RudS: bacterial desulfidase responsible for tRNA 4-thiouridine de-modification.

In this study, we present an extensive analysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a Domain of Unknown Function 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicating the enzyme's role in regulating photosensitive tRNA s4U modification. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses.

Conversion of ß-1,6-Glucans to Gentiobiose using an endo-ß-1,6-Glucanase PsGly30A from Paenibacillus sp. GKG.

A plethora of di- and oligosaccharides isolated from the natural sources are used in food and pharmaceutical industry. An enzymatic hydrolysis of fungal cell wall ß-glucans is a good alternative to produce the desired oligosaccharides with different functionalities, such as the flavour enhancer gentiobiose. We have previously identified PsGly30A as a potential yeast cell wall degrading ß-1,6-glycosidase. The aim of this study is to characterise the PsGly30A enzyme, a member of the GH30 family, and to evaluate its suitability for the production of gentiobiose from ß-1,6-glucans. An endo-ß-1,6-glucanase PsGly30A encoding gene from Paenibacillus sp. GKG has been cloned and overexpressed in Escherichia coli. The recombinant enzyme has been active towards pustulan and yeast ß-glucan, but not on laminarin from the Laminaria digitata, confirming the endo-ß-1,6-glucanase mode of action. The PsGly30A shows the highest activity at pH 5.5 and 50 °C. The specific activity of PsGly30A on pustulan (1262±82 U/mg) is among the highest reported for GH30 ß-1,6-glycosidases. Moreover, gentiobiose is the major reaction product when pustulan, yeast ß-glucan or yeast cell walls have been used as a substrate. Therefore, PsGly30A is a promising catalyst for valorisation of the yeast-related by-products.

Prebiotics Mitigate the Detrimental Effects of High-Fat Diet on memory, anxiety and microglia functionality in Ageing Mice.

Ageing is characterised by a progressive increase in systemic inflammation and especially neuroinflammation. Neuroinflammation is associated with altered brain states that affect behaviour, such as an increased level of anxiety with a concomitant decline in cognitive abilities. Although multiple factors play a role in the development of neuroinflammation, microglia have emerged as a crucial target. Microglia are the only macrophage population in the CNS parenchyma that plays a crucial role in maintaining homeostasis and in the immune response, which depends on the activation and subsequent deactivation of microglia. Therefore, microglial dysfunction has a major impact on neuroinflammation. The gut microbiota has been shown to significantly influence microglia from birth to adulthood in terms of development, proliferation, and function. Diet is a key modulating factor that influences the composition of the gut microbiota, along with prebiotics that support the growth of beneficial gut bacteria. Although the role of diet in neuroinflammation and behaviour has been well established, its relationship with microglia functionality is less explored. This article establishes a link between diet, animal behaviour and the functionality of microglia. The results of this research stem from experiments on mouse behaviour, i.e., memory, anxiety, and studies on microglia functionality, i.e., cytochemistry (phagocytosis, cellular senescence, and ROS assays), gene expression and protein quantification. In addition, shotgun sequencing was performed to identify specific bacterial families that may play a crucial role in the brain function. The results showed negative effects of long-term consumption of a high fat diet on ageing mice, epitomised by increased body weight, glucose intolerance, anxiety, cognitive impairment and microglia dysfunction compared to ageing mice on a control diet. These effects were a consequence of the changes in gut microbiota modulated by the diet. However, by adding the prebiotics fructo- and galacto-oligosaccharides, we were able to mitigate the deleterious effects of a long-term high-fat diet.

α-L-Fucosidases from an Alpaca Faeces Metagenome: Characterisation of Hydrolytic and Transfucosylation Potential.

In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N-acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and α-L-fucosidases. This work aimed to characterise α-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant α-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p-nitrophenyl-α-L-fucopyranoside and 2'-fucosyllactose. Furthermore, the enzymes' biochemical properties and kinetic characteristics were also determined. All four α-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of α-L-fucosidases for synthesising fucosylated amino acids.

Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates.

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.

