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.

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.

The Robust Self-Assembling Tubular Nanostructures Formed by gp053 from Phage vB_EcoM_FV3.

The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10⁻1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.

Functionalization of α-synuclein fibrils.

The propensity of peptides and proteins to form self-assembled structures has very promising applications in the development of novel nanomaterials. Under certain conditions, amyloid protein α-synuclein forms well-ordered structures - fibrils, which have proven to be valuable building blocks for bionanotechnological approaches. Herein we demonstrate the functionalization of fibrils formed by a mutant α-synuclein that contains an additional cysteine residue. The fibrils have been biotinylated via thiol groups and subsequently joined with neutravidin-conjugated gold nanoparticles. Atomic force microscopy and transmission electron microscopy confirmed the expected structure - nanoladders. The ability of fibrils (and of the additional components) to assemble into such complex structures offers new opportunities for fabricating novel hybrid materials or devices.

Studies of dimethylglycine oxidase isoenzymes in Arthrobacter globiformis cells.

Glycine betaine (GB) could be used by Arthrobacter globiformis cells as a sole carbon source. The cells took up this molecule in the low as well as in the high salinity medium. Addition of GB to the mineral medium with high salt concentration revealed that GB was also used as an osmoprotectant. Dimethylglycine oxidase (DMGO) was involved in the catabolism of GB. Two genes for DMGO were detected in a cloned 26267 bp fragment of A. globiformis DNA. The genes involved in the tetrahydrofolate-dependent assimilation of methyl groups were located nearby the two of DMGO genes. Both cloned A. globiformis DMGO were active. The activity of DMGO was detected in A. globiformis cells and it depended on the addition of GB and the salinity of the medium. Reverse transcription-PCR demonstrated that the addition of GB influenced the transcription of dmg genes.