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.

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.

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.

Bacterial amidohydrolases and modified 5-fluorocytidine compounds: Novel enzyme-prodrug pairs.

Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. However, it is limited by the lack of suitable enzyme variants and a scarce choice of chemical bonds that could be activated. Therefore, this study is aimed to determine the capability of bacterial amidohydrolases YqfB and D8_RL to activate novel prodrugs and the effect such system has on the viability of eukaryotic cancer cells. We have established cancer cell lines that stably express the bacterial amidohydrolase genes and selected several N4-acylated cytidine derivatives as potential prodrugs. A significant decrease in the viability of HCT116 human colon cancer cell lines expressing either the YqfB or the D8_RL was observed after exposure to the novel prodrugs. The data we acquired suggests that bacterial YqfB and D8_RL amidohydrolases, together with the modified cytidine-based prodrugs, may serve as a promising enzyme-prodrug system for gene-directed enzyme prodrug therapy.

TudS desulfidases recycle 4-thiouridine-5'-monophosphate at a catalytic [4Fe-4S] cluster.

In all domains of life, transfer RNAs (tRNAs) contain post-transcriptionally sulfur-modified nucleosides such as 2- and 4-thiouridine. We have previously reported that a recombinant [4Fe-4S] cluster-containing bacterial desulfidase (TudS) from an uncultured bacterium catalyzes the desulfuration of 2- and 4-thiouracil via a [4Fe-5S] cluster intermediate. However, the in vivo function of TudS enzymes has remained unclear and direct evidence for substrate binding to the [4Fe-4S] cluster during catalysis was lacking. Here, we provide kinetic evidence that 4-thiouridine-5'-monophosphate rather than sulfurated tRNA, thiouracil, thiouridine or 4-thiouridine-5'-triphosphate is the preferred substrate of TudS. The occurrence of sulfur- and substrate-bound catalytic intermediates was uncovered from the observed switch of the S = 3/2 spin state of the catalytic [4Fe-4S] cluster to a S = 1/2 spin state upon substrate addition. We show that a putative gene product from Pseudomonas putida KT2440 acts as a TudS desulfidase in vivo and conclude that TudS-like enzymes are widespread desulfidases involved in recycling and detoxifying tRNA-derived 4-thiouridine monophosphate nucleosides for RNA synthesis.

Exploring the enzymatic activity of depolymerase gp531 from Klebsiella pneumoniae jumbo phage RaK2.

Klebsiella pneumoniae poses a major global challenge due to its virulence, multidrug resistance, and nosocomial nature. Thus, bacteriophage-derived proteins are extensively being investigated as a means to combat this bacterium. In this study, we explored the enzymatic specificity of depolymerase gp531, encoded by the jumbo bacteriophage vB_KleM_RaK2 (RaK2). We used two different methods to modify the reducing end of the oligosaccharides released during capsule hydrolysis with gp531. Subsequent acidic cleavage with TFA, followed by TLC and HPLC-MS analyses, revealed that RaK2 gp531 is a ß-(1→4)-endoglucosidase. The enzyme specifically recognizes and cleaves the capsular polysaccharide (CPS) of the Klebsiella pneumoniae K54 serotype, releasing K-unit monomers (the main product), dimers, and trimers. Depolymerase gp531 remains active from 10 to 50 °C and in the pH 3-8 range, indicating its stability and versatility. Additionally, we demonstrated that gp531's activity is not affected by CPS acetylation, which is influenced by the growth conditions of the bacterial culture. Overall, our findings provide valuable insights into the enzymatic activity of the first characterized depolymerase targeting the capsule of the clinically relevant K54 serotype of K. pneumoniae.

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.

Geobacillus Bacteriophages from Compost Heaps: Representatives of Three New Genera within Thermophilic Siphoviruses.