Unraveling the Dynamics of Host-Microbiota Indole Metabolism: An Investigation of Indole, Indolin-2-one, Isatin, and 3-Hydroxyindolin-2-one.

The gut microbiota produces a variety of bioactive molecules that facilitate host-microbiota interaction. Indole and its metabolites are focused as possible biomarkers for various diseases. However, data on indole metabolism and individual metabolites remain limited. Hence, we investigated the metabolism and distribution of indole, indolin-2-one, isatin, and 3-hydroxyindolin-2-one. First, we orally administered a high dose of indole into C57BL/6J mice and measured the concentrations of indole metabolites in the brain, liver, plasma, large and small intestines, and cecum at multiple time points using HPLC/MS. Absorption in 30 min and full metabolization in 6 h were established. Furthermore, indole, indolin-2-one, and 3-hydroxiindolin-2-one, but not isatin, were found in the brain. Second, we confirmed these findings by using stable isotope-carrying indole. Third, we identified 3-hydroxyindolin-2-one as an indole metabolite in vivo by utilizing a 3-hydroxyindolin-2-one-converting enzyme, IifA. Further, we confirmed the ability of orally administered 3-hydroxyindolin-2-one to cross the blood-brain barrier in a dose-dependent manner. Finally, we detected upregulation of the CYP1A2 and CYP2A5 genes, confirming the importance of these cytochrome isoforms in indole metabolism in vivo. Overall, our results provide a basic characterization of indole metabolism in the host and highlight 3-hydroxyindolin-2-one as a potentially brain-affecting indole metabolite.

Characterization of Parageobacillus Bacteriophage vB_PtoS_NIIg3.2-A Representative of a New Genus within Thermophilic Siphoviruses.

A high temperature-adapted bacteriophage, vB_PtoS_NIIg3.2 (NIIg3.2), was isolated in Lithuania from compost heaps using Parageobacillus toebii strain NIIg-3 as a host for phage propagation. Furthermore, NIIg3.2 was active against four strains of Geobacillus thermodenitrificans, and it infected the host cells from 50 to 80 °C. Transmission electron microscopy analysis revealed siphovirus morphology characterized by an isometric head (~59 nm in diameter) and a noncontractile tail (~226 nm in length). The double-stranded DNA genome of NIIg3.2 (38,970 bp) contained 71 probable protein-encoding genes and no genes for tRNA. In total, 29 NIIg3.2 ORFs were given a putative functional annotation, including those coding for the proteins responsible for DNA packaging, virion structure/morphogenesis, phage-host interactions, lysis/lysogeny, replication/regulation, and nucleotide metabolism. Based on comparative phylogenetic and bioinformatic analysis, NIIg3.2 cannot be assigned to any genus currently recognized by ICTV and potentially represents a new one within siphoviruses. The results of this study not only extend our knowledge about poorly explored thermophilic bacteriophages but also provide new insights for further investigation and understanding the evolution of Bacilllus-group bacteria-infecting viruses.

Insights into the Alcyoneusvirus Adsorption Complex.

The structures of the Caudovirales phage tails are key factors in determining the host specificity of these viruses. However, because of the enormous structural diversity, the molecular anatomy of the host recognition apparatus has been elucidated in only a number of phages. Klebsiella viruses vB_KleM_RaK2 (RaK2) and phiK64-1, which form a new genus Alcyoneusvirus according to the ICTV, have perhaps one of the most structurally sophisticated adsorption complexes of all tailed viruses described to date. Here, to gain insight into the early steps of the alcyoneusvirus infection process, the adsorption apparatus of bacteriophage RaK2 is studied in silico and in vitro. We experimentally demonstrate that ten proteins, gp098 and gp526-gp534, previously designated as putative structural/tail fiber proteins (TFPs), are present in the adsorption complex of RaK2. We show that two of these proteins, gp098 and gp531, are essential for attaching to Klebsiella pneumoniae KV-3 cells: gp531 is an active depolymerase that recognizes and degrades the capsule of this particular host, while gp098 is a secondary receptor-binding protein that requires the coordinated action of gp531. Finally, we demonstrate that RaK2 long tail fibers consist of nine TFPs, seven of which are depolymerases, and propose a model for their assembly.

