Can Changes in Temperature or Ionic Conditions Modify the DNA

Article pubs.acs.org/JPCB

Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid? Marta de Frutos,*,† Amélie Leforestier,† Jéril Degrouard,† Nebraska Zambrano,† Frank Wien,‡ Pascale Boulanger,§ Sandrine Brasilès,§ Madalena Renouard,§ Dominique Durand,*,§ and Françoise Livolant*,† †

Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France Synchrotron SOLEIL, DISCO, L’Orme des Merisiers, 91190 St Aubin, France § Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS UMR 9198, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif sur Yvette Cedex, France ‡

S Supporting Information *

ABSTRACT: We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.

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INTRODUCTION DNA is highly confined and compressed in the bacteriophage capsid to reach near-crystalline densities that cannot be found otherwise except by strong dehydration of DNA solutions. The packaging inside the capsid is done by a powerful molecular motor able to drive DNA entry against large forces arising from DNA bending, repulsion between DNA segments, and entropy loss. As revealed initially by X-ray diffraction1−3 and visualized later on by cryoEM in several bacteriophages,4−6 the unique DNA chain appears densely packed and organized locally in such a way that DNA segments form an hexagonal lattice. It is established that the average interhelix spacing aH is close to 26−27 Å in most bacteriophages,7 but the 3D organization of DNA in the capsid or the path followed by the chain remains unknown. Another way to approach the question of the DNA packing state inside the capsid is to analyze the way it goes into or out of the capsid. DNA encapsidation has been analyzed by single molecule measurements using optical tweezers. Results on Φ29 reveal that DNA is kinetically constrained and undergoes out-of-equilibrium conformational dynamics during packaging in the capsid.8 As described by this group, with the relaxation time of the confined DNA being much longer than the time to package the viral genome, frequent pausing occurs during DNA translocation. Another consequence is the important heterogeneity observed in packaging rates of individual viruses. The structural analysis of full capsids also © XXXX American Chemical Society

indicates that there is not a unique deterministic DNA packaging pathway.9 Concerning DNA ejection, fluorescence microscopy experiments on a single T5 have shown that phages eject their DNA by stochastic bursts of partial ejection separated by transient pauses.10−12 A nonmonotonous variation of the velocity is also observed when ejection proceeds, both for T512 and λ phages.11 These results are consistent with recent theoretical approaches that describe dynamics of the packaging and ejection processes by including a continuous DNA reorganization within the capsid.13,14 The origin of the specificities observed for DNA packaging and ejection remains unclear. Different parameters distinguish phage species and could influence their DNA organization: shape and charge of the capsid, characteristics of the encapsidation motor, etc. All of these parameters can have an impact on the exact DNA organization within the phage and the energy balance, and consequently on the dynamics associated with their ejection and encapsidation. For example, it was proposed that the distance to DNA equilibrium state was related to the force developed by the motor to encapsidate DNA,15 and all phage motors do not exert the same force as Special Issue: William M. Gelbart Festschrift Received: February 22, 2016 Revised: April 21, 2016

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DOI: 10.1021/acs.jpcb.6b01783 J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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reviewed recently.16 Moreover, the average packaging speed was shown to scale with genome length (approximately 170 bp/s for Φ29, 600 bp/s for λ and 700 bp/s for T4) with strong variations between individuals of the same species.16 Other parameters like temperature or ionic conditions may have significant effects. Ions can enter the protein capsid and modify interactions between DNA strands. For example, the presence of Mg2+ cations changes the kinetics of DNA ejection as seen experimentally in T5 and λ11,12and also the kinetics of DNA packaging in Φ29.17 Simulations also predict a nonmonotonic speed for DNA ejection and encapsidation with a maximum value of the ejection speed that strongly depend on ionic conditions.14 Getting DNA to a configuration favorable to enter or to leave the capsid would depend on electrostatic interactions, and the presence of divalent cations would help by reducing the repulsive DNA−DNA interactions. The effect of trivalent and tetravalent cations is more complex. Low spermidine (3+) concentrations (below the threshold condensing concentration, resulting in maximally screened DNA− DNA repulsive interactions) accelerate DNA packaging whereas condensing concentrations of spermidine (which induce DNA−DNA attractive interactions) show an increased heterogeneity of behaviors.18 Most capsids show dramatic slowing, pausing and stalling at low filling that the authors attribute to an exaggerate formation of intracapsid nonequilibrium DNA conformations. On the other hand, the addition of 1.3 mM spermine (4+) to full wt phage λ suggests an increase of DNA ordering of the encapsidated DNA since the density cross-section of the genome reveals that two additional layers can be detected after cryoelectron microscopy (cryoEM) three-dimensional image reconstruction.19 Unfortunately, it is not specified whether this spermine concentration is below or above the condensing threshold concentration under their experimental conditions. It has also been proposed that encapsidated DNA undergoes a solid-to-fluid phase transition as a function of temperature, resulting locally in less densely packed DNA.19,20 All consequences of this high confinement on DNA ordering and mobility are not yet well-understood. A precise comparison of DNA packing in different phages is also lacking because they have been studied by different groups in different ionic environments and by different methods. 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. We used 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 interhelix distances, while controlling the conformation of the full capsids. CryoEM has been used to visualize DNA patterns in individual capsids. To check the effect of confinement on DNA arrangement, we have compared T5, λ, T7, and Φ29 bacteriophages. This selection let us explore the effect of extreme confinement that superimposes to high densities by comparing icosahedral capsids of different dimension, capsids of different shapes (prolate or isometric icosahedron), and capsids 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. We did not consider phages with DNA overpackaging since all bacteriophage species considered here do not use the headful strategies of packaging (as for example SPP1 or P22).

