Bacteriophages of Thermophilic 'Bacillus Group' Bacteria-A Systematic Review, 2023 Update.

Bacteriophages associated with thermophiles are gaining increased attention due to their pivotal roles in various biogeochemical and ecological processes, as well as their applications in biotechnology and bionanotechnology. Although thermophages are not suitable for controlling bacterial infections in humans or animals, their individual components, such as enzymes and capsid proteins, can be employed in molecular biology and significantly contribute to the enhancement of human and animal health. Despite their significance, thermophages still remain underrepresented in the known prokaryotic virosphere, primarily due to limited in-depth investigations. However, due to their unique properties, thermophages are currently attracting increasing interest, as evidenced by several newly discovered phages belonging to this group. This review offers an updated compilation of thermophages characterized to date, focusing on species infecting the thermophilic bacilli. Moreover, it presents experimental findings, including novel proteomic data (39 proteins) concerning the model TP-84 bacteriophage, along with the first announcement of 6 recently discovered thermophages infecting Geobacillus thermodenitrificans: PK5.2, PK2.1, NIIg10.1, NIIg2.1, NIIg2.2, and NIIg2.3. This review serves as an update to our previous publication in 2021.

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

Association Between the rs4680 Polymorphism of the COMT Gene and Personality Traits among Combat Sports Athletes.

Physical performance has been the focus of studies examining genetic influences in martial arts. There has been little quantitative analysis of the interaction between psychological traits and gene variants in athletes. This study aimed to determine whether the rs4680 polymorphism of the COMT gene (catechol-O-methyltransferase) was linked to other sports phenotypes such as temperament, mental toughness, and stress tolerance. In our study, we concentrated on the case-control analysis of athletes in the aspect of their personality traits in association with the COMT gene polymorphism. Participants comprised 258 combat sports athletes and 278 healthy male individuals as a control group. Psychometric properties were assessed with the Revised Temperament and Character Inventory (TCI-R). COMT polymorphism testing was performed using real-time PCR. We found a statistically significant effect of a complex factor COMT rs4680 genotype with combat athletes/controls and novelty seeking (F2,530 = 5.958, p = 0.0028, η2 = 0.022), self-management (F2,530 = 6.772, p = 0.0012, η2 = 0.025), and with self-transcendence skills (F2,530 = 9.387, p = 0.00009, η2 = 0.034). The results are important for encouraging further studies on the genetic makeup of athletes in conjunction with personality traits. Due to the multigene and multifactorial nature of determinants of sports predispositions, we propose to take into account also other features, especially when studying genes related to cerebral neurotransmission. It is a holistic departure, and it clearly illustrates the relationship between the given characteristics of an athlete.

A Method for Rapid Polyethyleneimine-Based Purification of Bacteriophage-Expressed Proteins from Diluted Crude Lysates, Exemplified by Thermostable TP-84 Depolymerase.

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA-protein complexes or PEI-acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme's properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion.

Effects of Vitamin D3 Supplementation and Aerobic Training on Autophagy Signaling Proteins in a Rat Model Type 2 Diabetes Induced by High-Fat Diet and Streptozotocin.

The aim of this study was to investigate the combined effects of vitamin D3 supplementation and aerobic training on regulating the autophagy process in rats with type 2 diabetic induced by a high-fat diet and streptozotocin. A total of 40 Wistar rats were divided into five groups: normal control (NC), diabetic control (DC), diabetic + aerobic training (DAT), diabetic + vitamin D3 (DVD), and diabetic + aerobic training + vitamin D3 (DVDAT). The rats underwent eight weeks of aerobic training with an intensity of 60% maximum running speed for one hour, along with weekly subcutaneous injections of 10,000 units of vitamin D3. The protein levels of different autophagy markers were assessed in the left ventricular heart tissue. The results showed that the protein levels of AMPK, pAMPK, mTOR, and pmTOR were significantly lower in the DC group compared to the NC group. Conversely, the levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D proteins were significantly higher in the DC group. However, the interventions of aerobic training and vitamin D3 supplementation, either individually or in combination, led to increased levels of AMPK, pAMPK, mTOR, and pmTOR, and decreased levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D (p < 0.05). Additionally, the aerobic capacity in the DAT and DVDAT groups was significantly higher compared to the NC, DC, and DVD groups (p < 0.05). These findings suggest that type 2 diabetes is associated with excessive autophagy in the left ventricle. However, after eight weeks of vitamin D3 supplementation and aerobic training, a significant reduction in excessive autophagy was observed in rats with type 2 diabetes.