Interaction between Phage T4 Protein RIII and Host Ribosomal Protein S1 Inhibits Endoribonuclease RegB Activation.

Lytic viruses of bacteria (bacteriophages, phages) are intracellular parasites that take over hosts' biosynthetic processes for their propagation. Most of the knowledge on the host hijacking mechanisms has come from the studies of the lytic phage T4, which infects Escherichia coli. The integrity of T4 development is achieved by strict control over the host and phage processes and by adjusting them to the changing infection conditions. In this study, using in vitro and in vivo biochemical methods, we detected the direct interaction between the T4 protein RIII and ribosomal protein S1 of the host. Protein RIII is known as a cytoplasmic antiholin, which plays a role in the lysis inhibition function of T4. However, our results show that RIII also acts as a viral effector protein mainly targeting S1 RNA-binding domains that are central for all the activities of this multifunctional protein. We confirm that the S1-RIII interaction prevents the S1-dependent activation of endoribonuclease RegB. In addition, we propose that by modulating the multiple processes mediated by S1, RIII could act as a regulator of all stages of T4 infection including the lysis inhibition state.

Depicting Temporal, Functional, and Phylogenetic Patterns in Estuarine Diazotrophic Communities from Environmental DNA and RNA.

Seasonally nitrogen-limited and phosphorus-replete temperate coastal waters generally host dense and diverse diazotrophic communities. Despite numerous studies in marine systems, little is known about diazotrophs and their functioning in oligohaline estuarine environments. Here we applied a combination of nifH transcript and metagenomic shotgun sequencing approaches to investigate temporal shifts in taxonomic composition and nifH activity of size-fractionated diazotrophic communities in a shallow and mostly freshwater coastal lagoon. Patterns in active nifH phylotypes exhibited a clear seasonal succession, which reflected their different tolerances to temperature change and nitrogen (N) availability. Thus, in spring, heterotrophic diazotrophs (Proteobacteria) dominated the nifH phylotypes, while increasing water temperature and depletion of inorganic N fostered heterocystous Cyanobacteria in summer. Metagenomic data demonstrated four main N-cycling pathways and three of them with a clear seasonal pattern: denitrification (spring) → N2 fixation (summer) → assimilative NO3- reduction (fall), with NH4+ uptake into cells occurring across all seasons. Although a substantial denitrification signal was observed in spring, it could have originated from the re-suspended benthic rather than planktonic community. Our results contribute to a better understanding of the realized genetic potential of pelagic N2 fixation and its seasonal dynamics in oligohaline estuarine ecosystems, which are natural coastal biogeochemical reactors.

The First Step of Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 Depends on an Alcohol Dehydrogenase-Type Enzyme.

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.

Pantoea Bacteriophage vB_PagS_MED16-A Siphovirus Containing a 2'-Deoxy-7-amido-7-deazaguanosine-Modified DNA.

A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using Pantoea agglomerans strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a gene encoding a preQ0 DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC-MS/MS analysis indicates 2'-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of Pantoea phages. Thus, the data presented in this study provide new information on Pantoea-infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages.

Structural Evidence for a [4Fe-5S] Intermediate in the Non-Redox Desulfuration of Thiouracil.

We recently discovered a [Fe-S]-containing protein with in vivo thiouracil desulfidase activity, dubbed TudS. The crystal structure of TudS refined at 1.5 Šresolution is reported; it harbors a [4Fe-4S] cluster bound by three cysteines only. Incubation of TudS crystals with 4-thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non-protein-bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe-5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site-directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information, together with modeling studies allow us to propose a mechanism for the unprecedented non-redox enzymatic desulfuration of thiouracil, in which a [4Fe-4S] cluster binds and activates the sulfur atom of the substrate.

Microbial Degradation of Pyridine: a Complete Pathway in Arthrobacter sp. Strain 68b Deciphered.

