(Received for publication, January 5, 1995; and in revised form, February 8, 1995)
From the
Extensive effort has been directed toward a quantitative evaluation of forces which operate between biomacromolecules since the characterization of such forces is essential to a thorough understanding of fundamental biological processes. However, all studies hitherto reported were conducted in vitro, using isolated species. Here we report the first quantitative characterization of forces operating between DNA molecules within living bacteria. Evaluation of x-ray scattering studies conducted on intact bacteria indicates that, at DNA-DNA surface separations characteristic of DNA assemblies, interactions are dominated by repulsive hydration forces which originate from the structuring of water molecules. The results support the notion that the mechanisms by means of which macromolecules function, fold, and interact with each other crucially depend upon their hydration properties.
Virtually all biological processes involve a close approach of the participating molecules, thus indicating the crucial role of those forces that operate between molecular surfaces at near contact. The issue is particularly significant when intracellular processes and assemblies are considered, in view of the very high macromolecular concentrations that prevail within the cellular systems(1, 2) . It has been proposed that interactions at small surface separation are dominated by solvation forces that arise from structuring of water molecules, and that these forces can be repulsive, attractive or oscillatory(3, 4, 5, 6, 7, 8) . Are such forces operative within intact cellular systems as well? Evidently, this question must be answered using non-invasive methods in order not to perturb intracellular superstructures and functions.
Escherichia coli (JM109) harboring BlueScript (2960 base pairs) were grown in LB medium to an optical density of 0.6 at 600 nm, then treated with chloramphenicol (150 µg/ml) for 12 h at 37 °C. Pellets were prepared by gentle centrifugation of 50-ml cultures, resuspended, and equilibrated against polyethylene glycol (average molecular weight 4000; Sigma) solutions of known osmotic pressures (9) for 3 h at 20 °C and transferred into x-ray capillaries.
X-ray measurements were
conducted on a low angle camera operating with Cu-K radiation
(
= 1.541 Å), monochromated with a nickel filter,
followed by a single Frank mirror. Scattering profiles were recorded
with a linear position-sensitive detector; data acquisition time was 12
h at 19 °C. Mean values of three independent determinations are
presented; reflections corresponding to the data points have been
determined with an error of ±0.4 Å. Interhelical spacings
are calculated on the basis of two-dimensional hexagonal
packing(10) .
In order to measure the forces operating between DNA molecules within intact cells, we applied the osmotic stress technique, which enables a rigorous evaluation of thermodynamic parameters through the measurement of interaxial distances as a function of the applied osmotic stress(11, 12) . Our recent observation, according to which plasmid DNA molecules spontaneously segregate into dense ordered clusters within the cells(13) , allows a direct measurement of the DNA interaxial spacing by means of small angle x-ray scattering. Such measurements were performed on intact E. coli cells carrying a high copy plasmid and equilibrated against polyethylene glycol solutions of known osmotic pressures.
In the
absence of an external osmotic pressure, plasmid assemblies within E. coli cells have been shown to exhibit a 51.5 Å x-ray
scattering peak(13) . Upon application of increasing external
osmotic pressure, a signal is observed which progressively shifts
toward higher scattering angles. A semilogarithmic plot of the osmotic
pressure versus interaxial separation of
DNA molecules within the plasmid assembly in E. coli cells is
shown in Fig. 1. The DNA interaxial spacing ranges from 32 to 25
Å; no further decrease in helix separation below 25 Å is
observed. Since the diameter of the physiological B-DNA conformation is
20 Å, the observed interaxial spacings correspond to surface
separations of 12-5 Å. Notably, low angle diffraction
maxima corresponding to distances greater than 32 Å were also
obtained but were too weak and diffuse to be used for the analysis.
These maxima strongly suggest that the 51.5 Å scattering peak
observed in the absence of an external stress (13) and the
diffractions reported here originate from a common source. The
observation that interwound DNA molecules may assume an interhelical
spacing as short as 25 Å is somewhat surprising. However, x-ray
scattering measurements, conducted with very short exposure times
(hence minimizing irradiation-induced DNA nicking) on isolated
supercoiled DNA plasmids under high osmolality conditions, conclusively
indicated that such short spacings are readily obtained. (
)In order to further eliminate the possibility that the
scattering profiles exhibited by the E. coli cells are
influenced by the presence of partially nicked or linearized plasmid
populations resulting from irradiation-induced damage, we have repeated
the experiments with various acquisition times, ranging from 3 to 12 h;
no alteration in the scattering patterns could be detected. Longer
exposures have indeed been shown to result in the deterioration of the
peaks, presumably due to a progressive DNA nicking(13) , but
not in the appearance of new signals.
