Structural Change of DNA Induced by Nucleoid Proteins: Growth Phase-Specific Fis and Stationary Phase-Specific Dps

Biophysical Journal Volume 105 August 2013 1037–1044

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Structural Change of DNA Induced by Nucleoid Proteins: Growth Phase-Specific Fis and Stationary Phase-Specific Dps Yuko T. Sato,† Shun Watanabe,‡ Takahiro Kenmotsu,‡ Masatoshi Ichikawa,† Yuko Yoshikawa,§ Jun Teramoto,{ Tadayuki Imanaka,§ Akira Ishihama,{ and Kenichi Yoshikawa‡* †

Department of Physics, Kyoto University, Kyoto, Japan; ‡Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan; Laboratory of Environmental Biotechnology, Ritsumeikan University, Kusatsu, Japan; and {Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan §

ABSTRACT The effects of nucleoid proteins Fis and Dps of Escherichia coli on the higher order structure of a giant DNA were studied, in which Fis and Dps are known to be expressed mainly in the exponential growth phase and stationary phase, respectively. Fis causes loose shrinking of the higher order structure of a genome-sized DNA, T4 DNA (166 kbp), in a cooperative manner, that is, the DNA conformational transition proceeds through the appearance of a bimodal size distribution or the coexistence of elongated coil and shrunken globular states. The effective volume of the loosely shrunken state induced by Fis is 30– 60 times larger than that of the compact state induced by spermidine, suggesting that cellular enzymes can access for DNA with the shrunken state but cannot for the compact state. Interestingly, Dps tends to inhibit the Fis-induced shrinkage of DNA, but promotes DNA compaction in the presence of spermidine. These characteristic effects of nucleotide proteins on a giant DNA are discussed by adopting a simple theoretical model with a mean-field approximation.

INTRODUCTION The chromosomal DNA of bacteria is associated with a number of nucleoid proteins, which are expected to play the essential role in global genetic regulation (1–7). The model organism Escherichia coli is a good system for studying the relationship between the genome conformation and its activities because its genome consists of a single molecule of circular DNA of 4.65 Mbp, and the function and regulation of the individual genes have been well characterized (8). The E. coli genome is associated with a set of DNA-binding proteins, which together form a nucleoid (9). The nucleoid proteins are classified into two groups: universal nucleoid proteins that are always associated with the nucleoid, and growth phase-specific nucleoid proteins (9,10). During the transition of E. coli cells from exponential growth to stationary phase, the morphology of the nucleoid changes from a fibrous state to a rod form (9,11). These changes are mainly attributable to changes in the composition of associated nucleoid proteins. In the exponential growth phase, Fis (factor for inversion stimulation) is the most abundant protein, and the order of abundance is Fis > Hfq > HU > StpA > H-NS > IHF > CbpB > Dps. In contrast, in the early stationary phase, Fis disappears and instead Dps (DNA-binding protein from starved cells) becomes the most predominant, and the order of abundance changes to Dps > IHF > HU > Hfq > HU > CbpA > StpA (12,13). This orderly change in the nucleoid protein composition on transition from exponential growth to stationary phase is accompanied by the silencing of genome functions. However, the higher ordered architectural features of the Submitted June 2, 2013, and accepted for publication July 15, 2013. *Correspondence: [email protected]

