Dynamic state of DNA topology is essential for genome condensation in bacteria
2006; Springer Nature; Volume: 25; Issue: 23 Linguagem: Inglês
10.1038/sj.emboj.7601414
ISSN1460-2075
AutoresRyosuke L. Ohniwa, Kazuya Morikawa, Joongbaek Kim, Toshiko Ohta, Akira Ishihama, Chieko Wada, Kunio Takeyasu,
Tópico(s)Escherichia coli research studies
ResumoArticle9 November 2006free access Dynamic state of DNA topology is essential for genome condensation in bacteria Ryosuke L Ohniwa Corresponding Author Ryosuke L Ohniwa Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kazuya Morikawa Kazuya Morikawa Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoh-dai, Tsukuba, Japan Search for more papers by this author Joongbaek Kim Joongbaek Kim Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Toshiko Ohta Toshiko Ohta Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoh-dai, Tsukuba, Japan Search for more papers by this author Akira Ishihama Akira Ishihama Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan Search for more papers by this author Chieko Wada Chieko Wada Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kunio Takeyasu Kunio Takeyasu Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Ryosuke L Ohniwa Corresponding Author Ryosuke L Ohniwa Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kazuya Morikawa Kazuya Morikawa Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoh-dai, Tsukuba, Japan Search for more papers by this author Joongbaek Kim Joongbaek Kim Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Toshiko Ohta Toshiko Ohta Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoh-dai, Tsukuba, Japan Search for more papers by this author Akira Ishihama Akira Ishihama Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan Search for more papers by this author Chieko Wada Chieko Wada Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kunio Takeyasu Kunio Takeyasu Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Author Information Ryosuke L Ohniwa 1, Kazuya Morikawa2, Joongbaek Kim1, Toshiko Ohta2, Akira Ishihama3, Chieko Wada1 and Kunio Takeyasu1 1Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan 2Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoh-dai, Tsukuba, Japan 3Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan *Corresponding author. Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. Tel./Fax: +81 75 753 7905; E-mail: [email protected] The EMBO Journal (2006)25:5591-5602https://doi.org/10.1038/sj.emboj.7601414 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In bacteria, Dps is one of the critical proteins to build up a condensed nucleoid in response to the environmental stresses. In this study, we found that the expression of Dps and the nucleoid condensation was not simply correlated in Escherichia coli, and that Fis, which is an E. coli (gamma-Proteobacteria)-specific nucleoid protein, interfered with the Dps-dependent nucleoid condensation. Atomic force microscopy and Northern blot analyses indicated that the inhibitory effect of Fis was due to the repression of the expression of Topoismerase I (Topo I) and DNA gyrase. In the Δfis strain, both topA and gyrA/B genes were found to be upregulated. Overexpression of Topo I and DNA gyrase enhanced the nulceoid condensation in the presence of Dps. DNA-topology assays using the cell extract showed that the extracts from the Δfis and Topo I-/DNA gyrase-overexpressing strains, but not the wild-type extract, shifted the population toward relaxed forms. These results indicate that the topology of DNA is dynamically transmutable and that the topology control is important for Dps-induced nucleoid condensation. Introduction It is intriguing that very long genomic DNA molecules ranging from ∼cm to ∼m (corresponding to ∼106 to ∼109 base pairs) are stored in small containers with ∼μm in diameter. Two strategies have been taken in the evolution of life to accommodate the genomes in cells: in bacteria, the genomic DNA is packed in a cell as a form of 'nucleoid' (Robinow and