Figure 1: Applied osmotic pressure versus plasmid DNA interhelical spacing. Each data point represents a mean value obtained from three independent x-ray measurements that were conducted on intact bacteria carrying the high copy BlueScript plasmid. The scattering peak used for the analysis was exhibited only by bacteria carrying the high copy BlueScript plasmid. Three additional diffraction maxima, at 54.1 ± 0.4, 46.6 ± 0.1, and 42.6 ± 0.3 Å, were exhibited under the conditions specified under ``Experimental Procedures'' by bacteria harboring the plasmid as well as by control (non-transformed) cells and hence are not associated with the plasmid assembly. The origin of these low angle maxima, found to be insensitive to the applied osmotic stress, is currently being investigated.
Linear regression analysis of the data points presented in Fig. 1indicates that the dependence of the measured DNA interhelical distances is accurately described by the relation shown in ,
where reflects the degree of water
perturbation at the interface (5, 6) and is found to
equal 2.5
10
dynes/cm
, and
is the
decay length, which, for the experimental range of interaxial
separation d
, is 3.8 Å.
The magnitudes of both the force and the decay constant are similar to the values reported for polyethylene glycol-induced in vitro condensed phases of linear DNA in the presence of NaCl (10) and are interpreted as indicating the predominance of a repulsive hydration term. Specifically, it has been argued that the magnitude of the decay length, as well as the lack of dependence of both the magnitude of the force and the decay length upon ionic strength, cannot be explained by repulsive electrostatic double-layer interactions for either fully or partially charged DNA molecules(10, 14) . The coexistence of an attractive hydration term is ruled out by the fact that such a term, observed in the presence of multivalent DNA-condensing agents such as cobalt hexamine or polyamines, results in a significantly smaller decay length of approximately 1.5 Å(14) .
The previously described
spontaneous assembly of plasmid DNA within bacteria which results, in
the absence of external stress, in surface separations of 30
Å(13) , probably reflects a balance between electrostatic
repulsion, excluded volume effects, and an intrinsic turgor stress. It
has been shown that the work of bringing double-stranded DNA molecules
from infinity to surface separations of 17 Å is approximately 0.1
kcal/mol base pair(15) . We have calculated the additional free
energy (G) associated with the interhelical compression
beyond the thermodynamically established equilibrium distance of the
spontaneously packed molecules. At a constant temperature, this
quantity can be evaluated by the relation shown in ,
where G is the free energy or work, and
dV is the change in lattice volume per
dinucleotide.
Thus, the change in free energy per base pair of moving the DNA
helices closer than the largest observed interaxial spacing is given by
integrating with respect to V
, normalized to the maximum volume V
, and is found to be quite small (Fig. 2), i.e. on the order of k
T. Summed over the plasmid molecule
(2960 base pairs), however, it becomes significant. Since the work of
water removal is a continuous function of the lattice volume, discrete
groups of bound water, like those associated with the inner hydration
shell of the DNA, cannot be discerned in this experiment.
Figure 2: Difference in free energy of compaction as a function of lattice volume. Changes in free energy were computed by numerical integration of the pressure versus total volume/(base pair) curve, normalized to the volume obtained at the lowest pressure applied. Since the system does not allow to distinguish between contributions from electrostatic interactions and those associated with water removal, only relative energy values are given.
The high macromolecular concentrations characteristic of cellular systems, in conjunction with the ability of polar surfaces to immobilize or organize water, underscore the central role of water molecules in determining the properties of subcellular assemblies and affecting processes such as cell-cell adhesion (16) and transport through biological channels(17) . The observations presented in this study provide the first example of the involvement of hydration forces in the process of DNA assembly in vivo. Similar effects are proposed to characterize all low protein DNA organizations, since in such assemblies, encountered in viruses, mitochondria, or dinoflagellate chromosomes(18) , the work associated with water removal during condensation is not likely to be strongly affected by structural DNA-binding proteins. The quantitative agreement between the results presented here and those derived from in vitro measurements implies that the osmotic stress technique may serve as a sensitive and reliable experimental tool for the evaluation of hydration forces in cellular compartments.
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