transcriptionally active and inactive nucleoid remain poorly understood. Fis is a DNA architectural protein that is 98 amino acid residues in length and has an a-helical core of four helices (14). It exhibits a total of 14 positive charges and 11 negative charges under physiological solution conditions (15). Fis stimulates DNA-associated functions such as DNA inversion and excision, and activates transcription of growth-related genes such as tRNA and rRNA genes (16,17). Fis binds DNA at poorly conserved 15 bp core sequences (18). Depending on the sequence bound, Fis induces DNA bending from 50 to 90 (19). When bound to a giant DNA, Fis stabilizes DNA loops as detected by the measurement of force extension with single DNA micromanipulation (20,21) and induces the dramatic compaction of DNA molecules, thereby forming branched plectonemes in the E. coli nucleoid (16). Dps, the major nucleoid protein in starved stationary-phase cells, is a small DNA-binding protein of 167 amino acid residues with a positive charge along the entire chain. Dps forms ferritin-like dodecamers (22) and plays a role in collecting free Fe(II) at its ferroxidase center (23). In resting bacterial cells, Dps protects DNA by means of a double mechanism, that is, by acting as a physical shield through the formation of Dps– DNA cocrystals and by binding and oxidizing Fe(II) (9,10,12,13,24). Dps plays an important role in the condensation and protection of the stationary-phase nucleoid from environmental stresses such as oxidative damage (9,22,25,26). To shed light on this long-standing problem regarding the growth-coupled changes in the structure and function of the E. coli nucleoid, we studied the higher order structures of DNA–protein complexes with two representative growth phase-specific nucleoid proteins, Fis and Dps. For this purpose, we used T4 phage DNA (166 kbp) as a model giant

Editor: Lois Pollack. Ó 2013 by the Biophysical Society 0006-3495/13/08/1037/8 $2.00

http://dx.doi.org/10.1016/j.bpj.2013.07.025

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DNA, the size of which is close to that of the minimum loop or domain of the E. coli nucleoid (7). In addition to these nucleoid proteins, we also analyzed the mode of DNA interactions for polyamines because polyamines, such as putrescine, spermidine, and spermine, are associated with the nucleoid, where their intracellular levels are millimolar concentrations in stationary phase and tend to be higher in growth phase (9,27,28).

A control

B 500 nM Fis

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Atomic force microscopy For the visualization of protein–DNA complexes by atomic force microscopy (AFM), T4 DNA (0.3–0.5 mM/bp) was incubated with and without Fis (250 nM) for 30 min at room temperature. The samples were fixed with 0.5% glutaraldehyde for 30 min on ice and were spotted onto a freshly cleaved mica substrate that had been pretreated with 10 mM SPD. After 10 min, the mica was gently washed with distilled water and dried under nitrogen gas. Multimode AFM (Veeco, Santa Barbara, CA) with a Nanoscope IIIa was used in air at room temperature under tapping mode with a 10-mm scanner. The probe, which consisted of a single silicon crystal on a cantilever (OMCL-AC160TS-W2, Olympus, Tokyo, Japan), 11 mm in length and with a spring constant of 53–68 N/m, was driven at a scanning rate of 1.5–2.5 Hz with an amplitude of 30–70 mV. Height mode data were collected into 512  512 pixel images and manipulated with WSxM v5.0 software to subtract background distortions (29).

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We prepared a solution of 0.1 mM/bp T4 DNA with 0.1 mM 40 ,6-diamino-2phenylindole and allowed it to sit for at least 10 min before the addition of Fis or Dps and/or SPD. All of the specimens consisted of a 10-mM TrisHCl aqueous solution at pH 8.0. For the observations, each sample was loaded onto a glass slip that had been cleaned (baked at 500 C for 5 h, sonicated in 0.1 M sodium hydroxide, sonicated in MilliQ-purified water, and then rinsed with ethanol). Fluorescence images were obtained with a fluorescent microscope (Axiovert 135TV, Carl Zeiss, Oberkochen, Germany) and recorded through an EBCCD camera (Hamamatsu Photonics, Hamamatsu, Japan).

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T4 GT7 DNA (166 kbp) was obtained from Nippon Gene (Toyama, Japan). DNA was stained by 40 ,6-diamino-2-phenylindole, which was purchased from DojinDo Molecular Technologies (Kumamoto, Japan). Spermidine (SPD) and glutaraldehyde were obtained from Nacalai Tesque (Kyoto, Japan). Nucleoid proteins Fis and Dps were purified as in a previous study (24).

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Change in the higher order structure of DNA induced by nucleoid proteins

FIGURE 1 Conformational transition of DNA with the addition of the nucleoid protein Fis and the influence of Dps on this transition. Fluorescence images of T4 DNA together with quasi three-dimensional representations of the fluorescence intensity, in solutions of (A) Tris-HCl buffer solution, (B) 500 nM Fis, and (C) 500 nM Fis and 50 nM Dps. Distribution of the long-axis length, L, of DNA at different Fis concentrations (D) without Dps and (E) with 50 nM Dps. (F) Schematic representation of the long-axis length L of DNA, as observed by fluorescence microscopy.