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EXPERIMENTAL SECTION Materials. Experiments have been performed in several Tris-buffer solutions, pH 7.5: (1) low salt buffer (LS) containing 10 mM Tris-HCl, 1 mM MgCl2, and 1 mM CaCl2; (2) high salt buffer (HS) with 10 mM Tris-HCl, 100 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2; (3) high salt− magnesium buffer (HS-Mg) with 10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, and 1 mM CaCl2; and (4) Tris-Mg buffer (TM) containing 50 mM Tris and 10 mM MgCl2. Phages T5, λ, T7, and Φ29 were purified on cesium chloride gradients, dialyzed, and stored at 4 °C in HS for T5st0, HS-Mg for λ, T5 Her28, and T5 amH231 and in TBT (100 mM Tris, 100 mM NaCl, 10 mM MgCl2, pH 7.5) for T7 and Φ29. T5st0, a heat stable mutant deleted of 8% of its genome (114 kbp, Genbank Acc AY692264)21 was compared to two strains containing the 121 kbp full length DNA, T5 Her28,22 and T5amH231.23 For our experiments, these two phages are equivalent (same capsid size and same genome length). To obtain empty T5 capsids, DNA ejection was triggered by mixing phages with the protein receptor FhuA (110 FhuA molecules per infective phage) in HS buffer + 0.03% LDAO (w/w) as described earlier.24,25 λ particles were produced by thermal induction of the lysogenic E. coli R594 (cI857 S7), a K12 strain. Phage solutions contaminated with free DNA were digested by DNase I (Invitrogen, 10 u/μL, 1 h, 37 °C) in samples prepared for circular dichroism experiments and for SAXS measurements in the presence of spermine. The DNase concentration was increased to 23 u/μL to digest the fully ejected DNA to record signals from the empty capsids. T5 DNA and 146 bp DNA were purified by phenol− chloroform extraction from T5 and from calf thymus chromatin, respectively, and stored at 4 °C in HS buffer. A 38 bp DNA fragment was constructed as shown in ref 26 and was buffered with 100 mM NaCl, 20 mM Tris pH 7.5, and 2.5% (v/v) glycerol. Samples were extensively dialyzed in the chosen buffer. Their concentration was adjusted to CDNA = 3−4 mg/mL, measured from the absorbance at 260 nm (molar extinction coefficient of phage approximated to pure DNA absorption, i.e., E260 = 6250 M−1 cm−1), using a nanodrop spectrophotometer (ThermoFisher). Spermine was added to the samples to reach final concentrations of 4, 40, or 100 mM. Synchrotron Radiation Circular Dichroism (SRCD). Measurements covering the UV−vis spectral range were carried out on the DISCO beamline at the SOLEIL synchrotron facility (Gif-sur-Yvette, France).27 For each sample, an aliquot of 4 μL was loaded in circular demountable CaF2 cells of 56 μm path length.28 Spectral acquisitions of 1 nm steps were performed at 10 s integration time, between 180 and 330 nm. (+)-Camphor10-sulfonic acid (CSA) was used to calibrate amplitudes and wavelength positions of the SRCD experiment.29 Data treatment including averaging, baseline subtraction, smoothing, scaling, and standardization was carried out with CDtool.30 For a comparison of DNA signals from the different samples, spectra were normalized by the pseudoabsorbance at 260 nm.31 Two separated series of data were acquired to check reproducibility. Cryoelectron Microscopy. The 3 μL aliquots of the phage suspension were deposited onto a glow-discharged holey carbon grid (Quantifoil R2/2). The grid was blotted with a filter paper for 4 s, and directly plunged into liquid ethane B


