the membrane proteins were the binding ligand of EnoM. Furthermore, an engineered ß-galactosidase (ß-Gal) was also successfully displayed on the cell surface of S. thermophilus CGMCC7.179 and the relative activity of the immobilized ß-Gal remained up to 64% after reused 8 times. Finally, we also demonstrated that EnoM could be used as an anchor for surface display in L. casei, L. bulgaricus, L. lactis and Leuconostoc lactis. CONCLUSION: To our knowledge, EnoM from S. thermophilus was firstly identified as an anchor and successfully achieved surface display in LAB. The EnoM-based surface display system provided a novel strategy for the enzyme immobilization.

Probiotic Validation of a Non-native, Thermostable, Phytase-Producing Bacterium: Streptococcus thermophilus.

Phytate-linked nutritional deficiency disorders have plagued poultry for centuries. The application of exogenous phytases in poultry feed has served as a solution to this problem. However, they are linked to certain limitations which include thermal instability during prolonged feed processing. Therefore, in this study, Streptococcus thermophilus 2412 based phytase stability was assessed at higher temperatures up to 90 °C. This was followed by probiotic validation of the same bacterium in an in vitro intestinal model. Bacterial phytase showed thermostability up to 70 °C with a recorded activity of 9.90 U. The bacterium was viable in the intestinal lumen as indicated by the cell count of 6.10 log(CFU/mL) after 16 h. It also showed acid tolerance with a stable cell count of 5.01 log(CFU/mL) after 16 h of incubation at pH 2. The bacterium displayed bile tolerance yielding a cell count of 6.36 log(CFU/mL) in the presence of 0.3% bile. Bacterial susceptibility was observed toward all tested antibiotics with a maximum zone of 20 mm against clindamycin. The maximum antagonistic activity was observed against Staphylococcus aureus, Serratia marcescens, and Escherichia coli with inhibition zone diameters up to 10 mm. The above characteristics prove that S. thermophilus 2412 can be used as an effective phytase-producing poultry probiotic.

Metabolic engineering of Parageobacillus thermoglucosidasius for the efficient production of (2R, 3R)-butanediol.

High-temperature fermentation using thermophilic microorganisms may provide cost-effective processes for the industrial production of fuels and chemicals, due to decreased hygiene and cooling costs. In the present study, the genetically trackable thermophile Parageobacillus thermoglucosidasius DSM2542T was engineered to produce (2R, 3R)-butanediol (R-BDO), a valuable chemical with broad industrial applications. The R-BDO biosynthetic pathway was optimized by testing different combinations of pathway enzymes, with acetolactate synthase (AlsS) from Bacillus subtilis and acetolactate decarboxylase (AlsD) from Streptococcus thermophilus yielding the highest production in P. thermoglucosidasius DSM2542T. Following fermentation condition optimization, shake flask fermentation at 55 °C resulted in the production of 7.2 g/L R-BDO with ~ 72% theoretical yield. This study details the microbial production of R-BDO at the highest fermentation temperature reported to date and demonstrates that P. thermoglucosidasius DSM2542T is a promising cell factory for the production of fuels and chemicals using high-temperature fermentation.

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

First Insight into the Probiotic Properties of Ten Streptococcus thermophilus Strains Based on In Vitro Conditions.

The aim of this study was to evaluate probiotic properties of ten Streptococcus thermophilus strains (st1 to st10) isolated from pickles in China. These strains all had ß-galactosidase activity, which laid foundation for studying their probiotic properties. In this study, the bile salt hydrolase activity, lysozyme resistance, tolerance to simulated gastric juice, bile salt tolerance, and bacterial adhesion capacity to the Caco-2 cells of these selected strains were detected in vitro conditions. The results indicated that the bile salt hydrolase activities of st2, st6, and st9 were higher than that for other strains. St10 showed the greatest lysozyme resistance (> 80% survival), followed by st9, st8, st7, st5, and st6. As for the tolerance to simulated gastric juice, st5 possessed the highest survival rate (35%), followed by st6 (30%). St6 was the best performer in both bile salt tolerance and bacterial adhesion capacity to the Caco-2 cells. The results of fluorescence microscope and electron microscope further confirmed previous studies and more intuitively demonstrated the st6 strain's tolerance to harsh environments. Overall, these strains were expected to possess beneficial properties and have the potentiality to be probiotics.