Correction: Lubkowska et al. Analysis of Industrial Bacillus Species as Potential Probiotics for Dietary Supplements. Microorganisms 2023, 11, 488.

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The ADORA2A TT Genotype Is Associated with Anti-Inflammatory Effects of Caffeine in Response to Resistance Exercise and Habitual Coffee Intake.

Caffeine is an adenosine A2A receptor (ADORA2A) antagonist with ergogenic and anti-inflammatory effects. Previous studies have reported that the ADORA2A gene regulates glutamate metabolism and immune responses, with the ADORA2A rs5751876 TT genotype (with high sensitivity to caffeine) showing larger ergogenic effect following caffeine ingestion. We therefore hypothesized that the TT genotype would be associated with greater anti-inflammatory effects of caffeine in response to exercise, and with higher coffee intake in physically active individuals. The aim of the present study was twofold: (1) to investigate the association of the ADORA2A variant with the anti-inflammatory effects of caffeine in response to intense resistance exercise (RE), and (2) to analyze the association of the rs5751876 with coffee intake in physically active individuals (n = 134). Fifteen resistance-trained athletes participated in a randomized, double-blind, placebo-controlled cross-over study, where they consumed 6 mg/kg of caffeine or placebo one hour prior to performing an RE protocol. Blood samples were taken immediately from the arterial vein before, immediately after, and 15 min after RE for the analysis of inflammatory markers myeloperoxidase (MPO) and acetylcholinesterase (AChE). We found that the ADORA2A TT genotype carriers experienced lower exercise-induced inflammatory responses (p < 0.05 for AchE) when compared to the C allele carriers (i.e., CC/CT) one hour following the ingestion of caffeine. Furthermore, the ADORA2A TT genotype was positively associated with coffee intake (p = 0.0143; irrespective of CYP1A2 rs762551 polymorphism). In conclusion, we found that the ADORA2A gene polymorphism is associated with anti-inflammatory effects of caffeine in response to resistance exercise, as well as with habitual coffee intake in physically active individuals.

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins.

BACKGROUND: In spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host's capsule. RESULTS: The TP84_26 'capsule depolymerase' (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC-MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion. CONCLUSIONS: The novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

Analysis of Industrial Bacillus Species as Potential Probiotics for Dietary Supplements.

So far, Bacillus species bacteria are being used as bacteria concentrates, supplementing cleaning preparations in order to reduce odor and expel pathogenic bacteria. Here, we discuss the potential of Bacillus species as 'natural' probiotics and evaluate their microbiological characteristics. An industrially used microbiological concentrates and their components of mixed Bacillus species cultures were tested, which may be a promising bacteria source for food probiotic preparation for supplementary diet. In this study, antagonistic activities and probiotic potential of Bacillus species, derived from an industrial microbiological concentrate, were demonstrated. The cell free supernatants (CFS) from Bacillus licheniformis mostly inhibited the growth of foodborne pathogenic bacteria, such as Escherichia coli O157:H7 ATCC 35150, Salmonella Enteritidis KCCM 12021, and Staphylococcus aureus KCCM 11335, while some of Bacillus strains showed synergistic effect with foodborne pathogenic bacteria. Moreover, Bacillus strains identified by the MALDI TOF-MS method were found sensitive to chloramphenicol, kanamycin, and rifampicin. B. licheniformis and B. cereus displayed the least sensitivity to the other tested antibiotics, such as ampicillin, ampicillin and sulfbactam, streptomycin, and oxacillin and bacitracin. Furthermore, some of the bacterial species detected extended their growth range from the mesophilic to moderately thermophilic range, up to 54 °C. Thus, their potential sensitivity to thermophilic TP-84 bacteriophage, infecting thermophilic Bacilli, was tested for the purpose of isolation a new bacterial host for engineered bionanoparticles construction. We reason that the natural environmental microflora of non-pathogenic Bacillus species, especially B. licheniformis, can become a present probiotic remedy for many contemporary issues related to gastrointestinal tract health, especially for individuals under metabolic strain or for the increasingly growing group of lactose-intolerant people.