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The Journal of Physical Chemistry B cooled down by liquid nitrogen, using a FEI Vitrobot operated at room temperature and 100% relative humidity. Frozen samples were transferred into a Gatan 626 cryostage and observed in a Cryo transmission electron microscope (JEOL 2010-UHR or JEOL 2010F) operated at 200 kV. Images were recorded on a Gatan Ultrascan 4K camera with a nominal defocus of −3 μm or on Kodak SO163 negative films at a magnification of 50 000 and a nominal defocus of −900 nm. The films were developed in full strength Kodak D19 for 12 min, and scanned with a Nikon Coolscan 9000 at a resolution of 4000 pixels per inch. Small Angle X-ray Scattering (SAXS). SAXS measurements were performed on the BM29 beamline at the ESRF (Grenoble, France)32 with 1012 photons/s and a cross-section of 700 × 700 μm2. The X-ray wavelength was equal to 0.992 Å and the sample to detector distance to 2.867 m, leading to an accessible q-range [0.004 Å−1, 0.5 Å−1] with q the momentum transfer (q = 4π sin θ/λ) and 2θ the scattering angle. A 40 μL aliquot of sample was flowing through a quartz capillary 1.8 mm in diameter with a speed adapted to record 8 × 1 s frames. The online data reduction and analysis software33 checks the perfect similarity of these frames and only keeps those frames that are strictly identical, ensuring that the final averaged pattern is free from the effects of any potential radiation damage. For measurements as a function of temperature, a new sample was loaded for each temperature value. The temperature was stabilized over 15 min before each measurement. Scattering of the buffer was recorded immediately before and after each sample and subtracted from the corresponding phage pattern. The greatest care was taken at the experimental level that no irradiation damage was incurred by our samples and that the temperature within the samples actually increased from 20 to 40 °C (this is testified by the value of the intensity scattered by the buffer, measured immediately before and after each sample and which duly increased according to theoretical predictions).

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Figure 1. 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 and (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.

RESULTS We present in Table 1 the main characteristics of phages analyzed in this study. T5 and λ belong to the Siphoviridae Table 1. Characteristics of the Phages

a

phage

LDNA (kpb)

family

T5st0 (92%) T5 Her28 (100%) T5 amH231 (100%) λa T7 Φ29

114 121 121 48.5 39.9 19.3

siphoviridae siphoviridae siphoviridae siphoviridae podoviridae podoviridae

capsid facilitate the DNA alignment and also (ii) because superimposition effects are reduced at the periphery compared to the center of the capsid. The lateral extension of such domains is correlated to the dimension of the icosahedral capsid. In T5, rows of up to 15 DNA segments can be seen along the capsid faces compared to a maximum of 8−9 in T7 or λ (Figure 1B). In the direction normal to the capsid faces, up to 5−6 layers define the thickness of the domain. Such domains, separated by twist and dislocation walls, have been described in T5st0.6 Circular Dichroism. CD spectra were recorded for phages T5st0, T7, and Φ29 in LS and HS buffer (Figure 2A). For wavelengths above 250 nm, where DNA absorption dominates (electronic transitions of the aromatic bases), no significant difference is observed between phages, in good agreement with previous results.35 Within experimental error, in this range, buffer composition and addition of spermine have negligible influence on CD spectra. A stronger difference is observed below 250 nm. The CD signal results from both amino acid (peptide bond) and DNA (sugars). Thus, the contribution from phage protein structure cannot be neglected (see CD spectrum from T5 capsids in Figure S1). The CD signal was

capsid T T T T T T

= = = = = =

13 13 13 7 7 3, Q = 5b

cl857 Sam7. bReference 34.

family (phages with a long noncontractile tail), and T7 and Φ29 to the Podoviridae (phages with a short tail). Except for Φ29, all phages possess an isometric icosahedral capsid. In all these phages, a long ds DNA chain is locally packed into hexagonally ordered domains as described long ago by cryoTEM and SAXS.3−6 All analyzed phages present multiple DNA patterns in 2D projection. A few CryoEM views have been selected to show the overall shape of the phages (Figure 1A) and to highlight the local hexagonal DNA lattice seen in the top view (domains framed in red in Figure 1B). The hexagonal lattice is better visualized at the periphery of the capsid for two reasons: (i) first because the protein faces of the C


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Figure 2. (A) CD spectra recorded for phages T5st0 (92%), T7, and Φ29 in LS buffer (dotted curves), in HS buffer (solid curves), and in LS buffer with 40 mM spermine (dashed curves). The signal for T5 was also recorded in LS buffer with 4 mM spermine (data not shown for clarity), and no difference was observed with 40 mM spermine. The standard deviation of the measured values (2% of the signal) is indicated on one curve. (B) CD spectra for full T5 (phages with confined DNA), T5 phages with released DNA, and purified DNA (DNA alone) in HS buffer.

also measured on DNA purified from T5 (Figure 2B). Differences are observed between purified DNA and T5 spectra below 250 nm (due to the phage protein contribution to the T5 spectrum) but also above 250 nm, in the DNA absorption region. No difference was observed between LS and HS buffers. LS buffer was initially preferred for spermine addition because it was expected to maximize spermine effect. Since no difference in CD spectra was detected at 4 or 40 mM spermine (Figure 2A), HS buffer has been chosen for subsequent measurements because DNA release from T5, triggered by interaction of the phage with its receptor FhuA, requires HS conditions.3624 For phage T5, DNA can be released from the phage by mixing the phage with its protein receptor FhuA.24,36 This property allows us to measure CD spectra in full T5 (phages with confined DNA) and in empty T5 (phages with released DNA around). The comparison with purified DNA (DNA alone) is presented in Figure 2B. Our data are in very good agreement with previous results published for T5.37 These results allow us to compare the CD signal corresponding to DNA “confined” inside the capsid with “unconfined” (purified) DNA. To do so, the phage protein contribution (details in Figure S1) was subtracted from full T5 signal (blue curve in Figure 2B). The resulting spectrum (green curve in Figure 3) was compared to “unconfined” DNA (red curve in Figure 3). Both DNA samples present a spectrum shape compatible with B-DNA conformation.38 In a comparison of confined DNA with unconfined (purified) DNA, the peak centered near 280 nm presents a slight decrease and red shift. A similar effect was observed in the CD spectrum for E. coli and calf thymus DNA in alcohol solutions. For these GC-rich DNA, methanol induces a gradual depression of the long-wavelength part of the CD spectrum39.40 The T5 genome has an average GC content of 39.3%,41 close to calf thymus DNA content (42%). This effect was also observed for lipid/DNA complexes42 and, to a lesser extent, for DNA in ethanol at concentrations below 70−80%.40 Above this ethanol concentration, the CD signal undergoes a strong change, from a typical B-type to a Ψ-type signal, characterized by either positive or negative CD signals with extremely large amplitudes. These types of CD signal can no longer be interpreted in terms of changes in DNA secondary structure and are associated with the formation of chiral condensates

Figure 3. Comparison between CD signal from purified DNA and from confined T5 DNA (full T5 signal after subtraction of the empty phage signal; see details in Supporting Information) in HS buffer.

possessing large-scale helicity.43 The so-called “Ψ-DNA” forms have been detected when polymers,44 ethanol,40 polycations,45 polypeptides,4647or basic proteins48 are added to DNA solutions inducing condensation. For long DNA, Ψ-type signals have been described when condensation is induced by polymer addition44 but not when it is induced by hexamminecobalt(III).45 In phage capsids, despite the high DNA concentration, we do not observe a change from a B to a Ψ-type signal. The moderate effect visible on the CD spectrum induced by the confinement is more likely associated with a dehydration process as also described for DNA solutions in the presence of hexamminecobalt(III) prior to condensation.45 SAXS. Scattering Patterns Analysis. As a first step in data analysis, we simulated the scattering pattern of an ensemble of DNA molecules to model DNA inside the viral capsid. This ensemble shown in Figure 4 comprises 19 DNA molecules 130 Å long with an intermolecular distance aH = 26.5 Å and a maximal dimension of the block normal to the DNA axis D = 128 Å. The choice of aH and D values was guided by the examination of electron micrographs of T5 bacteriophages showing a hexagonal order in the plane perpendicular to DNA molecules over 150 Å distances and aH values around 26−27 Å.6 The length L of DNA molecules is not critical provided it is D


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Figure 4. (A) Red curves show intensities I(q) scattered by the ensemble of 38 bp long DNA molecules shown in part B calculated using the program Crysol 3.0. The dashed (solid) line corresponds to the random (systematic) shift between two neighboring DNA fragments. Blue curves show structure factors S(qmax) obtained by dividing I(q) by the form factor F(q) (green curve) of a DNA molecule also calculated using Crysol 3.0. The vertical dashed gray line indicates the position q = 4π /aH / 3 corresponding to aH = 26.5 Å. The maximum of the structure factor S(q) is very close to this line. In contrast, the maximum of the scattering intensity I(q) is clearly shifted.

nation of the hexagonal parameter aH. This behavior was already noted in the case of collagen.51 Besides, the half-width at half-maximum of the peak (indicated in Figure 4A) is practically equal to π/D, where D is the characteristic size of the blocks of organized DNA molecules. We can therefore also derive an order of magnitude of the size of these blocks from our scattering data. Obviously, this analysis is an approximation since the above calculation does not take the capsid contribution into account. However, this is only important at small q-values, much smaller than that of the DNA peak. In the region of the DNA peak, the capsid will essentially contribute with a featureless scattering background that does not influence the position and width of the peak. Effect of Spermine. The effect of spermine has been studied for two spermine concentrations, 4 and 100 mM (conditions inducing DNA condensation) in HS buffer, since no difference was observed in CD experiments between spermine in LS and HS buffers. Figure 5A shows the scattering patterns of phage T5 in the absence of spermine and at both spermine concentrations. Other phages exhibit a similar behavior (not shown). We observe that the scattering intensity I(q) at small q-values does not vary with spermine addition. This region is largely determined by the capsid size and is not very sensitive to DNA organization since it corresponds to large distances, and does not vary with spermine addition. In contrast, a slight variation is observed for the DNA peak. The distance distribution function P(r) derived from I(q) (insert in Figure 5A) is the same with and without spermine, confirming that the capsid does not change when spermine is added. As explained above, quantitative information regarding the evolution of DNA organization within the capsid as a function of spermine addition is derived from the analysis of the structure factor S(q) = IDNA(q)/F(q), in the range q > 0.15 Å−1 where F(q) is the form factor of the DNA molecule and IDNA(q) is the contribution to scattering intensity from the DNA. IDNA(q) is extracted from the measured intensity I(q) by subtracting a contribution due to the proteins approximated by a power-law (q−2). We checked the validity of the power-law (q−2) by measuring the intensity scattered by T5 phages emptied from their DNA (data not shown but available). Clearly, this is a very crude approximation because I(q) is not the sum of both contributions, from DNA and from protein,

