Search Results (114)

Bacteriophage ΦIN93 has an icosahedral-like capsid that is believed to be composed of two putative capsid or coat proteins, namely open reading frame (ORF)13 and ORF14. In addition to the two capsid proteins, there are other proteins that may be associated with the structure of the virus. For example, five other proteins (ORF12, ORF16, ORF17, ORF19, and ORF20) in the virus have been identified as putative membrane-associated proteins. It is believed that membrane-associated proteins associate with coat proteins (serve as scaffolding proteins) to promote viral assembly. While the expression/co-expression of ORF13 and ORF14 have been done to assess if they can assemble to form virus-like particles (VLPs), the expression of any of the membrane-associated proteins and their contribution to assembly have never been attempted. In this study, we successfully co-expressed, for the first time, three membrane-associated proteins (ORF12, ORF16, ORF17) in addition to ORF13 and ORF14 in thermophilic bacteria (Thermus thermophilus, HB27:nar strain) and in mesophilic bacteria (BL21 Star). The expression levels of the proteins were higher in BL21 Star than in Thermus thermophilus, HB27:nar. Some of the expressed proteins (especially ORF17) migrated at sizes that were more than their deduced molecular weight (based on amino acid sequence). Co-expression of these proteins did not lead to the formation of structures that we believe are VLPs. Nevertheless, we believe co-expressing these proteins together from different plasmids is a good approach to assess which of them may be required to form VLPs. Full article

Black garlic, a fermented product of fresh garlic, has shown promising potential as a culinary ingredient and a medicinal remedy. This study examined the microbiological makeup, nutritional profile, and health advantages of black garlic to better understand its health-endorsing properties. Thermus, Corynebacterium, Streptococcus, and Brevundimonas were among the prominent taxa found when the microbial diversity in black garlic samples was investigated using Illumina MiSeq sequencing. This provided insight into the complex interactions between microorganisms during the fermentation process and clarified the distinctive qualities of black garlic. This study expanded its scope to include black garlic’s therapeutic potential, specifically in relation to liver function and hangovers caused by alcohol, in addition to its microbial complexity. Significant liver damage was revealed in alcohol-treated rats by serum biochemical indicators and histological stains; this damage was lessened by the administration of black garlic, particularly at higher dosages. Furthermore, black garlic showed hepatoprotective effects attributed to its high phenolic and flavonoid contents. These results offer a novel understanding of the medicinal qualities of black garlic as they lay out possibilities for the creation of functional drugs to treat alcohol-induced liver damage. Conclusively, black garlic’s diverse microbial composition also advances our knowledge of its nutritional makeup and health advantages. In summary, this research highlights the potential of black garlic as a flexible medical tool, having implications for both gastronomic and therapeutic uses. Full article

This study systematically evaluates the effects of dietary supplementation with phytogenic feed additive (0.2%, 0.4%, and 0.8%) on white-feathered broilers (n = 88) through a 42-day controlled trial with the weight of approximately 50 g. The experimental design incorporates a triplicate-group-replicated protocol with daily feed intake monitoring, culminating in comprehensive assessments of the growth performance, slaughter traits, meat quality, and cecal microbiome dynamics. The results demonstrated that the 0.8% supplementation significantly enhanced average daily weight gain (p < 0.05), optimized meat characteristics (elevated the redness of meat, reduced pH; p < 0.05), and restructured cecal microbiota by enriching Deinococcus-Thermus, Bacteroidetes, Actinobacteria, and Cyanobacteria (p < 0.05). Based on microbiota-based functional prediction analyses (COG/KEGG/MetaCyc), phytogenic feed additive significantly activated lipid metabolism pathways in broilers. The immunomodulatory correlations between Deinococcus/Thermus/Cyanobacteria and immune indicators suggested their potential immune-enhancing effects mediated through host immune regulation. The findings established the 0.8% phytogenic feed additive as a multifunctional phytogenic additive that synchronously improves zootechnical performance, meat quality, and microbiome homeostasis, offering a scientifically validated strategy for antibiotic-free precision nutrition in sustainable poultry production. Full article

Type IV pili (T4P) machinery is critical for bacterial surface motility, protein secretion, and DNA uptake. This review highlights the ecological significance of T4P-dependent motility in Thermus thermophilus, a thermophilic bacterium isolated from hot springs. Unlike swimming motility, the T4P machinery enables bacteria to move over two-dimensional surfaces through repeated cycles of extension and retraction of pilus filaments. Notably, T. thermophilus exhibits upstream-directed migration under shear stress, known as rheotaxis, which appears to represent an adaptive strategy unique to thermophilic bacteria thriving in rapid water flows. Furthermore, T4P contributes to the capture of DNA and phages, indicating their multifunctionality in natural environments. Understanding the T4P dynamics provides insights into bacterial survival and evolution in extreme habitats. Full article