Kellenberger, 1994; Poplawski and Bernander, 1997; Azam et al, 2000), whereas, in eukaryotic cells, the genomic DNA exists in a form of chromatin and is packed in a nucleus (Kornberg, 1974; Thoma et al, 1979; Widom and Klug, 1985). In either case, to organize the DNA into higher-order structures, a set of distinct structural DNA-binding proteins, such as histones in eukaryotic cells and Hu in bacteria, constitutively play major roles by utilizing the physical/chemical properties of DNA–protein interactions. A number of additional proteins, such as SMC (structural maintenance of chromosome) proteins and topoisomerases, also play crucial roles in the construction, maintenance and re-construction of well-organized higher-order structures of genomes (Hayat and Mancarella, 1995; Swedlow and Hirano, 2003). The formation of the higher order structures itself has a biological significance in the protection of the genomic DNA against the environmental stresses. In eukaryotes, DNA damage caused by UV light or oxidative stress accumulate less in nucleosomes than in naked DNA (Ljungman, 1991; Ljungman and Hanawalt, 1992; Yoshikawa et al, 2006). Furthermore, the formation of higher order chromatin dramatically decreases the DNA damage (Ljungman, 1991; Ljungman and Hanawalt, 1992). In bacteria, one of the proteins to transform the nucleoid into condensed state is Dps (DNA-binding protein from starved cells) (Almiron et al, 1992). Dps is a stress-induced protein with a molecular weight of 19 kDa and is known to be a member of the Fe-binding protein family that forms dodecameric complex in cells (Grant et al, 1998). Dps protects genomic DNA against oxidative stress (Martinez and Kolter, 1997), nuclease cleavage, UV light, thermal shock (Nair and Finkel, 2004) and acid (Choi et al, 2000), possibly by its DNA-binding ability to block the stress elements that attack DNA. Its Fe-chelating activity is also the important feature in the oxidative stress resistance, because iron (Fe2+) supplies an electron to produce the hydroxyl radicals via Fenton reaction (Zhao et al, 2002) and these radicals damage various critical macromolecules including the genomic DNA (Nunoshiba et al, 1999). Dps can reduce the intracellular level of Fe2+ and thus restricts the production of hydroxyl radicals (Zhao et al, 2002). Interaction between DNA and Dps results in a DNA–Dps co-crystal both in vitro and in vivo. In vitro, the mixture of naked DNA and Dps rapidly induces the crystalline state (Wolf et al, 1999; Frenkiel-Krispin et al, 2001). In Escherichia coli, Dps becomes the most abundant nucleoid component in the stationary phase (Azam and Ishihama, 1999) and causes a condensation of nucleoid (Wolf et al, 1999; Frenkiel-Krispin et al, 2001; Kim et al, 2004). Electron microscopy observations have revealed that the nucleoid in the stationary phase forms the biocrystal that is composed of the toroidally assembled Dps and DNA (Wolf et al, 1999; Frenkiel-Krispin et al, 2001, 2004). Even after the lysis of the cell, the nucleoid in the stationary phase is in a tightly condensed state (Kim et al, 2004). The effect of Dps on the nucleoid state can vary depending on the growth conditions or on the bacterial species. The overexpression of Dps in the log phase E. coli induces neither nucleoid condensation nor DNA–Dps co-crystallization (Frenkiel-Krispin et al, 2001), whereas Dps overexpression in the stationary phase caused nucleoid condensation (Kim et al, 2004). In contrast, the nucleoid in Staphylococcus aureus transforms its structure into a condensed state in both log and stationary phases by the induction of mrgA, a staphylococcal ortholog of dps (Morikawa et al, 2006). In addition, in S. aureus, the oxidative stress promotes the expression of MrgA and nucleoid condensation (Morikawa et al, 2006), whereas, in E. coli, as shown in this article, an induction of Dps by oxidative stress in the log phase did not result in the condensation of the nucleoid. Based on these differences between E. coli and S. aureus, we