Data storage based on the absence of nucleotides using a bacteriophage abortive infection system reverse transcriptase.

DNA molecules are a promising data storage medium for the future; however, effective synthesis of DNA using an enzyme that catalyzes the polymerization of natural nucleoside triphosphates in a user-defined manner, without the need for multiple injections of polymerase, remains a challenge. In the present study, we demonstrated that the bacteriophage abortive infection system reverse transcriptase AbiK from facilitates such an approach. We employed surface plasmon resonance to monitor the polymerization of the DNA strand with a user-defined sequence of multiple segments through a sequential buffer exchange process.

Autoregulation ensures vertical transmission of the linear prophage GIL01.

Betatectiviruses are prophages consisting of linear extrachromosomal genomes without obvious plasmid modules. It remains unclear how betatectiviruses are maintained in low-copy numbers in host cells and how they are vertically transmitted. Phage GIL01 is a model betatectivirus that infects the mosquito pathogen Bacillus thuringiensis serovar israelensis.

Targeting -Acetylglucosaminidase in with Iminosugar Inhibitors.

Bacteria are capable of remarkable adaptations to their environment, including undesirable bacterial resistance to antibacterial agents. One of the most serious cases is an infection caused by multidrug-resistant , which has unfortunately also spread outside hospitals. Therefore, the development of new effective antibacterial agents is extremely important to solve the increasing problem of bacterial resistance.

New Insights into Interactions between Mushroom Aegerolysins and Membrane Lipids.

Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus . Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain.

A single point mutation expands the applicability of ostreolysin A6 in biomedicine.

An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6 interacts with even higher affinity with ceramide phosphoethanolamine (CPE), a sphingolipid that dominates in invertebrate cell membranes. In the presence of pleurotolysin B, a protein bearing the membrane-attack complex/perforin domain, OlyA6 forms pores in insect midgut cell membranes and acts as a potent bioinsecticide.

A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors.

TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA.

Discovery of a fragment hit compound targeting D-Ala:D-Ala ligase of bacterial peptidoglycan biosynthesis.

Bacterial resistance is an increasing threat to healthcare systems, highlighting the need for discovering new antibacterial agents. An established technique, fragment-based drug discovery, was used to target a bacterial enzyme Ddl involved in the biosynthesis of peptidoglycan. We assembled general and focused fragment libraries that were screened in a biochemical inhibition assay.

A small bacteriophage protein determines the hierarchy over co-residential jumbo phage in Bacillus thuringiensis serovar israelensis.

Bacillus thuringiensis serovar israelensis is the most widely used biopesticide against insects, including vectors of animal and human diseases. Among several extrachromosomal elements, this endospore-forming entomopathogen harbors two bacteriophages: a linear DNA replicon named GIL01 that does not integrate into the chromosome during lysogeny and a circular-jumbo prophage known as pBtic235. Here, we show that GIL01 hinders the induction of cohabiting prophage pBtic235.

Ceramide Aminoethylphosphonate as a New Molecular Target for Pore-Forming Aegerolysin-Based Protein Complexes.

Ostreolysin A6 (OlyA6) is a 15 kDa protein produced by the oyster mushroom (). It belongs to the aegerolysin family of proteins and binds with high affinity to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with its partnering protein with the membrane-attack-complex/perforin domain, pleurotolysin B (PlyB), OlyA6 can form bicomponent 13-meric transmembrane pores in artificial and biological membranes containing the aegerolysin lipid receptor, CPE.

The Small DdrR Protein Directly Interacts with the UmuDAb Regulator of the Mutagenic DNA Damage Response in Acinetobacter baumannii.

Acinetobacter baumannii poses a great threat in health care settings worldwide, with clinical isolates displaying an ever-evolving multidrug resistance. In strains of A. baumannii, expression of multiple error-prone polymerase genes are corepressed by UmuDAb, a member of the LexA superfamily, and a small protein, DdrR.

Inhibition of the SARS-CoV-2 3CL main protease by plant polyphenols.

The abundance of polyphenols in edible plants makes them an important component of human nutrition. Considering the ongoing COVID-19 pandemic, a number of studies have investigated polyphenols as bioactive constituents. We applied in-silico molecular docking as well as molecular dynamics supported by in-vitro assays to determine the inhibitory potential of various plant polyphenols against an important SARS-CoV-2 therapeutic target, the protease 3CL.

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields.

The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1.

Surface plasmon resonance approach to study drug interactions with SARS-CoV-2 RNA-dependent RNA polymerase highlights treatment potential of suramin.

The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is essential for virus replication, therefore it is a promising drug target. Here we present a surface plasmon resonance approach to study the interaction of RdRp with drugs in real time. We monitored the effect of favipiravir, ribavirin, sofosbuvir triphosphate PSI-7409 and suramin on RdRp binding to RNA immobilized on the chip.

An anti-HER2 nanobody binds to its antigen HER2 via two independent paratopes.

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation.

Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine.

Aegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The most studied fungal aegerolysins share a common property of interacting with membranes enriched with cholesterol in combination with either sphingomyelin or ceramide phosphorylethanolamine (CPE), major sphingolipids in the cell membranes of vertebrates and invertebrates, respectively. However, genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibrio; these are human pathogens of high clinical relevance and can thrive in a variety of other species.

Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region.

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability.

A consensus protocol for the in silico optimisation of antibody fragments.

We present an in silico mutagenetic protocol for improving the binding affinity of single domain antibodies (or nanobodies, VHHs). The method iteratively attempts random mutations in the interacting region of the protein and evaluates the resulting binding affinity towards the target by scoring, with a collection of scoring functions, short explicit solvent molecular dynamics trajectories of the binder-target complexes. The acceptance/rejection of each attempted mutation is carried out by a consensus decision-making algorithm, which considers all individual assessments derived from each scoring function.

Comparative analysis of fusion tags used to functionalize recombinant antibodies.

Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody.

Specificity of Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography.

Diarrhea caused by enterotoxigenic (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea.