Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy

Background: We have recently developed a method to visualize the distribution of DNA in the cytoplasm of bacteria by analytical electron microscopy (EM), using the Phosphorus signal (dsDNA contains two phosphate groups per each nucleotide pair), that was detected and mapped onto the image of the cell (Danilova et al, 2020; Loiko et al, 2020). Here we applied this technique to study much smaller objects – the DNA packing inside bacteriophage heads. We studied phiEL, giant phiKZ-like bacteriophage of the Myoviridae family that infects Pseudomonas aeruginosa (Krylov et al, 2003). We have earlier demonstrated that this phage contains an ‘inner body’ inside its capsid, which is responsible for the specific DNA packing (Sokolova et al, 2014). Methods: The phage propagation was performed as described before (Sokolova et al, 2014). A 3 ul sample of purified bacteriophage phiEL was applied to the glow-discharged carbon-coated copper grid and stained with freshly prepared ammonium Molybdate 2% aquatic solution for 30 sec. Grids were loaded into Gatan cooling holder and the temperature of the specimen was kept at -180°C. EELS spectra and phosphorus elemental maps were obtained on JEOL2100 microscope, operating at 200 kV with the Gatan GIF Quantum ER spectrometer in STEM mode. Pixel size was set to 15-20 nm. STEM drift correction was applied after each 40-50 pixels. Each spectrum was obtained at a 6.0 mrad collection angle, 0.25 eV dispersion, and 132 eV energy shift. The spectra from different pixels were aligned to carbon K-edge. Results: Phosphorus mapping inside and outside the bacteriophage capsid was performed (Fig. 1). Outside the capsid, the phosphorus signal was practically absent, which corresponds to the presence of DNA only inside the capsid. The distribution of phosphorus inside the capsid was uneven: the rectangular area in the middle of the capsid contained a weak signal, while a more intense signal was detected on the periphery. This can be explained by the presence of an ‘inner body’ inside (Fig. 1C). Conclusion: Thus, our results justify the possibility of using the analytical EM technique to study the distribution of DNA by mapping Phosphorus in biological nano-objects at relatively low content of the element.

Phage phiKZ—The First of Giants

The paper covers the history of the discovery and description of phiKZ, the first known giant bacteriophage active on Pseudomonas aeruginosa. It also describes its unique features, especially the characteristic manner of DNA packing in the head around a cylinder-shaped structure (“inner body”), which probably governs an ordered and tight packaging of the phage genome. Important properties of phiKZ-like phages include a wide range of lytic activity and the blue opalescence of their negative colonies, and provide a background for the search and discovery of new P. aeruginosa giant phages. The importance of the phiKZ species and of other giant phage species in practical phage therapy is noted given their broad use in commercial phage preparations.

Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo

Chromatin organization is crucial for regulating gene expression. Previously, we showed that nucleosomes form groups, termed clutches. Clutch size correlated with the pluripotency grade of mouse embryonic stem cells and human induced pluripotent stem cells. Recently, it was also shown that regions of the chromatin containing activating epigenetic marks were composed of small and dispersed chromatin nanodomains with lower DNA density compared to the larger silenced domains. Overall, these results suggest that clutch size may regulate DNA packing density and gene activity. To directly test this model, we carried out 3D, two-color super-resolution microscopy of histones and DNA with and without increased histone tail acetylation. Our results showed that lower percentage of DNA was associated with nucleosome clutches in hyperacetylated cells. We further showed that the radius and compaction level of clutch-associated DNA decreased in hyperacetylated cells, especially in regions containing several neighboring clutches. Importantly, this change was independent of clutch size but dependent on the acetylation state of the clutch. Our results directly link the epigenetic state of nucleosome clutches to their DNA packing density. Our results further provide in vivo support to previous in vitro models that showed a disruption of nucleosome-DNA interactions upon hyperacetylation.

The Roles of Hippo Signaling Transducers Yap and Taz in Chromatin Remodeling

Hippo signaling controls cellular processes that ultimately impact organogenesis and homeostasis. Consequently, disease states including cancer can emerge when signaling is deregulated. The major pathway transducers Yap and Taz require cofactors to impart transcriptional control over target genes. Research into Yap/Taz-mediated epigenetic modifications has revealed their association with chromatin-remodeling complex proteins as a means of altering chromatin structure, therefore affecting accessibility and activity of target genes. Specifically, Yap/Taz have been found to associate with factors of the GAGA, Ncoa6, Mediator, Switch/sucrose nonfermentable (SWI/SNF), and Nucleosome Remodeling and Deacetylase (NuRD) chromatin-remodeling complexes to alter the accessibility of target genes. This review highlights the different mechanisms by which Yap/Taz collaborate with other factors to modify DNA packing at specific loci to either activate or repress target gene transcription.

A multiscale analysis of DNA phase separation: From atomistic to mesoscale level

ABSTRACTDNA condensation and phase separation is of utmost importance for DNA packing in vivo with important applications in medicine, biotechnology and polymer physics. The presence of hexagonally ordered DNA is observed in virus capsids, sperm heads and in dinoflagellates. Rigorous modelling of this process in all-atom MD simulations is presently difficult to achieve due to size and time scale limitations. We used a hierarchical approach for systematic multiscale coarse-grained (CG) simulations of DNA phase separation induced by the three-valent cobalt(III)-hexammine (CoHex3+). Solvent-mediated effective potentials for a CG model of DNA were extracted from all-atom MD simulations. Simulations of several hundred 100-bp-long CG DNA oligonucleotides in the presence of explicit CoHex3+ ions demonstrated aggregation to a liquid crystalline hexagonally ordered phase. Following further coarse-graining and extraction of effective potentials, we conducted modelling at mesoscale level. In agreement with electron microscopy observations, simulations of an 10.2-kbp-long DNA molecule showed phase separation to either a toroid or a fibre with distinct hexagonal DNA packing. The mechanism of toroid formation is analysed in detail. The approach used here is based only on the underlying all-atom force field and uses no adjustable parameters and may be generalized to modelling chromatin up to chromosome size.