large enough as compared to the diameter (hence, with respect to the intermolecular distance). If L ≫ aH, the scattering intensity profile I(q) in the q-range of interest, around 2π/aH, is practically independent of the length L. Finally, DNA molecules are mutually shifted along the molecule’s axis. Two kinds of shifts were considered: a random shift that corresponds to the absence of longitudinal correlation between two neighboring DNA fragments and a shift between two neighboring molecules of ±P/6 (where P is the helix pitch taken equal to 33.4 Å) as experimentally observed in DNA dense phases when the interhelical distance aH is lower than about 28−29 Å.49 The intensity I(q) scattered by the ensemble of DNA molecules was computed using the last version of CRYSOL (crysol 3.050) which takes into account the contribution of hydration water using explicit water molecules. The profiles corresponding to the two kinds of shifts are shown in red in Figure 4 (dashed line for random shift and solid line for the systematic shift). The position of the maximum of the scattering peak (measured, as is customary, after subtraction of a monotonically decreasing background), qmax = 0.263 Å−1, is practically independent of the kind of shift and yields a slightly overestimated value of aH = 27.6 Å. This is not surprising, since the hexagonal organization of DNA molecules yields a peak in the structure factor S(q) related, in a first approximation, to the scattering intensity I(q) by the relationship I(q) = S(q)F(q), where F(q) is the form factor of the DNA molecule. It is the position qmax of the peak of the structure factor S(q), and not that of the peak of the scattering intensity I(q), that is inversely proportional to the hexagonal parameter aH:

aH =

2 2π 3 qmax

F(q) was also computed using Crysol 3.0 (green curve in Figure 4), allowing us to derive the structure factor S(q) = I(q)/F(q) (dashed blue line for random shift and blue solid line for the systematic shift in Figure 4A). The position of the maximum of the peak of the structure factor, qmax = 0.272 Å−1, is quasi-independent of the kind of shift and yields a value aH = 26.7 Å, hardly 1% larger than the starting value of the model aH = 26.5 Å, very close to the experimental uncertainty. The use of the structure factor therefore yields a much better determiE


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Figure 5. (A) Scattered intensity I(q) and distance distribution P(r) (insert) for phage T5 (strain Her28) in the absence of spermine (red) and in the presence of 4 mM (blue) and 100 mM spermine (green). P(r) is derived from I(q) using the Gnom software.52 (B−F) Structure factors S(q) = IDNA(q)/F(q) for phages T5 Her28 (100%), T5st0 (92%), λ, T7, and Φ29 in the absence of spermine and in the presence of 4 mM and 100 mM spermine in HS buffer, with the same color code. Solid black line: Fit by a Lorentzian function over a q-range restricted on the large q-value side. This is obviously only an approximation, but an analogous fit performed with the simulated curves in Figure 4 shows that the resulting values obtained for qmax (and consequently aH and the half-width at half-maximum π/D) are very close to the original values of the model.

but this is adequate in the q-range of interest where the DNA contribution is preponderant. F(q) has been determined experimentally by measuring the intensity scattered by a monodisperse solution of 38 base pair DNA fragments and by a solution of free DNA extracted from T5st0 phages. The two curves are very similar, and we used the former curve of better statistical quality. One can wonder whether this form factor determined using isolated molecules in solution and used to extract the structure factor is a valid substitute of the encapsidated DNA form factor. Two arguments support this

view: (1) CD spectra of free DNA and encapsidated DNA only exhibit small differences, suggesting that encapsidated DNA is still in a form close to B-DNA, as determined also in several phages by wide angle RX diffraction,3,53 solid state NMR,54 or Raman spectroscopy.55 (2) The simulations presented above were performed again using DNA in A- and Z-form. Z-form DNA presents no difference from B-DNA. A-form DNA gives a very slight shift of the structure factor by 0.0005 Å−1, corresponding to a variation in aH of 0.05 Å, much below experimental uncertainty. F


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Table 2. Values of the Interhelix Distance aH and Dimensions D of the Domains Determined for Each Phage in HS Buffer for Three Spermine Conditions (no sp, 4 mM and 100 mM) no sp aH (Å) T5st0 (92%) T5 Her28 (100%) λ T7 Φ29

