Prokaryotic Research Articles

Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5–9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins.

B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

The production and release of small phospholipid membrane vesicles, or extracellular vesicles (EVs), is a trait of most prokaryotic and eukaryotic cells. EVs display heterogeneity in content, size, biogenesis, activity, and function. B cells uniquely express immunoglobulin and produce EVs; however, the relationship between these entities has not been clarified. Here, we used several methodologies to isolate large (11,000×g) and small (110,000×g) EVs and evaluate their IgM content, characteristics and activity. We found that B cells from multiple cell lines and primary B cells produce EVs that display monomeric IgM on the surface and contain encapsulated monomeric IgM, which is independent of secreted pentameric IgM. Our data indicate EV IgM can bind antigen specifically, and EV IgM can be incorporated intracellularly into secondary cells. These results suggest immunological activities different from secreted pentameric IgM that may constitute a separate and distinct antibody distribution system.

Insight into crustacean cathepsins: Structure-evolutionary relationships and functional roles in physiological processes.

Cathepsins belong to a group of proteins that are present in both prokaryotic and eukaryotic organisms and have an extremely high degree of evolutionary conservation. These proteins are functionally active in extracellular environments as soluble enzymatic proteins or attached to plasma membrane receptors. In addition, they occur in cellular secretory vesicles, mitochondria, the cytosol, and within the nuclei of eukaryotic cells. Cathepsins are classified into various groups based on their sequence variations, leading to their structural and functional diversification. The molecular understanding of the physiology of crustaceans has shown that proteases, including cathepsins, are expressed ubiquitously. They also contain one of the central regulatory systems for crustacean reproduction, growth, and immune responses. This review focuses on various aspects of the crustaceans cathepsins and emphasizes their biological roles in different physiological processes such as reproduction, growth, development, and immune responses. We also describe the bioactivity of crustaceans cathepsins. Because of the vital biological roles that cathepsins play as cellular proteases in physiological processes, they have been proposed as potential novel targets for the development of management strategies for the aquaculture industries.

Isolation and Identification of Thermophilic Bacteria from Lahendong Hot Spring, North Sulawesi

Thermophilic bacteria are a group of procaryotic organisms that can grow in high temperature around 450C until 900C. Thermophilic bacteria are able to produce lipase thermostable enzyme. Lipase thermostable enzymes produced from thermophilic bacteria are able to catalyze the hydrolysis of triglycerides into glycerol and free fatty acids and the synthesis of esters in organic solvents. Lipase thermostable enzymes are potential to be used in industrial production. Lipase enzymes have been used for the production of laundry detergents as additives or to replace chemical detergents because of their environmental friendliness. This research was aimed to isolates and identified themophilic bacteria from Lahendong hot springs, North Sulawesi and to conduct the morphological characterization, biochemical test, and molecular identification with 16S rRNA. Results showed that isolates obtained from the- Lahendong hot spring have high similarities with Geobacillus kaustophilus, Bacillus cereus, and Geobacillus lituanicus. The three isolates are able to produce lipase thermostbale enzyme.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

The role of Mycobacterium tuberculosis acetyltransferase and protein acetylation modifications in tuberculosis.

Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis (M. tb), which has been a significant burden for a long time. Post-translational modifications (PTMs) are essential for protein function in both eukaryotic and prokaryotic cells. This review focuses on the contribution of protein acetylation to the function of M. tb and its infected macrophages. The acetylation of M. tb proteins plays a critical role in virulence, drug resistance, regulation of metabolism, and host anti-TB immune response. Similarly, the PTMs of host proteins induced by M. tb are crucial for the development, treatment, and prevention of diseases. Host protein acetylation induced by M. tb is significant in regulating host immunity against TB, which substantially affects the disease's development. The review summarizes the functions and mechanisms of M. tb acetyltransferase in virulence and drug resistance. It also discusses the role and mechanism of M. tb in regulating host protein acetylation and immune response regulation. Furthermore, the current scenario of isoniazid usage in M. tb therapy treatment is examined. Overall, this review provides valuable information that can serve as a preliminary basis for studying pathogenic research, developing new drugs, exploring in-depth drug resistance mechanisms, and providing precise treatment for TB.

A Study on Yeast Using the Photoreactivation Process to Repair the Pyrimidine Dimer Mutations

Sunlight has an indispensable importance for living things in nature [1-3]. However, the direct absorption of UV will lead to the formation of pyrimidine dimers between adjacent pyrimidines in DNA strands usually in the form of cyclobutene pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) which causes great damage [4-6]. A DNA repair system, known as photoreactivation, can effectively repair the dimers using photolyase [7-9], which has currently been found in plants, prokaryotic and eukaryotic cells [10-12]. This study was carried out to determine whether photolyase DNA repair can be observed in yeast. Several yeast Petri dishes were treated with ultraviolet radiation, different treatments were then added to them, and the colonies were counted after culturing, hence verifying that yeasts can use the photoreactivation process.