Genome-Wide Association Study of Exercise-Induced Fat Loss Efficiency.

There is a wide range of individual variability in the change of body weight in response to exercise, and this variability partly depends on genetic factors. The study aimed to determine DNA polymorphisms associated with fat loss efficiency in untrained women with normal weight in response to a 12-week aerobic training program using the GWAS approach, followed by a cross-sectional study in athletes. The study involved 126 untrained young Polish women (age 21.4 ± 1.7 years; body mass index (BMI): 21.7 (2.4) kg/m2) and 550 Russian athletes (229 women, age 23.0 ± 4.1; 321 men, age 23.9 ± 4.7). We identified one genome-wide significant polymorphism (rs116143768) located in the ACSL1 gene (acyl-CoA synthetase long-chain family member 1, implicated in fatty acid oxidation), with a rare T allele associated with higher fat loss efficiency in Polish women (fat mass decrease: CC genotype (n = 122) -3.8%; CT genotype (n = 4) -31.4%; p = 1.18 × 10-9). Furthermore, male athletes with the T allele (n = 7) had significantly lower BMI (22.1 (3.1) vs. 25.3 (4.2) kg/m2, p = 0.046) than subjects with the CC genotype (n = 314). In conclusion, we have shown that the rs116143768 T allele of the ACSL1 gene is associated with higher fat loss efficiency in response to aerobic training in untrained women and lower BMI in physically active men.

A method for the transient inhibition of toxicity of secretory recombinant proteins, exemplified by bacterial alkaline phosphatase. Novel protocol for problematic DNA termini dephosphorylation.

Genes encoding proteins 'toxic' to recombinant host are difficult for cloning/expression and recombinant clones are unstable. Even tightly controlled inducible T7-lac, PBAD, PL, PR promoters are not totally silent in an uninduced state and thus not adequate for highly toxic proteins. An innovative approach to engineering and expression of the gene, encoding bacterial alkaline phosphatase (BAP) is proposed. The native precursor enzyme contains a signal peptide at the N-terminus and is secreted to the Escherichia coli (E. coli) periplasm. The signal peptide is then removed that allows oxidation and formation of active dimers. To decrease toxicity of the bap gene, its secretion leader coding section was replaced with a N-terminal His6-tag. The gene was expressed in E. coli in a PBAD vector, resulting in the accumulation of soluble His6-BAP in the cytoplasm. The His6-BAP was neutral to the cells, as no maturation was possible in the reducing cytoplasm. The purified homogenous protein was further reactivated in a redox buffer containing the protein structure stabilizing cofactors. The His6-BAP exhibited high activity. A dephosphorylation protocol for all types of DNA termini was developed.The method appears well suited for the industrial production of BAP and can be applied to other problematic proteins.• Efficient toxic gene expression • Novel approach to toxic gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme • Scaled-up production of ultrapure BAP • Improved protocol for all types of DNA termini dephosphorylation.

Bacteriophages of Thermophilic 'Bacillus Group' Bacteria-A Review.

Bacteriophages of thermophiles are of increasing interest owing to their important roles in many biogeochemical, ecological processes and in biotechnology applications, including emerging bionanotechnology. However, due to lack of in-depth investigation, they are underrepresented in the known prokaryotic virosphere. Therefore, there is a considerable potential for the discovery of novel bacteriophage-host systems in various environments: marine and terrestrial hot springs, compost piles, soil, industrial hot waters, among others. This review aims at providing a reference compendium of thermophages characterized thus far, which infect the species of thermophilic 'Bacillus group' bacteria, mostly from Geobacillus sp. We have listed 56 thermophages, out of which the majority belong to the Siphoviridae family, others belong to the Myoviridae and Podoviridae families and, apparently, a few belong to the Sphaerolipoviridae, Tectiviridae or Corticoviridae families. All of their genomes are composed of dsDNA, either linear, circular or circularly permuted. Fourteen genomes have been sequenced; their sizes vary greatly from 35,055 bp to an exceptionally large genome of 160,590 bp. We have also included our unpublished data on TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus). Since the TP-84 genome sequence shows essentially no similarity to any previously characterized bacteriophage, we have defined TP-84 as a new species in the newly proposed genus Tp84virus within the Siphoviridae family. The information summary presented here may be helpful in comparative deciphering of the molecular basis of the thermophages' biology, biotechnology and in analyzing the environmental aspects of the thermophages' effect on the thermophile community.