On the top of Fig. 1 are shown the examples of fluorescence microscopic images of single T4 DNA molecules exhibiting fluctuating Brownian motion in bulk aqueous solution. In the absence of nucleoid proteins, an elongated random coil conformation is observed, as in Fig. 1 A. With the addition of 500 nM Fis, DNA molecules exhibit the shrunken conformation shown in Fig. 1 B. In the presence Biophysical Journal 105(4) 1037–1044

DNA Structure With Nucleoid Proteins

750 µM SPD

D 250 µM SPD + 50 nM Dps

5 μm frequency / % frequency / % frequency / % frequency / % frequency / %

It has been well established that, with the addition of SPD(3þ), giant DNA molecules undergo a large discrete transition between an elongated coil state and folded compact state (30,31), where SPD exhibits þ3 charge under physiological conditions. In the present study, we confirmed the discrete transition on the level of individual DNA molecules, as shown in the histogram in Fig. 2 E. The manner of the conformational transition induced by Fis, as in Fig. 1 D, appears to be similar to that with SPD(3þ), as in Fig. 2 E; the transition proceeds through an intermediate state characterized by a bimodal distribution. A closer inspection of Fig. 2 E indicates that the size of L of the elongated coil state remains almost constant up to the SPD concentration in the bimodal distribution, whereas L of the coil state decreases gradually in the case of the Fis-induced conformational change, as in Fig. 1 D. We also examined the effects of Fis and Dps on the SPD-induced transition of DNA. The results shown in Fig. 2, F and G, indicate that both Fis and Dps accelerate the conformational transition into the compact state, in which Dps exhibits slightly larger enhancement. As for the size distributions in Fig. 2, E–G, the profile for coil state in the presence of 50 nM Fis seems to be somewhat different from that in 0 mM SPD and 50 nM Dps. It is noted that such a fluctuation of the size distribution reflects the very mild curve around the free energy minimum on the coil state as predicted by the theoretical expectations (see Fig. 4, C and D). Thus, we consider that the size distribution of the first row in Fig. 2, E–G, are essentially the same.

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Effects of Fis and Dps on the folding transition caused by SPD(3D)

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of both 50 nM Dps and 500 nM Fis, DNA shows an elongated conformation, as in Fig. 1 C, which suggests that Dps has an antagonistic effect on the Fis-induced shrinkage in DNA conformation. The changes in the conformation of DNA molecules are summarized in Fig. 1, D and E, as histograms of the long-axis length L, where the definition of L is schematically depicted in Fig. 1 F. L is the apparent long-axis length because of the optical blurring effect on the order of 0.3–0.5 mm, and it is impossible to evaluate the actual size of seemingly compact DNA molecules only from fluorescence images. The histogram in Fig. 1 D indicates that L decreases gradually up to 250 nM Fis, where a shrunken state appears in a bimodal distribution. With a further increase in the Fis concentration, almost all of the DNAs assume a shrunken conformation. However, as shown in Fig. 1 E, in the copresence of Dps, the transition to the shrunken state is apparently retarded, which indicates that Dps antagonizes the effect of Fis. We also measured the effect of Dps on giant DNA by fluorescence microscopy, and found that Dps has no apparent effect on the DNA conformation up to a concentration of 50 nM.

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FIGURE 2 Conformational transition of DNA with the addition of SPD, and the influence of the nucleoid proteins Fis and Dps on this transition. Fluorescence images of DNA, together with quasi three-dimensional images, in solutions of (A) 250 mM SPD, (B) 750 mM SPD, (C) 250 mM SPD with 50 nM Fis, and (D) 250 mM SPD with 50 nM Dps. Distribution of the long-axis length, L, of DNA at different SPD concentrations: (E) without nucleoid proteins, (F) with 50 nM Fis, and (G) with 50 nM Dps.