can postulate the presence of additional factors regulating the nucleoid condensation in E. coli. In this study, we show that Fis is the factor inhibiting the nucleoid condensation in the log phase. Fis is abundant in the log phase and is one of the major DNA-binding nucleoid proteins (Azam and Ishihama, 1999). It also acts as a transcription factor (Nilsson et al, 1990; Ross et al, 1990; Xu and Johnson, 1995; Hengen et al, 1997) to regulate a variety of genes. Our analyses show that Fis is one of the specific protein in gamma-Proteobacteria including E. coli and is not present in S. aureus, and that topoisomerase I (TopoI) and DNA gyrase, which are under the control of Fis in E. coli, facilitate the nucleoid condensation. Since Fis, Topo I and DNA gyrase can change the DNA superhelicity (Schneider et al, 1997; Schneider et al, 1999; Weinstein-Fischer et al, 2000), we propose that the control of DNA topology by these proteins is critical for the Dps-induced nucleoid condensation. Results Dps expression in E. coli Two different transcription factors are involved in the dps-gene regulation in E. coli (Figure 1A). First, IHF controls the accumulation of Dps in the stationary phase (Altuvia et al, 1994; Lomovskaya et al, 1994). Second, OxyR, an LysR-family transcription regulator, upregulates the dps gene under oxidative stress (such as in the presence of hydrogen peroxide (H2O2)) (Altuvia et al, 1994; Lomovskaya et al, 1994). Our Western blot analysis of wild-type (wt) E. coli showed that the levels of Dps expression in the stationary phase and under the oxidative stress were about 10 times and three times higher than that in the log phase, respectively (Figure 1B). On the other hand, in the Δdps strain lacking the dps gene, Dps was never detected under all the conditions examined (Figure 1B). In the ΔoxyR strain that lacks the oxyR gene, the expression of Dps stayed unchanged even after exposure to the oxidative stress, but was highly induced in the stationary phase (Figure 1B). A deletion of ihf (ΔhimD) diminished the induction of Dps during the transition into the stationary phase, but it still allowed the Dps induction under oxidative stress (Figure 1B). Thus, we conclude that the mechanisms of transcriptional regulation of dps gene are intact and physiologically functional in our experimental system. Figure 1.Dps expression under different growth conditions. (A) Schematic representation of the promoter region of the dps gene in E. coli. The IHF and OxyR binding sites exist upstream of the –35/–10 sequences (promoter of dps gene). (B) Western blot analyses against Dps in the wt (W3110) and the W3110 derived Δdps, ΔoxyR and ΔhimD strains, under oxidative stress (2 mM H2O2 treatment) in the log phase and in the stationary phase. Download figure Download PowerPoint Correlation between the Dps expression and the nucleoid condensation We have developed 'on-substrate lysis' procedure (see Materials and methods) for removing the cellular membrane and subsequent direct observation of the bacterial nucleoid and eukaryotic chromatin in cells by atomic force microscopy (AFM) (Yoshimura et al, 2003; Kim et al, 2004). This procedure can be applicable to evaluate the efficiency of nucleoid condensation in E. coli, because not-condensed nucleoid extends fibrous structures around the lysed cell, whereas the condensed nucleoid cannot release fibers upon lysis (Kim et al, 2004; Morikawa et al, 2006). In this study, to elucidate the regulatory factors involved in the Dps-induced nucleoid condensation in E. coli, we first assessed the degree of nucleoid condensation under various conditions that induced the Dps expression. After 'on-substrate lysis', the state of the nucleoid was observed by 4′,6-diamino-2-phenylindole (DAPI) staining. The number of DAPI-stained cells whose nucleoids remained compacted or spread out of the cell (termed 'lysed') was counted, and used as an indication of the degree of the nucleoid condensation. In the log phase, 95 and 93% of the wt and Δdps