A general overview of the multifactorial adaptation to cold: biochemical mechanisms and strategies.

Cold environments are more frequent than people think. They include deep oceans, cold lakes, snow, permafrost, sea ice, glaciers, cold soils, cold deserts, caves, areas at elevations greater than 3000m, and also artificial refrigeration systems. These environments are inhabited by a diversity of eukaryotic and prokaryotic organisms that must adapt to the hard conditions imposed by cold. This adaptation is multifactorial and includes (i) sensing the cold, mainly through the modification of the liquid-crystalline membrane state, leading to the activation of a two-component system that transduce the signal; (ii) adapting the composition of membranes for proper functions mainly due to the production of double bonds in lipids, changes in hopanoid composition, and the inclusion of pigments; (iii) producing cold-adapted proteins, some of which show modifications in the composition of amino acids involved in stabilizing interactions and structural adaptations, e.g., enzymes with high catalytic efficiency; and (iv) producing ice-binding proteins and anti-freeze proteins, extracellular polysaccharides and compatible solutes that protect cells from intracellular and extracellular ice. However, organisms also respond by reprogramming their metabolism and specifically inducing cold-shock and cold-adaptation genes through strategies such as DNA supercoiling, distinctive signatures in promoter regions and/or the action of CSPs on mRNAs, among others. In this review, we describe the main findings about how organisms adapt to cold, with a focus in prokaryotes and linking the information with findings in eukaryotes.

Evolutionary patterns of archaea predominant in acidic environment

BackgroundArchaea of the order Thermoplasmatales are widely distributed in natural acidic areas and are amongst the most acidophilic prokaryotic organisms known so far. These organisms are difficult to culture, with currently only six genera validly published since the discovery of Thermoplasma acidophilum in 1970. Moreover, known great diversity of uncultured Thermoplasmatales represents microbial dark matter and underlines the necessity of efforts in cultivation and study of these archaea. Organisms from the order Thermoplasmatales affiliated with the so-called “alphabet-plasmas”, and collectively dubbed “E-plasma”, were the focus of this study. These archaea were found predominantly in the hyperacidic site PM4 of Parys Mountain, Wales, UK, making up to 58% of total metagenomic reads. However, these archaea escaped all cultivation attempts.ResultsTheir genome-based metabolism revealed its peptidolytic potential, in line with the physiology of the previously studied Thermoplasmatales isolates. Analyses of the genome and evolutionary history reconstruction have shown both the gain and loss of genes, that may have contributed to the success of the “E-plasma” in hyperacidic environment compared to their community neighbours. Notable genes among them are involved in the following molecular processes: signal transduction, stress response and glyoxylate shunt, as well as multiple copies of genes associated with various cellular functions; from energy production and conversion, replication, recombination, and repair, to cell wall/membrane/envelope biogenesis and archaella production. History events reconstruction shows that these genes, acquired by putative common ancestors, may determine the evolutionary and functional divergences of “E-plasma”, which is much more developed than other representatives of the order Thermoplasmatales. In addition, the ancestral hereditary reconstruction strongly indicates the placement of Thermogymnomonas acidicola close to the root of the Thermoplasmatales.ConclusionsThis study has analysed the metagenome-assembled genome of “E-plasma”, which denotes the basis of their predominance in Parys Mountain environmental microbiome, their global ubiquity, and points into the right direction of further cultivation attempts. The results suggest distinct evolutionary trajectories of organisms comprising the order Thermoplasmatales, which is important for the understanding of their evolution and lifestyle.

Identification of serum immunoglobulin M (IgM) of crucian carp (Carassius auratus gibelio) and immune response to cyprinid herpesvirus 2 infection