Shrinkage and compaction of DNA As mentioned above, fluorescence microscopic observation of individual DNA molecules provides useful information regarding the conformation in solution. However, because fluorescence images are influenced by an optical blurring effect on the order of 0.3 mm, we cannot evaluate the actual size of the shrunken state induced by Fis in a precise Biophysical Journal 105(4) 1037–1044

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manner. To obtain information on the actual size of DNA in solution, we measured the diffusion constant from the Brownian motion of individual DNA molecules. The top-left pictures in Fig. 3 exemplify the trajectories of the center of mass (i.e., translational Brownian motion) of a single DNA molecule in the presence of 750 nM Fis (Fig. 3 A) and 750 mM SPD (Fig. 3 B) for 3 s. From the analysis of Brownian motion, we evaluated the translational diffusion constant D for individual DNA molecules observed by fluorescence microscopy. The D-value can be obtained from the mean-square displacement of the center of mass for DNA. During the observation of Brownian motion, a small but nonnegligible convective flow was present during the measurement, and this was possibly caused by the thermal effect of illumination. Because the rate and direction of convective flow were almost constant during the period of observation, we could eliminate the effect of convective flow by adopting the relationship in Eq. 1 (32–34),

2 ðRðtÞ  Rð0ÞÞ ¼ 4Dt þ At2 ;

B 750 μM SPD

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A 750 nM Fis

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0.5 μm FIGURE 3 Comparison of the higher order structures of DNA. Examples of the translational Brownian motion of DNA molecules observed by fluorescence microscopy: (A) 750 nM Fis and (B) 750 mM SPD. (C) Probability distribution of the hydrodynamic radius RH with 750 nM Fis and 750 mM SPD, as evaluated from the analysis of Brownian motion. AFM images of (D) T4 DNA without nucleoid protein and (E) with 250 nM Fis. Biophysical Journal 105(4) 1037–1044

where R(t) ¼ (Rx, Ry) is the position of the center of mass for DNA, h(R(t)  R(0))2i is the mean-square displacement, and A is a numerical constant that is related to the convective flow. Eq. 1 contains the implicit assumption that both the rate and direction of the convective flow are constant, which was actually the case in our measurement, at least for the period on the order of 10 s. The effective hydrodynamic radius RH of a single DNA molecule was calculated from the D-value based on the Stokes–Einstein equation given in Eq. 2 (35), RH ¼

kB T ; 6phs D

(2)

where kB is the Boltzmann constant and hs is the viscosity of the solvent (1.002 mPa , s for pure water at T ¼ 293 K). Fig. 3 C shows the distributions of RH for single DNA molecules with 750 nM Fis and with 750 mM SPD(3þ). The ensemble average over 50 DNA molecules, , of the compact state with SPD was 100 5 30 nm, whereas that for DNA shrunken with Fis was 410 5 100 nm. The effective volume of the shrunken state of DNA induced by Fis was ca. 70 times greater (z (410/100)3) than that of the compact state induced by SPD. The bottom pictures in Fig. 3 show AFM images of T4 DNA in the absence (Fig. 3 D) and presence (Fig. 3 E) of 250 nM Fis, respectively. The loosely shrunken conformation with Fis observed by AFM corresponds well to the above results regarding the hydrodynamic radius, RH. THEORETICAL From the observation of single DNA molecules in solution, it has become evident that Fis causes a conformational transition from an elongated coil state to a shrunken state through the appearance of a bimodal distribution of these states (Fig. 1 D). AFM observation revealed that the resulting condensed state is rather loose, where the hydrodynamic radius in the shrunken conformation induced by Fis is 3–4 times larger than that induced by SPD (Fig. 3 C), and this tendency was confirmed by the measurement of Brownian motion. The difference in the hydrodynamic radius indicates that the effective volume of the loosely shrunken state is 30–60 times greater than that of the compact state given that the volume scales as the cube of the radius. This unique effect of Fis at inducing a loosely shrunken state in the DNA conformation may be associated with the well-known trend that Fis stimulates the activity of genetic DNA. The ability of RNA polymerase and other working proteins to access DNA would be maintained in the loosely shrunken state, but not the tightly compacted state. It has been reported that Fis binds to DNA in a largely sequence-neutral fashion and generates loops along a long DNA chain (20,21). Based on a consideration of these experimental observations, we propose a simple theoretical model to describe the