Thermus thermophilus HB27 laccase (Tth-Lac) is a thermostable enzyme that contains a β-hairpin (Ala292-Gln307) covering the substrate entrance. We analyzed the role of this β-hairpin in the enzymatic activity of Tth-Lac through three β-hairpin mutants: two variants without the β-hairpin (C1Tth-Lac and C2Tth-Lac) and one with a partially modified β-hairpin (P1Tth-Lac). Enzymatic activity was assayed with different substrates with and without copper. C1Tth-Lac showed a higher dependency on copper, increasing its activity by 1600-fold for syringaldazine (SGZ). All mutants presented a higher activity than Tth-Lac with phenolic substrates in the presence of copper. The position of the signal associated with CuT2 also changed, as shown in EPR spectra. Elucidation of the crystal structure of P1Tth-Lac mutant (PDB: 9CPM) showed that the partial deletion of the β-hairpin did not significantly affect the overall tertiary structure compared to the wild-type (PDB: 2xu9) nor the coordination of the four internally bound Cu atoms. Higher B-factors of the residues downstream of the deletion indicate increased flexibility (Q307, G308, P309, S310) that were otherwise more ordered in the Tth-Lac structure. Redox potential experiments on platinum electrodes have shown that all proteins have high redox potential, a finding that could have significant implications in the field of protein research. Full article

Tt72 DNA polymerase is a newly characterized PolA-type thermostable enzyme derived from the Thermus thermophilus phage vB_Tt72. The enzyme demonstrates strong 3′→5′ exonucleolytic proofreading activity, even in the presence of 1 mM dNTPs. In this study, we examined how the exonucleolytic activity of Tt72 DNA polymerase affects the fidelity of DNA synthesis. Using a plasmid-based lacZα gene complementation assay, we determined that the enzyme’s mutation frequency was 2.06 × 10⁻³, corresponding to an error rate of 1.41 × 10⁻⁵. For the exonuclease-deficient variant, the mutation frequency increased to 6.23 × 10⁻³, with an associated error rate of 4.29 × 10⁻⁵. The enzyme retained 3′→5′ exonucleolytic activity at temperatures up to 70 °C but lost it after 10 min of incubation at temperatures above 75 °C. Additionally, we demonstrated that Tt72 DNA polymerase efficiently processes 3′/5′-overhangs and removes a single-nucleotide 3′-dA overhang from PCR products at 55 °C. These characteristics make Tt72 DNA polymerase well suited for specialized molecular cloning applications. Full article

The increasing antibiotic resistance among bacteria challenges the biotech industry to search for new antibacterial molecules. Endolysin TP84_28 is a thermostable, lytic enzyme, encoded by the bacteriophage (phage) TP-84, and it effectively digests host bacteria cell wall. Biofilms, together with antibiotic resistance, are major problems in clinical medicine and industry. The challenge is to keep antibacterial molecules at the site of desired action, as their diffusion leads to a loss of efficacy. The TP84_28 endolysin gene was cloned into an expression-fusion vector, forming a fusion gene cbd_tp84_28_his with a cellulose-binding domain from the cellulase enzyme. The Cellulose-Binding Thermostable TP84_Endolysin (CBD_TP84_28_His) fusion protein was biosynthesized in Escherichia coli and purified. Thermostability and enzymatic activities against various bacterial species were measured by a turbidity reduction assay, a spot assay, and biofilm removal. Cellulose-binding properties were confirmed via interactions with microcellulose and cellulose paper-based immunoblotting. The high affinity of the CBD allows for a high concentration of the fusion enzyme at desired target sites such as cellulose-based wound dressings, artificial heart valves and food packaging. CBD_TP84_28_His exhibits a lytic effect against thermophilic bacteria Geobacillus stearothemophilus, Thermus aquaticus, Bacillus stearothermophilus, and Geobacillus ICI and minor effects against mesophilic Bacillus cereus and Bacillus subtilis. CBD_TP84_28_His retains full activity after preincubation in the temperatures of 30–65 °C and exhibits significant activity up to its melting point at 73 °C. CBD_TP84_28_His effectively reduces biofilms. These findings suggest that integrating CBDs into thermostable endolysins could enable the development of targeted antibacterial recombinant proteins with diverse clinical and industrial applications. Full article