Pyridine and its derivatives constitute the majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to environmental pollution. It is known that they can be degraded by various bacteria in the environment; however, the degradation of unsubstituted pyridine has not yet been completely resolved. In this study, we present data on the pyridine catabolic pathway in Arthrobacter sp. strain 68b at the level of genes, enzymes, and metabolites. The pyr gene cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid, p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by the formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalyzed by a two-component flavin-dependent monooxygenase system, encoded by pyrA (pyridine monooxygenase) and pyrE genes. The genes pyrB, pyrC, and pyrD were found to encode (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation. The metabolites of these enzymatic reactions were identified, and this allowed us to reconstruct the entire pyridine catabolism pathway in Arthrobacter sp. 68b.IMPORTANCE The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-borne pyr gene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism of N-heterocyclic compounds in nature.

Complete genome sequence of Buttiauxella phage vB_ButM_GuL6.

We present here the results of the analysis of the complete genome sequence of a lytic bacteriophage, vB_ButM_GuL6, which is the first virus isolated from Buttiauxella. Electron microscopy revealed that vB_ButM_GuL6 belongs to the family Myoviridae, order Caudovirales. The genome of vB_ButM_GuL6 is a linear, circularly permuted 178,039-bp dsDNA molecule with a GC content of 43.4%. It has been predicted to contain 282 protein-coding genes and two tRNA genes, tRNA-Met and tRNA-Gly. Using bioinformatics approaches, 99 (36%) of the vB_ButM_GuL6 genes were assigned a putative function. Genome-wide comparisons and phylogenetic analysis indicated that vB_ButM_GuL6 represents a new species of the subfamily Tevenvirinae and is most closely related to Escherichia virus RB43. These phages, together with Cronobacter phages Miller, CfP1, and IME-CF2, likely form a new genus within the subfamily Tevenvirinae.

Complete genome analysis of Pantoea agglomerans-infecting bacteriophage vB_PagM_AAM22.

A novel myovirus, vB_PagM_AAM22 (AAM22), was isolated in Lithuania using Pantoea agglomerans as the host for phage propagation. The 49,744-bp genome of AAM22 has a G + C content of 48.4% and contains 96 probable protein-encoding genes and no genes for tRNA. In total, 34 ORFs were given a putative functional annotation, including genes associated with virion morphogenesis, DNA metabolism, and phage-host interactions. Based on comparative phylogenetic analysis, AAM22 cannot be assigned to any genus currently recognized by the ICTV and is a potential candidate to form a new genus within the family Myoviridae.

N4-acyl-2'-deoxycytidine-5'-triphosphates for the enzymatic synthesis of modified DNA.

A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of pyrimidines and C7 of purines. Unfortunately, by using these two positions only, one cannot expand a repertoire of modified nucleotides to a maximum. Here, we demonstrate the synthesis and enzymatic incorporation of novel N4-acylated 2'-deoxycytidine nucleotides (dCAcyl). We find that a variety of family A and B DNA polymerases efficiently use dCAcylTPs as substrates. In addition to the formation of complementary CAcyl•G pair, a strong base-pairing between N4-acyl-cytosine and adenine takes place when Taq, Klenow fragment (exo-), Bsm and KOD XL DNA polymerases are used for the primer extension reactions. In contrast, a proofreading phi29 DNA polymerase successfully utilizes dCAcylTPs but is prone to form CAcyl•A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N4-acylated-deoxycytidine nucleotides. These data reveal novel N4-acylated deoxycytidine nucleotides as beneficial substrates for the enzymatic synthesis of modified DNA, which can be further applied for specific labelling of DNA fragments, selection of aptamers or photoimmobilization.

Transient N4 -Acyl-DNA Protection against Cleavage by Restriction Endonucleases.

A set of five N4 -acyl-modified 2'-deoxycytidine 5'-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N4 -acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N4 -acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N4 modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N4 -modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.

Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions.

While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria.IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.

Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy.

BACKGROUND: The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. METHODS: Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. RESULTS: 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 µM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. CONCLUSIONS: We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.