cells, respectively, were lysed and appeared to have a non-condensed nucleoid (Figure 2A, B and H). Close observations by AFM demonstrated the existence of nucleoid fibers around the lysed cells (Figure 2C and I). In the stationary phase, only 12% of the wt cells were lysed (i.e. condensed) (Figure 2 A, F and G). Although 44% of the Δdps cells were not apparently lysed (Figure 2A), dispersed nucleoid fibers were still observed in the Δdps strain (Figure 2L and M). Namely, the nucleoid becomes condensed during the transition into the stationary phase in a Dps-dependent manner. Figure 2.The lysis efficiencies and the nucleoid architectures of the wt, Δdps and ΔoxyR strains under three different conditions. (A) The efficiency of lysis. The number of lysed cells that dispersed fibers was divided by the total cell number and indicated as percent. The cells were observed by DAPI staining (B, D, F, H, J, L, N, P, R) or by AFM (C, E, G, I, K, M, O, Q, S). The numbers of the cells examined are following (each value separated by slash in parentheses represents the number of cells counted at each independent experiment); wt in log phase (218/154/64/34/54), wt under 2 mM H2O2 (235/77/51/135), wt in stationary phase (72/43/109), Δdps strain in the log phase (73/88/17/26), Δdps strain under 2 mM H2O2 (102/91/52), Δdps strain in the stationary phase (37/62/75), ΔoxyRs strain in the log phase (77/47/51/50), Δdps strain under 2 mM H2O2 (74/49/30/53/39), Δdps strain in the stationary phase (74/138/98/165/106). (B, C) wt in the log phase, (D, E) wt in the log phase treated with 2 mM H2O2, (F, G) wt in the stationary phase, (H, I) Δdps strain in the log phase, (J, K) Δdps strain in the log phase treated with 2 mM H2O2, (L, M) Δdps strain in the stationary phase, (N, O) ΔoxyR strain in the log phase, (P, Q) ΔoxyR strain in the log phase treated with 2 mM H2O2, (R, S) ΔoxyR strain in the stationary phase. Scale bars: 10 μm (B, D, F, H, J, L, N, P, R) and 500 nm (C, E, G, I, K, M, O, Q, S). Download figure Download PowerPoint In contrast, the oxidative stress given in the log phase induced the expression of Dps but did not condense the nucleoid. Eighty-five percent of the wt cells were efficiently lysed even after the induction of Dps by 2 mM H2O2, and the nucleoid fibers were observed around the lysed cells (Figure 2A, D and E). Negative control strains, Δdps and ΔoxyR strains, exhibited 81 and 72% efficiency of lysis after the treatment with H2O2, respectively (Figure 2A, J, K, P and Q). Therefore, we concluded that the induction of Dps by oxidative stress does not induce the nucleoid condensation in E. coli. This implies, in turn, the existence of a log phase specific factor(s) that inhibits the nulceoid condensation under oxidative stress. Role of Fis on the Dps-induced nucleoid condensation In E. coli, Fis is the most abundant nucleoid component in the log phase (∼60 000 molecules/cell), whereas its expression becomes undetectable at the stationary phase (Azam and Ishihama, 1999). When Dps is overexpressed in the wt (fis+) strain in the log phase, the nucleoid was not condensed (Kim et al, 2004). S. aureus, in which the expression of MrgA (Dps ortholog) is directly coupled with the nucleoid condensation (Morikawa et al, 2006), does not possess fis nor its homologous genes (Takeyasu et al, 2004). Therefore, we suspect Fis to be an inhibitory factor against the nucleoid condensation. To test this hypothesis, Dps was transiently overexpressed in the log phase of a Δfis strain of E. coli using a Dps-expression plasmid, and possible changes of the nucleoid structure were examined by DAPI staining and AFM observation. As expected, the induction of Dps by isopropyl-β-D-thiogalactopyranoside (IPTG) condensed the nucleoid even in the log phase, and the efficiency of cell lysis was decreased to 16% (Figure 3A, B and F) from the original value of 80% (Figure 3A and D). Close observations by AFM revealed that the Δfis strain exhibited the fibrous and condensed nucleoid in the absence and presence