Actin homolog MreB affects chromosome segregation by regulating topoisomerase IV in Escherichia coli - PubMed (original) (raw)
Actin homolog MreB affects chromosome segregation by regulating topoisomerase IV in Escherichia coli
Ram Madabhushi et al. Mol Cell. 2009.
Abstract
In Escherichia coli, topoisomerase IV, a type II topoisomerase, mediates the resolution of topological linkages between replicated daughter chromosomes and is essential for chromosome segregation. Topo IV activity is restricted to only a short interval late in the cell cycle. However, the mechanism that confers this temporal regulation is unknown. Here we report that the bacterial actin homolog MreB participates in the temporal oscillation of Topo IV activity. We show that mreB mutant strains are deficient in Topo IV activity. In addition, we demonstrate that, depending upon whether it is in a monomeric or polymerized state, MreB affects Topo IV activity differentially. In addition, MreB physically interacts with the ParC subunit of Topo IV. Together, these results may explain how dynamics of the bacterial cytoskeleton are coordinated with the timing of chromosome segregation.
Figures
Figure 1. Dumbbell-shaped Nucleoids Are Intermediates of Chromosome Segregation
Compact nucleoids were purified from C600 and C600_parC1215_, visualized by fluorescence microscopy after staining with DAPI, and the prevalence of dumbbells was assessed. Approximately 300 nucleoids were examined for each experiment. (A) and (B) Two morphologically distinct classes are present in nucleoids purified from E.coli cells: (A) Singlets and (B) dumbbells. (C) Dumbbell-shaped nucleoids are enriched in parC mutants and can by resolved by Topo IV in vitro.
Figure 2. mreB Mutant Strains Are Deficient in Topo IV Activity
Compact nucleoids were isolated from the indicated strains, visualized by DAPI staining, their areas were measured, and the prevalence of dumbbells was assessed. Approximately 300 nucleoids were examined for each experiment. (A) Distribution of nucleoid sizes in preparations from either wild-type (wt) or Δmre cells that either had or had not been treated with Topo IV; as well as from wild-type cells where MreB had been overexpressed. (B) Incidence of dumbbell-shaped nucleoids from (A). (C) Nucleoid size distribution after Topo IV overexpression in Δmre strains. (D) Nucleoids isolated from PA340(pTK509) after 2 h of induction of the ectopic mreBD165V gene. (E) Nucleoids from (D) after treatment with Topo IV (F) Size distribution of nucleoids from (D) and (E).
Figure 3. MreB Specifically Inhibits Decatenation of kDNA by Topo IV
kDNA decatenation reaction mixtures were incubated with either 0.5 nM Topo IV or 30 nM gyrase and the indicated amounts of MreB for 4 min at 37 °C. The reaction products were electrophoresed through 1.4% agarose gels, stained with SYBR Gold, and quantified as described under Experimental Procedures. The amount of kDNA minicircles produced by Topo IV or Gyrase in the absence of MreB was assigned a value of 1 and decatenation in the presence of MreB was expressed relative to this number. (A) and (B) Effect of increasing concentrations of MreB on kDNA decatenation catalyzed by (A) Topo IV and (B) gyrase. (C) Quantification of data shown in panels (A) and (B). Shown is an average of three measurements each for Topo IV and gyrase. m.c., minicircles; sc. m.c., supercoiled minicircles.
Figure 4. Analysis of MreB Polymerization Under kDNA Decatenation Conditions
(A) MreB polymerization as a function of total monomer concentration. MreB was diluted to the indicated concentrations in kDNA reaction buffer and incubated at 37°C for either 30 min or overnight. Light scattering counts were then measured as described in Experimental Procedures. The inset gives an exploded view of the results for 6.25 nM – 100 nM MreB. (B) Kinetics of MreB polymerization. MreB was diluted to a concentration of 200 nM in kDNA reaction buffer and incubated at 37 °C for the indicated times. Light scattering counts were then measured as in (A). (C) – (H) Electron microscopic ananlysis of MreB filaments. MreB was diluted to a concentration of either 100 nM (D and E), 1 μM (C, F, and G), or 4 μM (H) in kDNA reaction buffer, incubated at 37 °C for either 5 min (C) or 30 min (D–H), and filaments were visualized by electron microscopy as described in Experimental Procedures. (I) Distribution of filament length at 100 nM, 1 μM, and 4 μM MreB after 30 min of polymerization at 37 °C. About 40 filaments were measured at 100 nM and about 200 filaments were measured at 1 μM and 4 μM MreB respectively.
Figure 5. The Oligomeric State of MreB Determines its Effect on Topo IV Decatenation Activity
(A) Two sets of reactions were performed. In one set (lanes marked “M”), MreB was titrated as in Figure 3. In the second set (lanes marked “P”), the indicated amounts of MreB were pre-incubated with the kDNA reaction mix for 30 min at 37 °C prior to the addition of Topo IV. (B) Quantification of (A). Relative activity was measured as in Figure 3.
Figure 6. MreB Physically Interacts with ParC
HA-ParC (A) or HA-GyrA (B) was incubated with an eight-fold excess of the indicated proteins and analyzed as described under Experimental Procedures. MreB marker lanes are direct loads of the protein to the gel. h.c., IgG heavy chain; l.c., IgG light chain.
Figure 7. Interaction between MreB and Topo IV May Help Coordinate Late Cell Cycle Events
(1) Topo IV activity is spatially and temporally regulated during the bacterial cell cycle. (2) Active Topo IV is assembled only late in the cell cycle. Meanwhile, remodeling of MreB filaments during the cell cycle causes the accumulation of a ring of polymerized MreB at the cell center. (3) Polymerized MreB stimulates the decatenation activity of Topo IV. (4) MreB filaments are further remodeled to facilitate their segregation to the two daughter cells. This remodeling produces monomeric MreB. (5) Inhibition of Topo IV activity by monomeric MreB restores a state of low Topo IV activity at the onset of a new cell cycle.
References
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