Crucian carp (Carassius auratus gibelio), an extensively cultivated freshwater fish, was one of the model species for the study of fish immunology. Polyclonal antibodies were advantageous molecular tools for studying teleost immune system. Specifically, polyclonal antibodies reacting with immunoglobulins (Ig) were used successfully in studies of the teleost fishes. In the present study, we produced polyclonal antibody against CH2 domains of crucian carp IgM, and measured the in vivo dynamics of IgM mRNA caused by CyHV-2 infection. The recombinant protein IgM with relative molecular weight about 53 KD was correctly expressed in prokaryotic cells. The specificity of the polyclonal antibody was evaluated by Western blotting and results revealed that the antibody not only specifically recognized crucian carp serum but also cross-reacted with grass carp serum. Quantitative RT-PCR analysis demonstrated the expression of IgM mRNA changed significantly after CyHV-2 infection. The expression of IgM in the kidney increased and reached a maximum at 6 h post-infection (hpi), while dropped to a low level at 5 days post-infection (dpi). In conclusion, the expression of IgM was significantly upregulated in the kidney of crucian carp infected with CyHV-2, indicating that IgM played a potential role in systemic immunity against viral infection. Polyclonal antibody against crucian carp IgM had certain clinical relevance, which might provide insight into the early stage of virus infection and prevention of the disease.

A Microfluidic Approach for Synthesis of Silver Nanoparticles as a Potential Antimicrobial Agent in Alginate-Hyaluronic Acid-Based Wound Dressings.

The recognized antimicrobial activity of silver nanoparticles is a well-studied property, especially when designing and developing biomaterials with medical applications. As biological activity is closely related to the physicochemical characteristics of a material, aspects such as particle morphology and dimension should be considered. Microfluidic systems in continuous flow represent a promising method to control the size, shape, and size distribution of synthesized nanoparticles. Moreover, using microfluidics widens the synthesis options by creating and controlling parameters that are otherwise difficult to maintain in conventional batch procedures. This study used a microfluidic platform with a cross-shape design as an innovative method for synthesizing silver nanoparticles and varied the precursor concentration and the purging speed as experimental parameters. The compositional and microstructural characterization of the obtained samples was carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Four formulations of alginate-based hydrogels with the addition of hyaluronic acid and silver nanoparticles were obtained to highlight the antimicrobial activity of silver nanoparticles and the efficiency of such a composite in wound treatment. The porous structure, swelling capacity, and biological properties were evaluated through physicochemical analysis (FT-IR and SEM) and through contact with prokaryotic and eukaryotic cells. The results of the physicochemical and biological investigations revealed desirable characteristics for performant wound dressings (i.e., biocompatibility, appropriate porous structure, swelling rate, and degradation rate, ability to inhibit biofilm formation, and cell growth stimulation capacity), and the obtained materials are thus recommended for treating chronic and infected wounds.

Engineered bacterial orthogonal DNA replication system for continuous evolution.

Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can efficiently mutate long DNA fragments in an easy-to-operate manner. However, such a system is lacking in bacteria. Therefore, we developed a bacterial orthogonal DNA replication system (BacORep) for continuous evolution. We achieved this by harnessing the temperate phage GIL16 DNA replication machinery in Bacillus thuringiensis with an engineered error-prone orthogonal DNA polymerase. BacORep introduces all 12 types of nucleotide substitution in 15-kilobase genes on orthogonally replicating linear plasmids with a 6,700-fold higher mutation rate than that of the host genome, the mutation rate of which is unchanged. Here we demonstrate the utility of BacORep-based continuous evolution by generating strong promoters applicable to model bacteria, Bacillus subtilis and Escherichia coli, and achieving a 7.4-fold methanol assimilation increase in B. thuringiensis. BacORep is a powerful tool for continuous evolution in prokaryotic cells.

Immunomodulation, Bioavailability and Safety of Bacteriocins.

The rise of antibiotic-resistant bacteria and the emergence of new pathogens have created a need for new strategies to fight against infectious diseases. One promising approach is the use of antimicrobial peptides produced by a certain species of bacteria, known as bacteriocins, which are active against other strains of the same or related species. Bacteriocins can help in the treatment and prevention of infectious diseases. Moreover, bacteriocins can be obtained in prokaryotic organisms, and contribute s to their widespread use. While the use of bacteriocins is currently limited to the food industry (for example, nisin is used as a preservative, E234), a large number of studies on their microbicidal properties suggest that their use in medicine may increase in the foreseeable future. However, for the successful use of bacteriocins in medicine, it is necessary to understand their effect on the immune system, especially in cases where immunity is weakened due to infectious processes, oncological, allergic, or autoimmune diseases. Studies on the immuno-modulatory activity of bacteriocins in animal models and human cells have revealed their ability to induce both pro-inflammatory and anti-inflammatory factors involved in the implementation of innate immunity. The influence of bacteriocins on acquired immunity is revealed by an increase in the number of T-lymphocytes with a simultaneous decrease in B-lymphocyte levels, which makes them attractive substances for reducing inflammation. The widespread use of bacteriocins in the food industry, their low toxicity, and their broad and narrow specificity are reasons for researchers to pay attention to their immunomodulatory properties and explore their medical applications. Inflammation regulation by bacteriocins can be used in the treatment of various pathologies. The aim of the review was to analyze scientific publications on the immunomodulatory activity, bioavailability, and safety of bacteriocins in order to use the data obtained to organize preclinical and clinical studies.