of Dps overexpression, respectively (Figure 3E and G). Figure 3.The lysis efficiency and the nucleoid architecture of the Δfis strain. (A) The efficiency of lysis. The number of lysed cells that dispersed fibers was divided by the total cell number and indicated as percent. The numbers of the cells examined are following (each value separated by slash in parentheses represents the number of cells counted at each independent experiment); the log phase (58/39/45/43), under 2 mM H2O2 (56/21/45/35), the stationary phase (125/59/77), the overexpression (76/50/74/40/29). (B) Overexpression of Dps by IPTG in the Δfis strain. The expression was detected by antibody against Dps. (C) Western blot against Dps in the Δfis strain under three different conditions. (D) DAPI image of the Δfis strain in the log phase. (E) AFM image of the Δdps strain in the log phase. (F) DAPI image of the Δfis strain under overexpression of Dps in the log phase. (G) AFM image of the Δfis strain under overexpression of DPS in the log phase. (H) DAPI image of the Δfis strain in the log phase treated with 2 mM H2O2, (I) AFM image of the Δfis strain in the log phase treated with 2 mM H2O2, (J) DAPI image of the Δfis strain in the stationary phase, (K) AFM image of the Δfis strain in the stationary phase. Scale bars: 10 μm (D, F, H, J) and 500 nm (E, G, I, K). Download figure Download PowerPoint We further investigated whether or not the nucleoid condensation could be induced in the Δfis strain by oxidative stress. Western blot analyses against Dps showed that a treatment of the Δfis strain with 2 mM H2O2 induced as much amount of Dps as in the wt treated with 2 mM H2O2 (Figure 3C). On-substrate-lysis treatment of the cells and the subsequent DAPI staining demonstrated that only 18% cells were lysed (Figure 3A and H). A close observation by AFM found that the nucleoid in the Δfis cells was condensed under H2O2 treatment (Figure 3I). In the stationary phase of the Δfis cells, Dps was normally expressed (Figure 3C), and the nucleoid was condensed (Figure 3A, J and K). Thus, we concluded that Fis is the inhibitory factor for the Dps-dependent nucleoid condensation in the log phase, but it does not participate in the regulation of the dps-gene expression. Effects of Topo I and DNA gyrase on the Dps-induced nucleoid condensation Fis is known to work as a transcription regulator for the topA, gyrA and gyrB genes, each coding for Topoisomerase I (Topo I), DNA gyrase subunit A (GyrA) and subunit B (GyrB), respectively (Schneider et al, 1999; Weinstein-Fischer et al, 2000). Indeed, our Northern blot analysis indicated that the expression of these genes were upregulated in the Δfis strain (Figure 4). Topo I is known to relax supercoiled DNA, whereas DNA gyrase causes negative supercoiling of DNA. Therefore, the repression of nucleoid condensation by Fis might be due to the changes in intracellular levels of Topo I, GyrA and GyrB, ultimately leading to the changes in DNA topology. This interpretation is in good agreement with the fact that the topology control of DNA is critical for achieving the higher-order structures of DNA (Yoshimura et al, 2000; Hizume et al, 2004; Hizume et al, 2005). We examined this possibility by monitoring the in vivo status of DNA topology in the wt and Δfis strain. Agarose gel electrophoresis containing chloroquine showed that, in the log phase, the population distribution of supercoiled plasmids was larger in the wt than in Δfis strain, being consistent with our interpretation (Supplementary Figure 1). Figure 4.Northern blot analyses of top A-, gyrA-, gyrB- and dps-mRNAs. The levels of (A) topA-, (B) gyrA- and (C) gyrB-mRNAs in the wt and Δfis strains. (D) The levels of dps-mRNAs in the wt cells in the log phase and in the log phase treated with 2 mM H2O2. (A–C) do not show tight bands, whereas (D) shows a single tight band. In bacteria, many mRNAs have been detected as broad smears by Northern blot analyses (Maruyama et al, 2003). Download figure Download PowerPoint We constructed