27.29 26.51 26.96 25.66 24.80

± ± ± ± ±

0.2 0.2 0.2 0.2 0.5

no sp domain D (Å) 141 139 111 111 96

4 mM sp aH (Å) 27.01 26.29 26.62 25.50 24.72

± ± ± ± ±

0.2 0.2 0.2 0.2 0.5

4 mM sp domain D (Å) 151 154 121 127 101

100 mM sp aH (Å) 26.98 26.28 26.60 25.54 24.74

± ± ± ± ±

0.2 0.2 0.2 0.2 0.5

100 mM sp domain D (Å) 155 156 123 128 110

Figure 6. (A−E) Scattering curves I(q) measured for phages T5st0 (92%), T5 amH231 (100%), λ, T7, and Φ29 in HS-Mg buffer. In each panel, four practically indistinguishable I(q) curves are superimposed with the following color code: T = 20 °C (black), T = 25 °C (blue), T = 35 °C (dark red), and T = 40 °C (green). (F) Distance distribution function P(r) derived from I(q) shown in panel B using the Gnom software at two temperatures T = 20 °C (black) and T = 40 °C (green). The two P(r) profiles are indistinguishable.

G


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buffer, less sensitive to temperature variations, may be used to explore a larger temperature range. For all phages, the results are the same in the three buffers: the temperature increase from 20 to 40 °C has no effect regarding the capsid structure and the DNA organization. In Figure 6, panels A−E present experimental curves for each phage at four temperature values: 20, 25, 35, and 40 °C. The curves at the various temperatures were simply scaled to take into account the very slight increase in scattered intensity with temperature, of the order of 4% between 20 and 40 °C, and likely also due to a minute evaporation of samples at 40 °C. However, within this scaling factor, the curves appear to be strictly identical both in the small q-region that contains the capsid contribution and in the q-range [0.2−0.35 Å−1] of the DNA organization peak. Panel F of Figure 6 shows an example of the distance distribution function P(r) (T5 phage, T = 20, and 40 °C) derived from the scattering pattern I(q). The examination of both I(q) and P(r) curves demonstrates that the phage has undergone no global modification upon temperature increase. A very slight difference was observed, though: the value of the hexagonal parameter aH exhibits a systematic reduction when increasing the MgCl2 concentration from 1 to 10 mM (data not shown but available) regardless of the temperature. This effect is very close to experimental uncertainty and warrants a more systematic study as a function of Mg2+ ion concentration.

Figure 5 shows the structure factors, using the following color code: red curve, no spermine; blue curve, 4 mM spermine; green curve, 100 mM spermine. The structure factors were adjusted so as to reach the theoretical asymptotic value S(q) = 1 for large q-values. As explained in the Scattering Patterns Analysis section, the interhelix distance is evaluated with an accuracy better than 1% from the position qmax of the maximum of the structure factor peak, and the order of magnitude of the extension D of ordered domains in the plane perpendicular to DNA helices is derived from the half-width at half-maximum of this peak. Scattering intensity profiles I(q) and, of course, structure factors S(q) as well exhibit oscillations in the region of the DNA peak. They are due to the form factor of capsids, and their period is related to the internal dimensions of the capsid. However, these oscillations can be smoothed out when fitting the structure factor peak (see fits in Figure 5) and do not perturb the determination of both parameters qmax and D. aH and D values are summarized in Table 2. An approximate value of DNA concentration CDNA within the phages is obtained using the following relation: CDNA ≈ MDNA/σh, where MDNA is the molecular weight of a base pair (MDNA ≈ 660 g mol−1), σ the area of the unit cell of the two3

dimensional hexagonal lattice (σ ≈ aH2 / 2 ), and h the distance between two base pairs (h = 3.36 Å). This expression gives an average value of the concentration of the densely packed DNA in the whole capsid. CDNA ≈ 510, 540, 525, 575 ± 20, and 615 ± 50 mg/mL for the phages T5st0, T5, λ, T7, and Φ29, respectively. In the case of Φ29, smaller than other phages and with a different and complex architecture, the determination of the parameters CDNA and D is less precise because it is difficult to separate the contributions to the scattering intensity from the phage proteins and from DNA. In summary, all five investigated phages are seen to behave in the same way upon spermine addition: spermine enters inside the capsid and causes a more ordered DNA organization as judged by the increase in the size of ordered domains. The experimental signature of this more ordered organization is the reduction in the half-width at half-maximum of the structure factor and the increase in its height. A very slight reduction of the interhelix distance aH can also be observed, corresponding to a very slight increase of qmax. This is very close to the experimental uncertainty and could be due to the fact that the expression above is an approximation. Indeed, it has been shown that, for a given aH interdistance of the 2D hexagonal lattice, an increase of the local disorder produces a very slight decrease of the value of qmax and thus an increase of the calculated aH value (see ref 56 and Figure III.7 in ref 57). Consequently, the higher value calculated here for aH without spermine could be due to the fact that the organization is less ordered without spermine. Effect of Temperature. The effect of temperature was investigated for five phages: T5st0, T5 (strain amH231), λ, T7, and Φ29 in HS, HS-Mg, and TM buffers (λ was studied only in HS-Mg and TM buffers). The latter one was used to be exactly under conditions used in previous reports from another group.19,20 In view of the very strong temperature dependence of the pH in Tris buffer (dpH/dT = −0.03), the explored temperature range was restricted to 20−40 °C, to limit the pH reduction to 0.6. Increasing temperature beyond 40 °C would be essentially meaningless since any effect could be attributed (at least in part) to the resulting pH variation. A different

