As revealed initially by X-ray diffraction and visualized later on by cryoEM in several bacteriophages, the DNA chain confined in the capsid is densely packed and organized locally in such a way that DNA segments form an hexagonal lattice with an average inter-helix spacing aH close to 26 – 27 Å. The 3D organization of DNA in the capsid or the path followed by the chain remains unknown. DNA is kinetically constrained and undergoes out-of-equilibrium conformational dynamics during packaging in the capsid. The structural analysis of full capsids also indicates that there is not a unique deterministic DNA packaging pathway. All consequences of this high confinement on DNA ordering and mobility are not yet well understood.
Our aim is here to explore how confinement determines DNA organization in the full capsid and to what extent spermine and temperature may affect this ordering. CryoEM has been used to visualize DNA patterns in individual capsids, Circular Dichroism (CD) to detect possible changes of the conformation of DNA and of its supramolecular chiral organization and Small Angle X-ray Scattering (SAXS) to detect possible variations of inter-helix distances, while controlling the conformation of the full capsids. We have compared T5, λ, T7, and Φ29 bacteriophages. This selection let us explore the effect of extreme confinement and compare capsids of different shape (prolate or isometric icosahedron) and size, with or without an internal core. We also compared T5 strains containing the full-length genome to the mutant T5st0 containing a shorter DNA chain, to detect whether possible effects may be increased or reduced at lower densities.
Selected CryoEM views show the overall shape of the phages (Figure 1A) and highlight the local hexagonal DNA lattice seen in top view (domains framed in red in Figure 1B). In the direction normal to the capsid faces, up to 5-6 layers define the thickness of the domain. Figure 1C presents structure factors recorded for all phages.
– First, we did not detect any change of DNA organization as a function of temperature between 20 to 40°C, as opposed to previous results (1, 2).
– Second, the presence of spermine (4+) enlarges the size of the hexagonal domains determined by the width of the diffraction peak: from 139 to 156 Å in T5, from 111 to 123 Å in λ, from 111 to 128 Å in T7, and from 96 to 110 Å in Φ29 when 100 mM sp is added in HS buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2). 4 mM spermine is enough to produce this effect. We interpret this increase of the domain size detected for all phages as an increase of the DNA ordering upon spermine addition in agreement with (3). The addition of spermine, by reducing the repulsive interactions between DNA strands would help the local hexagonal order to expand and slightly move the defects further apart. In this hypothesis, DNA reorganization would require very minor sliding of the DNA chain inside the capsid. The enlargement of the DNA hexagonal domains by spermine does not modify the CD spectrum of the encapsidated DNA that stays close to the typical B-type spectrum. Our estimations of the DNA concentrations based on SAXS measurements (540 ± 20 mg/ml in T5, 525 ± 20 mg/ml in λ, 575 ± 20 in T7 and 615 ± 50 in Φ29) seem to indicate that the smallest phages (T7 and Φ29) are the most densely packed. The comparison between λ and T7 (both icosahedral, with dimensions and DNA length in the same range) suggest that the presence of an internal core in T7 facilitate DNA organization by wounding part of the chain around it in a toroidal organization, thus reducing the defect lattice required to solve the frustration arising from the confinement of the hexagonal lattice in an icosahedral capsid (4).
(1) Liu, T. et al, Proc Natl Acad Sci U S A 2014, 111, 14675–14680 (2) Li, D.et al, A. Nucleic Acids Res 2015, 43, 6348–6358 (3) Lander, G. C. et al, Nucleic Acids Res 2013, 41, 4518–4524 (4) De Frutos et al, JPhysChemB in press.
Figures:
Figure 1: A-B: CryoEM of bacteriophage T5 Her28 (100%), T5st0 (92%), λ, T7 and Φ29, imaged in HS buffer at high defocus (A) to visualize the entire phage with its tail and at low defocus (B) to visualize details of the DNA packing. Monocrystalline domains showing the hexagonal lattice are underlined in red. Arrows point to striated patterns corresponding to oblique views of the lattice. C: Structure factors S(q) = I DNA(q)/F(q) for phages T5 Her28 (100%), λ, T7 and Φ29 in the absence of spermine (red) and in the presence of 4 mM (blue) and 100 mM spermine (green). Black line: fit by a lorentzian function over a q-range restricted on the large q-value side.
To cite this abstract:
Marta DE FRUTOS, Amélie LEFORESTIER, Jeril DEGROUARD, Dominique DURAND, Françoise LIVOLANT; Can DNA Organization in the Full Bacteriophage Capsid be modified by changes in Temperature or Ionic Conditions?. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/can-dna-organization-in-the-full-bacteriophage-capsid-be-modified-by-changes-in-temperature-or-ionic-conditions/. Accessed: December 2, 2023« Back to The 16th European Microscopy Congress 2016
EMC Abstracts - https://emc-proceedings.com/abstract/can-dna-organization-in-the-full-bacteriophage-capsid-be-modified-by-changes-in-temperature-or-ionic-conditions/