We report a detailed characterization of five thermophilic bacteriophages (phages) that were isolated from compost heaps in Vilnius, Lithuania using Geobacillus thermodenitrificans strains as the hosts for phage propagation. The efficiency of plating experiments revealed that phages formed plaques from 45 to 80 °C. Furthermore, most of the phages formed plaques surrounded by halo zones, indicating the presence of phage-encoded bacterial exopolysaccharide (EPS)-degrading depolymerases. Transmission Electron Microscopy (TEM) analysis revealed that all phages were siphoviruses characterized by an isometric head (from ~63 nm to ~67 nm in diameter) and a non-contractile flexible tail (from ~137 nm to ~150 nm in length). The genome sequencing resulted in genomes ranging from 38,161 to 39,016 bp. Comparative genomic and phylogenetic analysis revealed that all the isolated phages had no close relatives to date, and potentially represent three new genera within siphoviruses. The results of this study not only improve our knowledge about poorly explored thermophilic bacteriophages but also give new insights for further investigation of thermophilic and/or thermostable enzymes of bacterial viruses.

Nanotubes from bacteriophage tail sheath proteins: internalisation by cancer cells and macrophages.

Bionanoparticles comprised of naturally occurring monomers are gaining interest in the development of novel drug transportation systems. Here we report on the stabilisation, cellular uptake, and macrophage clearance of nanotubes formed from the self-assembling gp053 tail sheath protein of the vB_EcoM_FV3 bacteriophage. To evaluate the potential of the bacteriophage protein-based nanotubes as therapeutic nanocarriers, we investigated their internalisation into colorectal cancer cell lines and professional macrophages that may hinder therapeutic applications by clearing nanotube carriers. We fused the bacteriophage protein with a SNAP-tag self-labelling enzyme and demonstrated that its activity is retained in assembled nanotubes, indicating that such carriers can be applied to deliver therapeutic biomolecules. Under physiological conditions, the stabilisation of the nanotubes by PEGylation was required to prevent aggregation and yield a stable solution with uniform nano-sized structures. Colorectal carcinoma cells from primary and metastatic tumours internalized SNAP-tag-carrying nanotubes with different efficiencies. The nanotubes entered HCT116 cells via dynamin-dependent and SW480 cells - via dynamin- and clathrin-dependent pathways and were accumulated in lysosomes. Meanwhile, peritoneal macrophages phagocytosed the nanotubes in a highly efficient manner through actin-dependent mechanisms. Macrophage clearance of nanotubes was enhanced by inflammatory activation but was dampened in macrophages isolated from aged animals. Altogether, our results demonstrate that gp053 nanotubes retained the cargo's enzymatic activity post-assembly and had the capacity to enter cancer cells. Furthermore, we emphasise the importance of evaluating the nanocarrier clearance by immune cells under conditions mimicking a cancerous environment.

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.

Cytidine deaminases catalyze the conversion of N(S,O)4-substituted pyrimidine nucleosides.

Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2'-deoxycytidine to uridine and 2'-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N4-acyl-cytidines, N4-alkyl-cytidines, and N4-alkyloxycarbonyl-cytidines, and S4-alkylthio-uridines and O4-alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible ß3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs.

Characterization of Paenibacillus sp. GKG Endo-ß-1, 3-Glucanase, a Member of Family 81 Glycoside Hydrolases.

Paenibacillus sp. GKG was isolated based on its ability to produce hydrolysis zones on agar plates containing yeast cell wall substrate as the single carbon source. The extracellular enzymes secreted into the culture medium were identified by LC-MS/MS proteomics. Endo-ß-1,3-glucanase PsLam81A containing GH81 catalytic and the CBM56 carbohydrate-binding modules was selected for heterologous expression in Escherichia coli. The identity of the recombinant PsLam81A was confirmed by LC-MS/MS proteomics. The PsLam81A showed the highest activity at 60 °C, and the optimal pH range was between 6.5 and 8.0. The analysis of the full-length PsLam81A and truncated PsLam81AΔCBM56 enzymes showed that the CBM56 module improved the hydrolytic activity towards linear ß-1,3-glucans-curdlan and pachyman but had no effect on hydrolysis of ß-1,3/ß1,6-branched glucans-laminarin and yeast ß-glucan. The characterization of PsLam81A enzyme broadens current knowledge on the biochemical properties and substrate specificity of family 81 glycoside hydrolases and allows prediction of the necessity of CBM56 module in the process of designing new truncated or chimeric glycosidases.