a wt (fis+) strain that can transiently express His-tagged Topo I, as described in Materials and methods. The induction of His-Topo I expression by IPTG was evidenced by Western blot analysis (Figure 5A). In these His-Topo I expressing cells, the nucleoid was easily dispersed as in the wt cells (Figure 5C). In contrast, when Dps was induced by the treatment with 2 mM H2O2 after Topo I induction, the nucleoid became highly condensed (Figure 5D–F). In the cells expressing His-tagged DNA gyrase (GyrA and GyrB) (Figure 5B), the nucleoid without H2O2 was easily dispersed (Figure 5G), whereas the nucleoids were also condensed when treated with 2 mM H2O2 (Figure 5H–J). Figure 5.Nucleoid dynamics in Topo I- or DNA gyrase-overexpressing cells. The expression of His-tagged Topo I (A) or His-tagged DNA gyrase (B) was detected by an antibody against His-tag. (C) AFM image of Topo I-overexpressing cell in the log phase, (D–F) AFM images of Topo I-overexpressing cells treated with 2 mM H2O2, (G) AFM image of DNA gyrase-overexpressing cell in the log phase, (H–J) AFM image of DNA gyrase-overexpressing cells treated with 2 mM H2O2. Scale bars: 500 nm. Download figure Download PowerPoint Cell extracts from Δfis, Topo I++ and DNA Gyrase++ cells control DNA topology The expression of gyrA and gyrB is induced by the decrease in negative supercoil in cells (Menzel and Gellert, 1987; Peter et al, 2004), and the increase in negative supercoil facilitates topA expression (Menzel and Gellert, 1983; Mizushima et al, 1993; Ogata et al, 1994). Thus, DNA topology can be controlled by Topo I and GyrA/GyrB in cells. Northern blot analyses indicated that the overexpression of GyrA and GyrB upregulated the expression level of Topo I, but that the overexpression of Topo I did not change the amount of gyrA/gyrB mRNA in our experimental system (Figure 6A–C). In any case, Topo I and DNA gyrase coexist in these overexpressing cells. Figure 6.Northern blot analyses of topA-, gyrA-, gyrB- and fis-mRNAs under the overexpression of Topo I and DNA gyrase. The levels of (A) topA-mRNA in DNA gyrase++, (B, C) gyrA- and gyrB-mRNAs in Topo I++, and (D) fis-mRNA in Topo I++ or DNA gyrase++. Download figure Download PowerPoint To clarify the biological significance, we further examined how efficiently different cell extracts derived from the wt, Δfis and Topo I-/DNA gyrase-overexpressing strains (Topo I++ and Gyrase++) could change the topology of DNA in vitro. The agarose gel electrophoresis containing chloroquine showed that, whereas wt extract never changed the population of topoisomers of DNA, the extract from Δfis, Topo I++ and Gyrase++ changed the population toward relaxed form (Figure 7A). These results indicate that the topology of DNA is dynamically transmutable in the Δfis, Topo I++ and Gyr ase++ strains, but not in the wt strain. Also, it is interesting to note that the cell extract from Gyrase++ had a very similar effect to that from Topo I++. It seems that the effect of Topo I in the extract predominate over the effect of DNA gyrase, and the effects of DNA gyrases in the extract may be very subtle and involved a local topology control. Indeed, when the extract from Gyrase++ was added to the extract from Topo I++, the topoisomers were not shifted toward supercoiling but toward relaxed form (Figure 7B, also see Supplementary Figure 2). Figure 7.DNA topology assay using Δfis, Topo I++ and Gyrase ++ cell extracts. (A) The plasmid DNA (pBSII SK−) was incubated with cell extracts prepared from wt, fis-deletion cells (Δfis), Topo I-overexpressed cells (Topo I ++) and DNA gyrase-overexpressed cells (Gyrase ++) for 15 min at 37°C, and then loaded onto agarose gels containing 2.5 mg/ml chloroquine. (B) The extract from Gyrase++ was added to the extract from Topo I++, and was incubated with pBSII for 15 min at 37°C. The electrophoresis was performed without chloroquine. M represents the marker DNA (λ DNA digested by HindIII (BioLabs)), and the numbers above the gels indicate the amounts of proteins in each