In recent years, the study of antimicrobial peptides (AMPs) has garnered considerable attention due to their potential in combating antibiotic-resistant pathogens. Mass spectrometry-based proteomics provides valuable information on microbial stress responses induced by AMPs. This work aims to unravel the proteomic alterations induced by the amyloidogenic antimicrobial peptide R23I, encompassing both inhibitory and non-inhibitory concentrations. This study investigates the effects of the R23I peptide on the protein abundance of Thermus thermophilus (T. thermophilus) at different concentrations (20, 50, and 100 μg/mL). We found 82 differentially expressed proteins, including 15 upregulated and 67 downregulated proteins. We also compared the protein identification results between the PEAKS and IdentiPy programs. Our proteomic analysis revealed distinct patterns of protein expression, suggesting compensatory mechanisms in response to the R23I peptide. Notably, the alterations predominantly affected membrane and cytoplasmic proteins that play a central role in critical cellular processes such as transcription, translation, and energy conversion. This study sheds light on the complex interactions between antimicrobial peptides and bacterial responses, offering insights into microbial adaptability and potential implications for antimicrobial strategies and the understanding of microbial physiology. Full article

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis—unlike pyrimidine NPs (PyNPs, UP, and TP)—multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60–90 °C) and within a pH range of 6–8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80–100% of their activity when incubated at 60–80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2′-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2′-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis. Full article

Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system. Full article

Hot springs worldwide can be a source of extremophilic microorganisms of biotechnological interest. In this study, samplings of a hot spring in Hidalgo, Mexico, were conducted to isolate, identify, and characterize morphologically, biochemically, and molecularly those bacterial strains with potential industrial applications. In addition, a physicochemical and geochemical examination of the hot spring was conducted to fully understand the study region and its potential connection to the strains discovered. The hot spring was classified as sulfate-calcic according to the Piper Diagram; the hydrogeochemical analysis showed the possible interactions between minerals and water. Eighteen bacterial strains were isolated with optimal growth temperatures from 50 to 55 °C. All strains are Gram-positive, the majority having a rod shape, and one a round shape, and 17 produce endospores. Hydrolysis tests on cellulose, pectin, and xylan agar plates demonstrated enzymatic activity in some of the strains. Molecular identification through the 16S rDNA gene allowed classification of 17 strains within the Phylum Firmicutes and one within Deinococcus-Thermus. The bacterial strains were associated with the genera Anoxybacillus, Bacillus, Anerunibacillus, Paenibacillus, and Deinococcus, indicating a diversity of bacterial strains with potential industrial applications. Full article

The blotched snakehead Channa maculata is an important economical freshwater species in East Asia. However, there has been relatively little research conducted on the correlation between gender and gut microbes. In this study, 36 of 1000 blotched snakeheads were randomly selected for growth performance measurement and gut microbiota high-throughput sequencing. Results showed that microbial diversity, composition, and metabolic functions were altered by gender and growth performance except the microbial network. In our study, Proteobacteria were the most abundant phylum, with Fusobacteria showing enrichment in males and Bacteroidetes in females. Notably, phylum Deinococcus-Thermus was identified as a significant biomarker. The Cetobacterium was the most abundant genus-level taxon. Furthermore, gut microbes specializing in the production of gut-healthy substances, such as coenzymes and vitamins, were identified as biomarkers in the fast-growing group. Our investigation highlighted the impact of gender on the composition and abundance of gut microbial biomarkers in both males and females, thereby influencing differential growth performance through the modulation of specific metabolic functions. Full article

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich’s ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients’ cells, and this prompted us to further characterize both the Nqo15 solution’s behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15’s folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15’s iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function. Full article

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus, at concentrations ranging from 3 to 12 μM, exhibited enhanced antimicrobial activity against B. cereus. TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa, at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli (E. coli) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus, MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development. Full article

The thermostable protease TTHA0724 derived from Thermus thermophilus HB8 is an ideal industrial washing enzyme due to its thermophilic characteristics; although it can be expressed in Escherichia coli via pET-22b, high yields are difficult to achieve, leading to frequent autolysis of the host. This paper details the development of a signal peptide library in the expression system of B. subtilis and the optimization of signal peptides for enhanced extracellular expression of TTHA0724. When B. subtilis was used as the host and the optimized signal peptide was used, the expression level of TTHA0724 was 16.7 times higher compared with E. coli. B. subtilis as an expression host does not change the characteristics of TTHA0724. The potential application fields of TTHA0724 are studied. TTHA0724 can be used as a detergent additive at 60 °C, which can sterilize and eliminate mites while thoroughly cleaning protein stains. Soybean meal enzymatic hydrolysis with TTHA0724 at a high temperature produced a higher content of antioxidant peptides. These results indicate that TTHA0724 has great potential for industrial applications. Full article