Calorimetric analysis of AdcR and its interactions with zinc(II) and DNA

Zinc(II) ions play critical roles in all known life as structurally important stabilizing ions in proteins, catalytically active metals in enzymes, and signaling agents impacting physiological changes. To maintain homeostasis, the intracellular concentration of zinc(II) is strictly controlled by a family of metal-regulatory proteins in both prokaryotic and eukaryotic organisms. In S. pneumoniae, there are two proteins that share responsibility for Zn2+ homeostasis, one of them is the Adhesin Competence Repressor (AdcR) and it binds to a specific double-stranded DNA binding domain (dsDNA). AdcR has been structurally characterized containing two zinc(II) metal centers per monomeric unit. Here we report data collected from differential scanning calorimetry (DSC) experiments aimed to measure the structural stability of AdcR, the fully complimented Zn2AdcR complex, and the protein/DNA complex Zn2AdcR/dsDNA. Thermograms collected from DSC experiments yielded endothermic unfolding events for AdcR, Zn2AdcR, and Zn2AdcR/dsDNA complex at 55.6, 70.2, and 56.6 °C, respectively. A non-two state unfolding model best fits the data, giving ΔH terms associated with these thermal unfolding events of 5.1, 7.1, and 4.9 kcal/mol. These data allow for the development of a thermodynamic cycle connecting both zinc(II) and DNA binding to AdcR. Furthermore, pairing this newly reported data with known association constants for zinc(II) and DNA binding allowed for the generation of thermodynamic profiles for both zinc(II) binding to AdcR and Zn2AdcR binding to DNA, which show both are decisively entropy-driven processes.

Unlocking the Stability of Reduced Graphene Oxide Nanosheets in Biological Media via Use of Sodium Ascorbate

AbstractGraphene oxide and reduced graphene oxide (RGO) are carbon bidimensional nanomaterials largely exploited in biomedicine. Their unique interactions with eukaryotic and prokaryotic cells are used to obtain precise intracellular delivery, to create device coatings, and to design theranostic materials for both therapeutic and imaging applications, mainly in the cancer research field. It is known, however, that the hydrophobic behavior of RGO limits its stability in biological media. Here, the employment of sodium ascorbate (NaA) as a reducing agent for the preparation of RGO to provide a nanomaterial with remarkable suitability for applications in cell culture media is proposed. It is demonstrated via a combined experimental and theoretical approach that NaA is able to yield a peculiar RGO derivative, exerting a twofold effect, that is, C sp2 network restoration upon epoxide reduction and RGO edge functionalization via H‐bonding, lending RGO a so far unexampled dispersibility in aqueous‐based media. The kinetic stability of the bidimensional layers of RGO obtained from NaA is demonstrated together with its superior biocompatibility for drug delivery, unlocking outstanding potentialities for biological applications.

Effects of selective RNA processing and stabilization enzymes on carbon sequestration by photosynthesis of Synechococcus sp. PCC7002

Synechococcus is one of the most abundant prokaryotic photosynthetic organisms on Earth and plays a key role in oceanic carbon fixation and transformation. To improve the photosynthetic efficiency of synechococcus, a post-transcriptional regulatory mechanism - Selective RNA Processing and Stabilization (SRPS) was considered. We inactivated the SRPS-enzymes, executor of the SRPS mechanism, to explore their regulation rule of photosynthetic carbon fixation efficiency in Synechococcus. The results showed that the inactivation of SRPS-enzymes mainly affected the growth rate or growth phase. It significantly alters the photosynthetic oxygen evolution rate, pigment content, chlorophyll fluorescence, carbon and nitrogen content, as well as the composition and biological activity of the dissolved organic matter derived from Synechococcus (SOM). Inactivating SRPS-enzymes results in an increase in the expression level of most subunits of the Cytochrome b6-f complex, while the expression levels of most subunits of PSI, PSII, RuBisCO, and NDH decrease. All SRPS-enzymes are involved in the expression regulation of basilic protein complexes in photosynthesis, such as PSI, PSII, Cytochrome b6-f complex, ATP synthase, and RuBisCO. Our results indicate that the inactivation of SRPS-enzymes have a significant influence on carbon sequestration by photosynthesis of Synechococcus sp. PCC7002.