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DISCUSSION To our knowledge, this is the first series of experiments performed under identical conditions (salt buffer, spermine concentrations, SAXS equipment, method to analyze the spectra, etc.) with a series of bacteriophage species. These parallel analyses make it possible to compare DNA organization between phages. We want to point out that the method taking into account the form factor (F(q)) leads to smaller values of the interhelix distance compared to those reported earlier by SAXS. This decrease leads to a better agreement between SAXS and cryoEM measurements of DNA parameters inside the capsids, a point that remained intriguing until now. We discuss below the consequences of changes in temperature and ionic conditions. No Temperature Effect on DNA Organization. In our temperature experiments, great care was taken to avoid any radiation damage of the samples. A new sample was taken for each temperature measurement, and the sample heated from 20 to 40 °C was returned to 20 °C and checked again to verify the absence of any global modification of the I(q) and P(r) curves. We observed no temperature effect on the DNA organization inside the capsid in the 20−40 °C temperature range, for any of the phages that have been studied. In particular, regarding λ, our results are in disagreement with previous observations19 since no significant evolution of the area of the DNA diffraction peak was observed, even in the Mg-HS buffer used by these authors. We observed only a very slight increase of the whole scattering curve due in part to the variation with temperature of density and compressibility (we see that the intensity scattered by the buffer increases by 2% in perfect agreement with the theoretical expression: I is proportional to TχTρ2, where χT is the compressibility and ρ the density) and in part to evaporation. In contrast, the authors in ref 19 report a reduction of the DNA peak of more than 20% in the 20−40 °C temperature range. As a consequence, the hypotheses suggesting that a temperature raise around 37 °C H


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increase of the DNA ordering upon spermine addition. This effect was also detected by cryoEM in λ: the concentric DNA layering that comes out by averaging a large number of capsids extends further toward the center of the capsid, and their number is increased from 7 to 9 in the presence of 1 mM spermine.19 How can these changes be understood, and how can DNA ordering be increased under such confinement? At the high degree of packing encountered in full bacteriophages, the entire structure becomes hexagonal apart from faults caused by frustration. Frustration arises because the hexagonal packing cannot be perfect in a quasispherical geometry.62 Ordering is thus mostly dominated by defects rather than bulk properties. Along these faults/defects, DNA interhelix spacing is slightly larger than in the hexagonal domains because of the lack of perfect alignment. These defects thus contribute to enlarge the DNA diffraction peak, to decrease its intensity, and to increase slightly the calculated aH value. The more defects there are, the larger the diffraction peak is. By the way, the DNA peak observed in bacteriophages is significantly larger than the peaks recorded from DNA phases at the same concentration.49 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 question of a possible reptation of DNA chain when ionic conditions are changed has been discussed earlier62 and remains open. 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. Like others,37 we never detected in the capsids the Ψ-type spectrum that characterizes the long-range supramolecular chiral organization of DNA.44,63 Similarly, no Ψspectra are evident in samples of DNA collapsed with spermidine or other polyamines that form toroids with dimensions (≈ 50 nm) close to dimensions of phages. Their CD differs in negligible details from that characteristic of Bform DNA.64,65 On the other hand, solutions of small toroidal structures (≈ 30 nm) formed by interaction of DNA with polylysine show a Ψ-type spectrum.46,47 Anomalous CD spectra are not a necessary consequence of DNA packing or collapse.65 Calculations from Keller and Bustamante66 demonstrated that the amplitude of the signal is proportional to the overall size and long-range chiral nature of the aggregates. The chiral structures with dimensions (pitch) close to the wavelength of the incident light will usually be much more able to discriminate left from right, circularly polarize, and give a signal. The absence of the CD signal in capsids suggests that there is no long-range chiral ordering of DNA in the capsid. The twist walls where the twist frustration arising from the hexagonal packing is relaxed (described earlier in T56) are probably not organized regularly to form a TGB-like structure67 that would produce a long-range chiral organization. We suspect that the length of the DNA chain that is known to restrict the long-range chiral organization68 and the constraints imposed by capsid faces on DNA alignment prevents this regular distribution of twist walls. Comparison between Bacteriophages. If we compare the bacteriophage species, the domains (measured in 100 mM spermine for example) are significantly larger in T5 (155 Å) compared to λ (123 Å), T7 (128 Å), and Φ29 (110 Å). It makes sense that larger domains are found in larger capsids