Comparative Analysis of Mesophilic YqfB-Type Amidohydrolases.

The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N4-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications.

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.

Glucose-to-Resistor Transduction Integrated into a Radio-Frequency Antenna for Chip-less and Battery-less Wireless Sensing.

To maximize the potential of 5G infrastructure in healthcare, simple integration of biosensors with wireless tag antennas would be beneficial. This work introduces novel glucose-to-resistor transduction, which enables simple, wireless biosensor design. The biosensor was realized on a near-field communication tag antenna, where a sensing bioanode generated electrical current and electroreduced a nonconducting antenna material into an excellent conductor. For this, a part of the antenna was replaced by a Ag nanoparticle layer oxidized to high-resistance AgCl. The bioanode was based on Au nanoparticle-wired glucose dehydrogenase (GDH). The exposure of the cathode-bioanode to glucose solution resulted in GDH-catalyzed oxidation of glucose at the bioanode with a concomitant reduction of AgCl to highly conducting Ag on the cathode. The AgCl-to-Ag conversion strongly affected the impedance of the antenna circuit, allowing wireless detection of glucose. Mimicking the final application, the proposed wireless biosensor was ultimately evaluated through the measurement of glucose in whole blood, showing good agreement with the values obtained with a commercially available glucometer. This work, for the first time, demonstrates that making a part of the antenna from the AgCl layer allows achieving simple, chip-less, and battery-less wireless sensing of enzyme-catalyzed reduction reaction.

Functionalized Protein Nanotubes Based on the Bacteriophage vB_KleM-RaK2 Tail Sheath Protein.

We report on the construction of functionalized nanotubes based on tail sheath protein 041 from vB_KleM-RaK2 bacteriophage. The truncated 041 protein (041Δ200) was fused with fluorescent proteins GFP and mCherry or amidohydrolase YqfB. The generated chimeric proteins were successfully synthesized in E. coli BL21 (DE3) cells and self-assembled into tubular structures. We detected the fluorescence of the structures, which was confirmed by stimulated emission depletion microscopy. When 041Δ200GFP and 041Δ200mCherry were coexpressed in E. coli BL21 (DE3) cells, the formed nanotubes generated Förster resonance energy transfer, indicating that both fluorescent proteins assemble into a single nanotube. Chimeric 041Δ200YqfB nanotubes possessed an enzymatic activity, which was confirmed by hydrolysis of N4-acetyl-2'-deoxycytidine. The enzymatic properties of 041Δ200YqfB were similar to those of a free wild-type YqfB. Hence, we conclude that 041-based chimeric nanotubes have the potential for the development of delivery vehicles and targeted imaging and are applicable as scaffolds for biocatalysts.

Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 µA mM-1cm-2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

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.

Low-Temperature Virus vB_EcoM_VR26 Shows Potential in Biocontrol of STEC O26:H11.

Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging foodborne pathogen of growing concern. Since current strategies to control microbial contamination in foodstuffs do not guarantee the elimination of O26:H11, novel approaches are needed. Bacteriophages present an alternative to traditional biocontrol methods used in the food industry. Here, a previously isolated bacteriophage vB_EcoM_VR26 (VR26), adapted to grow at common refrigeration temperatures (4 and 8 °C), has been evaluated for its potential as a biocontrol agent against O26:H11. After 2 h of treatment in broth, VR26 reduced O26:H11 numbers (p < 0.01) by > 2 log10 at 22 °C, and ~3 log10 at 4 °C. No bacterial regrowth was observed after 24 h of treatment at both temperatures. When VR26 was introduced to O26:H11-inoculated lettuce, ~2.0 log10 CFU/piece reduction was observed at 4, 8, and 22 °C. No survivors were detected after 4 and 6 h at 8 and 4 °C, respectively. Although at 22 °C, bacterial regrowth was observed after 6 h of treatment, O26:H11 counts on non-treated samples were >2 log10 CFU/piece higher than on phage-treated ones (p < 0.02). This, and the ability of VR26 to survive over a pH range of 3-11, indicates that VR26 could be used to control STEC O26:H11 in the food industry.