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conformational transition of a giant DNA molecule induced by Fis. Physicochemical aspects of the conformational transition of DNA Double-stranded DNA exhibits a persistence length on the order of 50 nm: lp z 50 nm. This implies that DNA molecules smaller than several kbp behave as a stiff polymer, whereas giant DNA molecules larger than several tens of kbp have the characteristics of a semiflexible polymer. Here, the term semiflexible chain refers to a linear macromolecule where L ¼ lp , N (contour length) >> lp (persistence length) >> a (diameter of the polymer chain) (30,36). In our experiment, we used T4 DNA as a DNA chain that was long enough to act as a semiflexible polymer, that is, its size of 166 kbp roughly corresponds to N ¼1000. According to a theoretical treatment by mean field theory in polymer physics, the free energy of a polymer chain F(a) is given as follows:

FðaÞ 3 ¼ Fela þ Fint z a2 þ a2 þ Br2 V þ Cr3 Vþ O r4 ; kB T 2 (3) where Fela and Fint are due to the conformational entropy and the interaction between segments, respectively. a ¼ R/R0 is the relative size of the polymer, where R is the size of the polymer chain and R0 ~ lpN1/2 is that of the Gaussian chain. The constants, B and C, are the second and third virial coefficients, respectively; V is the volume occupied by a polymer chain; r is the density of the segments (r z N/V), and kB is the Boltzmann constant. To interpret the characteristics that are inherent to a semiflexible chain, such as a long DNA chain, we have to modify the free energy expression from Eq. 3. By introducing angular dependence on the binary interaction between the segments, the free energy is reformulated as follows (37,38):
FðaÞ 3 2 z a þ a2 þ C a6 þ ½Higher order; kB T 2

(4)

 3 a Cz ; lp

(5)



where C* is the effective third virial coefficient. We may consider that the effective diameter of the double-strand DNA is ca. 4 nm (a ¼ 4 nm) by taking into account of the Coulomb repulsive interaction between the segments. Thus, we simply adopt the value C* ~ 5  104 for double-strand DNA in solution through the calculation of (a/lp )3 z (4/50)3. Our experimental observation indicates that the effective volume of shrunken DNA induced by Fis is several 10-fold larger than that of compact DNA caused

by SPD. Thus, we can consider that, for the case of Fis-induced shrinkage, the last term of the excluded volume in Eq. 4 is negligibly small. Fis bridges pairs of segments by looping (21), which suggests that shrinkage of a giant DNA molecule is due to an increase in looping. Such bridging or looping effect of Fis may be interpreted in terms of the effective negative second-virial coefficient B*, the absolute value of which increases with an increase in the Fis concentration. Now, we can consider the effect of Fis on the free energy expression of a single giant DNA molecule:
FðaÞ 3 2 z a þ a2 þ B a3 þ C a6 : kB T 2

(6)

Fig. 4 A shows the free energy profile based on Eq. 6 with different B* values, where the negative larger values of B* correspond to the increase of the pairwise attraction between the DNA segments. By assuming a Boltzmann distribution at T ¼ 300 K, probability distributions, P(a)s, are also obtained, as in Fig. 4 C, where we have shown the distribution as the histogram for the convenience of the comparison with the experiments. The histogram reproduces the experimental trend in a satisfactory manner, that is, the shrinking transition is generated through the appearance of a bimodal distribution accompanied by the gradual decrease on the size of the unfolded conformation. Thus, we can consider that the looping effect of Fis is the main cause of the shrunken state for long DNA molecules, through a scenario that involves an increase in the effective attractive interaction between segments with an increase in the concentration of Fis. The retardation of the conformational shrinkage in the presence of Dps, as in Fig. 1 E, suggests that Dps has an antagonistic effect on Fis-induced looping along the DNA chain. Whereas, the weak promotion of Fis on SPD-induced compaction (histogram in Fig. 2 F) implies that there is some cooperative effect between the charge neutralization induced by SPD and the looping induced by Fis. Upon the addition of SPD(3þ), individual DNA molecules undergo a large discrete transition as shown in the left diagram in Fig. 2 E, being apparently different from the effect of Fis as in Fig. 1 D. Concerning the physicochemical action of SPD(3þ) on the conformational transition of DNA, we have presented rather detailed theoretical discussions in previous papers (37,38), and therefore skip the theoretical details and present here only a short explanation. In contrast to the case of shrinking by Fis, DNA is tightly compacted by SPD, that is, there is a 30- to 60-fold difference in the DNA segment density between the shrunken and compact states (see, e.g., Fig. 3 C). It has been shown that the effective attraction between segments can be incorporated into an effective third virial coefficient C* by introducing a reduced temperature, t: t ¼ (T  TC)/TC). Biophysical Journal 105(4) 1037–1044