cell extract (ng). The graphs represent microdensitometric tracings corresponding to the lanes. Download figure Download PowerPoint It is well known that DNA gyrase requires ATP and Mg2+ for its action. Consequently, we examined the effect of ATP (1∼10 mM) and Mg2+ (1∼100 mM) in the DNA gyrase++ cell extracts on the DNA topology. Unexpectedly, the populations of topoisomers were shifted toward the relaxed forms in a similar extent to the effect in the absence of ATP and Mg2+ (data not shown). It seems that the effect of Topo I predominates over the effect of DNA gyrase. Discussion We have previously reported that, in S. aureus, the induction of Dps ortholog, MrgA, by oxidative stress induced a nucleoid condensation. The Dps-dependent nucleoid condensation was postulated as the genome protection system against oxidative stress. However, in this study, we demonstrated that the oxidative stress did not simply lead to the nucleoid condensation in E. coli due to the effects of Fis as the regulator of the topA and gyrA/B gene expression that is required for the control of DNA topology. DNA topology maintenance by Fis, Topo I and DNA gyrase Fis preferably binds to the 15 bp consensus sequence, but when it exists in excess amount, its DNA-binding becomes sequence nonspecific. In vitro, Fis changes the overall shape of supercoiled DNA in a sequence-independent manner (Schneider et al, 2001; Hardy and Cozzarelli, 2005), and prevents the topological changes caused by DNA gyrase and Topo I (Schneider et al, 1997). In addition, our results showed that Fis repressed the expressions of gyrA, gyrB and topA (Figure 4). These facts suggest that Fis plays a role to sustain the DNA superhelicity at the steady-state level using two different pathways: one is acting as a physical barrier and buffering the change in the DNA superhelicity, and the other is repressing the expression of the factors that change the DNA superhelicity (Figure 8A). In fact, the cell extract from the wt (fis+) strain could not affect the population distribution of topoisomers, whereas the cell extract from the Δfis strain changed the population distribution of the topoisomers (Figure 7). Figure 8.A model of the topology control of genome DNA required for the Dps-dependent nucleoid condensation. (A) In the presence of Fis, DNA topology is maintained statically by the repression of Topo I and DNA gyrase expressions, and also by the physical blocking against the topological changes of DNA topoisomers. Therefore, the expression of Dps does not lead to a nucleoid condensation. (B) In the absence of Fis, Topo I and DNA gyrase are induced and may easily change the DNA topology. Thus DNA topology is dynamic. This status allows the nucleoid to be condensed under the expression of Dps (Topo I and DNA gyrase coexist). (C) Overexpression of Topo I or DNA gyrase may overcome the barrier of Fis, cause the DNA topology dynamic, and facilitate the nucleoid condensation as in (B). Download figure Download PowerPoint The DNA topology should be always balanced in a cell. In this sense, all of the following may be critical: (i) direct binding of Fis to DNA and forming a physical barrier (Schneider et al, 1997, 2001; Hardy and Cozzarelli, 2005); (ii) controlling gene expression including topA and others (Schneider et al, 1999; Weinstein-Fischer et al, 2000); (iii) ionic environment that directly affect DNA compaction (Iwataki et al, 2004). In the Δfis strain, DNA gyrases and Topo I actively change the DNA superhelicity without the barrier of Fis, and the topology of DNA turns into the dynamic state, in which the expression of Dps would easily results in the nucleoid condensation (Figure 8B). This is the case for S. aureus, whose MrgA expression and the nucleoid condensation are completely correlated (Morikawa et al, 2006). Database search showed that S. aureus lacks fis gene (Table I), but possess the orthologs of topA, gyrA and gyrB. Namely, the nucleoid dynamics in the wt
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