Methods for Interactome Analysis of Microproteins Encoded by Small Open Reading Frames

Recent studies have shown that small open reading frames (sORFs, 100 codons) can encode peptides or microproteins that perform important functions in prokaryotic and eukaryotic cells. It has been established that sORF translation products are involved in the regulation of many processes, for example, they modulate the activity of the mitochondrial respiratory chain or the functions of muscle cells in mammals. However, the identification and subsequent functional analysis of peptides or microproteins encoded by sORFs is a non-trivial task and requires the use of special approaches. One of the critical steps in functional analysis is identification of protein partners of the peptide under study. This review considers the features of the interactome analysis of short protein molecules and describes the approaches currently used for studies in the field.

Structure and assembly of type VI secretion system cargo delivery vehicle

Type VI secretion system is widely used in Gram-negative bacteria for injecting toxic effectors into neighboring prokaryotic or eukaryotic cells. Various effectors can be loaded onto the T6SS delivery tube via its core components: Hcp, VgrG, or PAAR. Here, we report 2.8-Å resolution cryo-EM structure of intact T6SS Hcp5-VgrG-PAAR cargo delivery system and crystal structure of unbound Hcp5 from B.fragilis NCTC 9343. Loading of Hcp5 hexameric ring onto VgrG causes expansion of its inner cavity and external surface, explaining how structural changes could be propagated to regulate co-polymerization and surrounding contractile sheath. High-affinity binding between Hcp and VgrG causes entropically unfavorable structuring of long loops. Furthermore, interactions between VgrG trimer and Hcp hexamer are asymmetric, with three of the six Hcp monomers exhibiting a major loop flip. Our study provides insights into the assembly, loading, and firing of T6SS nanomachine that contributes to bacterial inter-species competition and host interactions.

The Prokaryotic Community Structure of Oil-Contaminated Chernozem during the Introduction of Nitrate and Potassium Chloride

The effect of nitrate and potassium chloride salts, on the structure of the metabolically active prokaryotic community of oil-contaminated chernozem has been studied. Molecular biological approaches and bioinformatic methods of analysis were used in the study. The objects of the study were samples of chernozem selected in the Voronezh region (N 51°1′41″, E 40°43′31″). The phylogenetic and functional diversity of the prokaryotic complex of oil-contaminated chernozem was considered when introducing nitrate and potassium chloride under conditions of a slightly alkaline reaction of the medium. Contamination of chernozem with oil in an amount of 5% of the soil mass led to alkalinization of the medium from 7.1 to 7.9. The introduction of nitrate and potassium chloride, both separately and together in a total dose of 2 mmol/100 g of soil removed this negative effect. The combined addition of nitrate and potassium chloride led to a more than twofold increase in the biomass of metabolically active prokaryotic cells and the number of copies of functional genes responsible for the synthesis of alkanmonooxygenase enzymes involved in the decomposition of oil. In the presence of oil, the formation of a specific complex of bacteria was revealed, in which representatives of A-ctinobacteria (Rhodococcus erythropolis) and Alphaproteobacteria (Bradyrhizobium japonicum) prevailed. Rhodococcus erythropolis and Bradyrhizobium japonicum, being autochthonous organisms in uncontaminated soil, began to occupy dominant positions in oil-contaminated samples, and the introduction of nitrates enhanced this effect.

Similarities and Differences of Expression of Gene in Prokaryotic and Eukaryotic Cells: A Critical Review

This article is totally based on the literature review about expression of gene in prokaryotic and eukaryotic cells. Gene is the part of the DNA that is responsible for the formation of the genotype and then a phenotype. Gene can only be expressed in case of formation of protein. The process of the gene expression is accomplished in the two steps; Transcription and translation. Transcription is the process of the formation of the mRNA. Transcription proceeds in the three steps. In initiation step the RNA polymerase moves on the unwind DNA strand until the promoter sequence is reached, next is the elongation step in the newly synthesized RNA strand elongates, the process terminates as the RNA polymerase realize the termination sequence. After the mRNA is formed it is released into the cytoplasm without any alterations in case of prokaryotes, but in case of eukaryotes post transcriptional modifications takes place which include capping, tailing and splicing. Translation is the process of the formation of protein from mRNA. Translation proceeds in the four steps in which the t RNA is charged, the initiation complex is formed with Met-tRNA at the P site start codon is recognized after binding of the ribosomal subunits to mRNA, in elongation step the peptide bond is formed and the polypeptide chain grows, stop codons enters at the A site and thus the process is terminated. Post translational modications takes place which the initiation amino acid methionine is removed.