Dynamic state of DNA topology is essential for genome condensation in bacteria

Abstract

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 Deltafis strain, both topA and gyrA/B genes were found to be upregulated. Overexpression of Topo I and DNA gyrase enhanced the nucleoid condensation in the presence of Dps. DNA-topology assays using the cell extract showed that the extracts from the Deltafis 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.

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.

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).

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).

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).

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.

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⁺⁺.

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.

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).