Short communication: Lactose utilization of Streptococcus thermophilus and correlations with ß-galactosidase and urease.

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 ß-galactosidase and urease has rarely been investigated. In this study, 10 strains of S. thermophilus isolated from fermented yak milk exhibited a diversity of ß-galactosidase and urease activities, growth, and acid production in de Man, Rogosa, and Sharpe-lactose. Among the strains, 15G5 possessed the highest ß-galactosidase activity and showed the highest cell growth, lactic acid production, and titratable acidity during fermentation. In contrast, 7G10, with the weakest ß-galactosidase activity, produced the lowest lactic acid content and change in titratable acidity. Further investigation indicated that ß-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 ß-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.

Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

Modeling, stability and the activity assessment of glutathione reductase from Streptococcus Thermophilus; Insights from the in-silico simulation study.

Antioxidant enzymes (AEs) are the main parts of the natural barriers of the body which deactivate the oxidant factors. To discover and understand their structures and function will deserve a deeper investigation. Accordingly, as an AE of probiotic strains, glutathione reductase of Streptococcus thermophilus (GRst), is characterized and modeled by in-silico methods. The investigation indicated the physicochemical properties of the enzyme and estimated its half-life of being more than 10 h. The analysis revealed that the enzyme is composed of 86 strands, 123 helices, and 241 random coils. Homology modeling of the GRst led to the construction of the enzyme's 3D model that 62% of which is analogous to the glutathione reductase of Escherichia Coli (GRec), and which is qualitatively high in terms of Molpdf, ERRAT, Verify-3D and Ramachandran scores. Moreover, the structural stability of the model was substantiated within 10 and 20 ns at 400 and 300 K, respectively. Interestingly, these data showed that the enzyme is more stable than GRec at 400 K. In other words, the active cavity of the constructed model is characteristic of 38 amino acid residues within 4 Šaround the NADPH and GSSG as corresponding ligands of GRst. Noteworthy, herein is the fact that, CYS40 and CYS45 are specified as the active site residues of this enzyme. Furthermore, the interaction assays of the model support its antioxidant capability which is even more than that of GRec. In general, these data provide a new model of AEs being inclusive of high antioxidant capacity and thermostability.

A mutation in the methionine aminopeptidase gene provides phage resistance in Streptococcus thermophilus.

Streptococcus thermophilus is a lactic acid bacterium widely used by the dairy industry for the manufacture of yogurt and specialty cheeses. It is also a Gram-positive bacterial model to study phage-host interactions. CRISPR-Cas systems are one of the most prevalent phage resistance mechanisms in S. thermophilus. Little information is available about other host factors involved in phage replication in this food-grade streptococcal species. We used the model strain S. thermophilus SMQ-301 and its virulent phage DT1, harboring the anti-CRISPR protein AcrIIA6, to show that a host gene coding for a methionine aminopeptidase (metAP) is necessary for phage DT1 to complete its lytic cycle. A single mutation in metAP provides S. thermophilus SMQ-301 with strong resistance against phage DT1. The mutation impedes a late step of the lytic cycle since phage adsorption, DNA replication, and protein expression were not affected. When the mutated strain was complemented with the wild-type version of the gene, the phage sensitivity phenotype was restored. When this mutation was introduced into other S. thermophilus strains it provided resistance against cos-type (Sfi21dt1virus genus) phages but replication of pac-type (Sfi11virus genus) phages was not affected. The mutation in the gene coding for the MetAP induces amino acid change in a catalytic domain conserved across many bacterial species. Introducing the same mutation in Streptococcus mutans also provided a phage resistance phenotype, suggesting the wide-ranging importance of the host methionine aminopeptidase in phage replication.