would induce a liquid crystalline phase transition in the core of the bacteriophage capsid,19 resulting in less densely packed DNA, must be reconsidered. It is well-known that temperature strongly affects kinetics of DNA ejection as shown for T5, λ, and SPP1 bacteriophages.58 For each phage, the shape of the ejection curves stays unchanged for temperatures ranging from 5 to 41 °C and the averaged characteristic time, over the phage population, follows an Arrhenius law. The strong temperature dependence of the kinetics of ejection is associated with an activation energy barrier that must be overcome for the opening of the connector-tail channel. These conformational changes occur prior to DNA release and constitute the limiting step of the DNA ejection kinetics. The same effect was observed with λ in comparison with the ejection at 22 and 31 °C by fluorescence microscopy (Figure 4 in ref 19). We will not discuss such effects here because they deal with protein conformation changes58 and do not concern DNA changes in any respect. The exploration of possible effects of temperature on DNA translocation (masked by the protein effect in refs 19, 25) would require the analysis of individual translocation DNA events, by using single phage assays as a function of temperature, as it has been done for T512 and λ59 for other parameters. To our knowledge such experiments have not been done. Reorganization of the Defect Lattice. The decrease of the calculated aH values upon addition of spermine is very small and close to the experimental error. Thus, although it is observed in all five phages, this effect must be discussed with great care. The effect of spermine remained undetected in previous experiments with λ when 1.3 to 100 mM spermine (4+) or CoHex (3+) was added in TM buffer.60,61 The presence of 10 mM divalent cations in TM buffer was probably masking the spermine effect. Indeed, in our experiments, we noticed that replacing HS buffer by TM buffer slightly shifts the DNA peak in the same direction as spermine. Earlier experiments showed no effect on the measured spacing when 1 mM MgCl2 was substituted by 1 mM putrescine (2+), 1 mM spd (3+),3 or 1 mM sp (4+),7 suggesting that, at this concentration, all of these cations play the same role in slightly reducing the repulsive interactions. This effect is more significant in the deletion mutant T5st0 as already noticed in the deletion mutants of λ.60 Thus, the slight decrease of the calculated aH values cannot be interpreted as a reduction of the volume occupied by the DNA chain, for example by creation of a core of lower density as suggested earlier by theoretical approaches.62 This hypothesis was not supported by any CryoEM observation from different teams working on different bacteriophage species. We cannot exclude that changes in the ionic conditions modify marginally the capsid−DNA interactions and facilitate a closer packing along the capsid faces (and thus increase the volume accessible to DNA while keeping constant the capsid volume itself). We support instead the idea that our measurements detect slight reorganizations of DNA in the capsid as shown by the rise and shrinking of the diffraction peak. Indeed, the presence of spermine enlarges the size of the domains D 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. A concentration of 4 mM spermine is enough to produce this effect in HS buffer (values in Table 2). We interpret this increase of the domain size detected for all phages as an I



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since DNA chains are aligned parallel to the internal protein faces. As a consequence, DNA domains can extent further when the capsid dimensions increase. More surprising is the comparison of the DNA concentrations estimated from the measured interhelix distances. We found 540 ± 20 mg/mL in T5, 525 ± 20 mg/mL in λ, 575 ± 20 in T7, and 615 ± 50 in Φ29. These differences are above the calculated error bars (even in Φ29 where the concentration cannot be calculated so accurately). Both λ and T7 have icosahedral capsid, and their dimensions and DNA length are in the same range. The main difference comes from the presence of an internal core in T7. As suggested by others,5 the core may facilitate DNA organization by winding part of the chain around it in a toroidal organization. This facilitated organization would reduce or modify the defect lattice required to solve the frustration arising from the confinement of the hexagonal lattice in an icosahedral capsid. Our estimations of the DNA concentrations seem to indicate that the smallest phages (T7 and Φ29) are the most densely packed. For small phages, the higher value of this concentration may result either from a denser DNA compared to other capsids or from a better DNA organization, with fewer defects. In the first hypothesis, small capsids would encapsidate larger amounts of DNA and sustain a higher internal pressure. To decipher and progress further, we would need accurate independent measurements of the volume accessible to DNA in each capsid from high resolution cryoEM reconstructions. Defects themselves and DNA domains would need to be characterized by recording tomograms of individual phages for each phage species.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b01783. CD spectra of empty T5 and purified T5 capsids (PDF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank P. Tavares, C. Knobler, and M. Feiss for providing phage strains. CD experiments were performed on the DISCO beamline at SOLEIL Synchrotron, France (Project 20120717). The SAXS experiments were performed on beamline BM29 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We are grateful to B. Calisto Machado and M. Brennich at the ESRF for providing assistance in using beamline BM29. This work has been supported by CNRS, the French Research Agency [ANR-12-BSV5-0023], and Investissements Avenir-LabEx PALM [ANR-10-LABX-0039-PALM]. D.D., M.d.F., A.L., and F.L. designed and performed most of the experiments and discussed the results. M.d.F., P.B., N.Z., S.B., A.L., M.R., and F.L. prepared phages or FhuA. J.D. and A.L. performed cryoEM experiments. F.W. helped collect CD data at SOLEIL. M.d.F. analyzed CD data. D.D. analyzed X-ray data. M.d.F., D.D., and F.L. wrote the article. J


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Can Changes in Temperature or Ionic Conditions Modify the DNA

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