Probiotics in the times of COVID-19.

The time of COVID-19 pandemic focused the attention of scientist to recognise the complex medical symptoms of the disease, modes of infection and possible therapies. The organisms' response towards SARS-CoV-2 infection depends on many individual factors and the course of disease is described as unprecedented and complex. Numerous symptoms from the respiratory system, abnormalities in the gastrointestinal tract, stroke, liver damage and coagulopathy, among others, are accompanied by negative side effects of the pandemic lifestyle, including immunity depletion, overall fitness impairment, skin condition worsening, psychological and psychiatric consequences. There is an urgent need to seek all possible routes for assuring favouring conditions to build and support the organisms' microbiological barriers and enhance immunity, which will also help during the ongoing vaccination action. Probiotic Lactic Acid Bacteria (LAB) and environmental Bacillus species are microorganisms typically found in food products or dietary supplements, but also applied on body surfaces or technological surfaces at home and in the industry. Since the contemporary definition of probiotics points to positive health effects, it is of highest importance to follow strict regulations and standards of product manufacturing, especially in the times of biohazard risks and rising public distrust of therapies. There is an urgent need to seek all possible routes for assuring the favouring conditions to build and support the organisms' microbiological barriers and enhance the immunity, that will serve also during the ongoing vaccination action. Probiotic LAB and environmental Bacillus species are microorganisms typically found in food products or dietary supplements, but also applied on body surface or technological surfaces in household and industry. Since the contemporary definition of probiotics points out the positive health effects, it is of highest importance to follow strict regulations and standards of product manufacturing, especially in the times of biohazard and rising public distrust of therapies.

Boosting toxic protein biosynthesis: transient in vivo inactivation of engineered bacterial alkaline phosphatase.

BACKGROUND: The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts' metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host's viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli (E. coli) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors' background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation. RESULTS: We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal His6-tag, cloned and expressed in E. coli in a PBAD promoter expression vector. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn2+, Mg2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3'-protruding). CONCLUSIONS: The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

Synthesis of α-collagen fragments and research of their influence on the degree of hydration of a model of epidermis.

INTRODUCTION: In recent years the interest into areas of science, such as cosmetology, dermatology, pharmacology or aesthetic medicine has increased significantly. Scientists are more frequently looking for ingredients that affect the skin's condition and slow down the aging process. Practically every year, the scientists discover a number of new chemical substances (both natural and synthetic) that can be potentially used to manufacture cosmetics. AIM: To evaluate the influence of selected peptides derived from α-collagen fragments on the degree of hydration of a model of epidermis isolated from a pig. MATERIAL AND METHODS: The synthesis of selected cosmetic oligopeptides were performed manually, on the solid medium, using procedure of SPPS (solid phase peptide synthesis). Following components: aqua, carbomer, glycerine, phenonip, D-panthenol, dimethicone and triethanolamine were used to prepare a reference hydrogel masks. Both the number of components and the composition of hydrogels have been developed individually for the purposes of this research. For this study the skin from a domestic pig was used. The degree of the skin hydration was measured with the SKINTEST plus camera, which uses the latest semiconductor technology. RESULTS: During the study the absorption of hydrogels with peptides was faster than that of the reference hydrogel mask. The combination of hydrophilic properties of the peptide with hydrophobic properties of Palm enabled receiving an amphiphilic structure. Such molecules are considered to be able to penetrate the corneum barrier with the greatest ease. CONCLUSIONS: The results showed that the modified compounds have contributed to water retention in the cells, thereby increasing the degree of hydration of the biological material.