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FIGURE 4 Free energy profiles with respect to the logarithmic scale with swelling/shrinking ratio, a, as deduced from theoretical models (A) for the shrinkage induced by Fis and (B) for the compaction induced by SPD. Conformation of a ¼ 1 is the ideal Gaussian chain. Corresponding size distributions of the DNA chain, (C) and (D), depicted as the histograms. In the theoretical calculation, the number of persistence segments of the DNA is taken as N ¼ 1000; the contour length is ca. 180 kbp.

   a 1 þ st : Cz lp lp 

(7)

Thus, we can change the solvent quality (C* > 0 good; C* < 0 poor) by changing a single control parameter t. The physicochemical effects on the interaction between a DNA chain as a highly charged polyelectrolyte and SPD(3þ) can be renormalized into a single parameter while maintaining the essential property of the conformational transition. Based on the framework in Eq. 4 together with the higher order term, [Higher order] ¼N ln½1  a=ðN 1=2 lp a3 Þ (37,38), we evaluated the free energy for a DNA chain, with the parameter of Biophysical Journal 105(4) 1037–1044

a ¼ 2 nm (diameter of the double-strand DNA), lp ¼ 50 nm (persistence length of DNA), and N ¼ 1000 (same as in the model of Fis-induced shrinking). Fig. 4 B shows free energy profiles with a change in the size distribution at different values of the effective third virial coefficient C* as a theoretical model of DNA compaction induced by SPD(3þ), where C* < 0 exhibits the physical meaning of attraction between the aligned DNA segments (37,38). Fig. 4 D shows the change in the size distribution. Consistent with the characteristics of DNA as a semiflexible polymer, the conformational transition is largely discrete and the coil conformation in the region of coexistence maintains its size regardless of the change in C*, that is, the a-value

DNA Structure With Nucleoid Proteins

remains essentially the same as that for the elongated coil conformation at C* ¼ 0. DISCUSSION With a transition in cell growth of E. coli from exponential growth to stationary phase, there is a marked reduction in the overall level of genome transcription (39). This change in genome activity is mainly attributable to the change in the shape of the nucleoid as observed by electron microscopy and the composition of associated nucleoid proteins (9,10). The nucleoid of growing cells is composed of as many as 50–100 topologically isolated domains, corresponding to loops or branched projections, to which DNAinteracting functional proteins such as transcription-related proteins can make contact. However, the higher ordered structure of nucleoids has remained unclear. In the present study, we have shown, for the first time as far as we aware, that the effective volume of the shrunken state induced by Fis is 30–60 times larger than that of the compact state induced by SPD. We may expect that the tightly packed compact state prevents the access of enzymes and substrates, such as RNA polymerase and nucleoside triphosphates, to DNA in the nucleoid. Thus, we propose that proteins that are needed for the expression of DNA functions can access the loosely shrunken state of the exponential-phase nucleoid, but not the compact state of the stationary-phase nucleoid. The large difference in the architectural roles of Fis and Dps with respect to the degree of compaction/shrinkage will play a primary role in the difference in the overall activity of genome DNA between exponential growth and stationary phase. The authors thank T. Kanbe (Nagoya University) for his helpful discussion. The authors also thank A. Kori and K. Yamada (Hosei University) for the purification of Fis and Dps. This work was supported by Grants-in-Aid for Scientific Research (A) 23240044 and 21241047 to K.Y. and A.I., respectively.

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