Low-Force DNA Condensation and Discontinuous High-Force Decondensation Reveal a Loop-Stabilizing Function of the Protein Fis

We report single-DNA-stretching experiments showing that the protein Fis, an abundant bacterial chromosome protein of E. coli, mediates a dramatic DNA condensation to zero length. This condensation occurs abruptly when DNA tension is reduced below a protein-concentration-dependent threshold $f^{*}<1\mathrm{pN}$. Following condensation, reopening under larger forces proceeds via a series of discrete jumps, indicating that Fis is able to stabilize DNA crossings. Our experiments suggest that Fis may play a role in vivo stabilizing the “loop-domain” structure of the bacterial chromosome.

Figure 1

(a) Collapse of DNA against applied force by Fis. After incubation with $13\mu \mathrm{M}$ Fis in buffer (20 mM HEPES, 100 mM potassium glutamate, $p\mathrm{H}$ 7.6) at a force of 14 pN, DNA extension decreased from 16.5 to $15.5\mu \mathrm{m}$, consistent with the action of a DNA-bending protein. As force was subsequently reduced to 5 pN and later to 1 pN, DNA length decreased over a few seconds to stable final values (horizontal black lines), expected given the known DNA-bending function of Fis. By contrast, at 0.6 pN force, the DNA-Fis complex condensed (red squares); between 3 and 7 min DNA was gradually compacted down to zero extension. At this force, naked DNA extension would be $14.5\mu \mathrm{m}$. (b) Proposed model for DNA condensation by Fis against applied force. Thermally excited DNA loops can be stabilized by Fis molecules (red circles). Fis likely coats the DNA at concentrations where looping is observed; the red circles show only those complexes capable of stabilizing loops.

Figure 2

Force-extension behavior of Fis-DNA complexes. (a) Open circles indicate naked DNA in buffer: as in other studies, a nonlinear and reversible polymer elastic response is observed. Squares show DNA after reaction with $13\mu \mathrm{M}$ Fis at a force of 14 pN. Fis-induced DNA bends result in shortening of the DNA length from 16.5 to $15.5\mu \mathrm{m}$; for forces above 0.6 pN (extensions above $12.5\mu \mathrm{m}$) the force-extension is shifted to slightly larger forces, and is reversible. However, at the threshold force of 0.6 pN abrupt DNA condensation occurs. In other experiments with $6\mu \mathrm{M}$ (solid circles) and $1\mu \mathrm{M}$ Fis (solid diamonds), abrupt condensation was again observed, with the difference that at lower protein concentrations the threshold force is gradually shifted down towards smaller forces. At or below 200 nM Fis concentration, abrupt condensation no longer occurs (open diamonds) and the Fis-DNA complex displays reversible elasticity over the entire force range. Note that data points for forces above collapse represent averages of stable series of extension measurements. (b) Relation between Fis concentration and threshold force $f^{*}$. At high protein concentration (left) loop-forming complexes are in close proximity, allowing small, thermally excited loops to be captured; these loops form at relatively high forces. At lower concentrations (right) loop-forming sites are further apart, so condensation can only occur via formation of larger loops; these loops form only at relatively low forces.

Figure 3

Decondensation of Fis-condensed DNA shows discrete jump opening events, consistent with opening of Fis-mediated DNA loops. Extension versus time recorded during the opening driven by 9 pN force, following 3 min condensation by $5\mu \mathrm{M}$ Fis at $<0.2\mathrm{pN}$ (note condensation reaction is not shown). (a) Entire opening time series shows opening of molecule from near zero to near the full contour length of $16.5\mu \mathrm{m}$. Note that the small discontinuities between points correspond to small jump events. (b) Expanded view of first 0.1 min of (a) shows discrete extension jumps more clearly. (c) Expanded view of later part of (a) shows similar jumps.