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, 18 (5), 555-64

SlmA, a Nucleoid-Associated, FtsZ Binding Protein Required for Blocking Septal Ring Assembly Over Chromosomes in E. Coli

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SlmA, a Nucleoid-Associated, FtsZ Binding Protein Required for Blocking Septal Ring Assembly Over Chromosomes in E. Coli

Thomas G Bernhardt et al. Mol Cell.

Abstract

Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.

Figures

Figure 1
Figure 1. The slmA (ttk) Locus and Phenotypes of slmA Mutants
(A–D) DIC micrographs showing cells of TB43/pTB8 [ΔlaclZYA::frtΔminCDE::frt/Plao::minCDE::JacZ] (A and B) and its slmA127 derivative (C and D). Cells were grown to OD600 = 0.6–0.7 in liquid LB-Amp with (A and C) or without (B and D) 500 µM IPTG and fixed. Bar = 5 µm. (E) Diagram of the slmA locus indicating the location and orientation of the slmA127 and slmA267 EZTnKan-2 insertions (black triangles, inserted after bp 313 or 88 of slmA, respectively). SwissProt annotation P06969 was used to number amino acid residues. Potential DNA binding (HTH) and coiled-coil (CC) regions are indicated. The latter was identified by using COILS (Lupas et al., 1991). The double-headed arrow indicates the endpoints of the ΔslmA::aph and ΔslmA::frt deletion-replacement alleles. (F) Overnight cultures of TB28 [wt], TB57 [Para::minCDE], TB66 [slmA127], and TB68 [slmA127 Para::minCDE] were grown to equal density in LB-0.2% arabinose and serially diluted (10−1-10–6) in LB. Aliquots (5 µl) were spotted on LB agar without arabinose and incubated overnight at 30°C.
Figure 2
Figure 2. Septal Ring and Nucleoid Positioning in SlmA and SlmA Min Mutants
Shown are representative cells of the following λCH151 [Plac::zipA-gfp] lysogens: TB28 [wt] (A), TB85 [ΔslmA::aph] (B), TB43 [ΔminCDE::frt] (C), TB86 [ΔslmA::aph ΔminCDE::frt] (D), TB43/pTB63 (E), and TB86/pTB63 (F). Plasmid pTB63 [ftsQAZ] is a low-copy, pSC101-derivative expressing ftsQAZ from native promoters. Cells were grown to OD600 = 0.6–0.7 at 30°C in LB with 37 µM IPTG, fixed, stained with DAPI (0.25 µg/ml), and imaged with optics specific for GFP (A1, B1, C1, D1, E1, and F1), DAPI (A2, B2, C2, D2, E2, and F2), and DIC (A4, B4, C4, D4, E4, and F4). A3, B3, C3, D3, E3, and F3 show a digital overlay of the GFP and DAPI signals. Arrowheads point to examples of ZipA-GFP rings, and other types of accumulations, in regions with significant DAPI staining. Bar = 2 µm.
Figure 3
Figure 3. Nucleoid Cutting in SlmA DnaA Cells
Cells of TB104 [cI857 λPR::dnaA] (A) and TB105 [ΔslmA::frt cI857 λPR::dnaA] (B and C) were grown in LB for 3.5 hr at 30°C to deplete DnaA. Cells were fixed, stained with DAPI, and imaged with DAPI- and DIC-specific optics. A1, B1, and C1 show a digital overlay of the DIC and DAPI images, and A2, B2, and C2 show the DIC image only. Bar = 2 µm. Several parameters of randomly selected cells from each strain were measured, and the results are summarized in (D).
Figure 4
Figure 4. Distribution of GFP-SlmA on the Nucleoid
Shown are live cells of TB85(HKTB99) [ΔslmA::frt (Plac::gfp-slmA)] grown to OD600 = 0.5–0.6 at 30°C in LB with 250 µM IPTG. DAPI was added to 0.25 µg/ml 30 min prior to imaging. Cells in (F) and (G) were treated with chloramphenicol (100 µg/ml) and grown for an additional 30 min prior to viewing. Panels show GFP-SlmA (1), DAPI (2), merged (3), and DIC (4) images. Bar = 2 µm.
Figure 5
Figure 5. slmA Overexpression Blocks Z Ring Formation
Shown are cells of strain TB28/pDB407 [wt/λPR::gfp-ftsZ] harboring pJF118EH [vector] (A), pTB67 [Ptac::slmA] (B), pDR144 [Plac::sfiA] (C), or pTB108 [Ptac::slmA(58–198)-h] (E). D1–D4 show a cell of strain TB28/pLL14/pTB67 [wt/λPR::gfp-minC(141–231)/Ptac::slmA]. Cells were grown to OD600 = 0.25–0.3 at 39°C in LB-Amp-Spc. DAPI (to 0.25 µg/ml) and IPTG (to 50 µM) were added, and growth was continued for 30 min prior to imaging. Cells were imaged live, and panels show GFP (1), DAPI (2), merged (3), and DIC (4) images. Bar = 2 µm. Note that continued incubation of the cells in panels B1–E4 led to the formation of very long nonseptate filaments, indicative of a complete division block.
Figure 6
Figure 6. Formation of FtsZ-SlmA Ribbons
(A–D) 90° angle light scattering was monitored in reactions containing 3 µM FtsZ before and after the addition of HT-SlmA storage buffer and GTP (1 mM) (A), HT-SlmA (0.3 µM) and GDP (1 mM) (B), HT-SlmA (0.3 µM) and GTP (1 mM) (C), and HT-YcdC (0.3 µM) and GTP (1 mM) (D). (E–H) Electron micrographs of structures formed in reactions containing 3 µM FtsZ and 1 mM GTP with the addition of 0.6 µM HT-SlmA (E and F), 0.6 µM HT-YcdC (G), or HT-SlmA storage buffer (H). Bar = 500 nm (E) or 200 nm (F–H). (I)SDS-PAGE analysis of supernatant (S) and pellet (P) fractions after centrifugation of reactions containing nucleotide (1 mM) and proteins (6 µM each) as indicated. Note that the pellet fractions were twice as concentrated as the supernatant fractions (see Experimental Procedures).
Figure 7
Figure 7. Model for SlmA Function
DNA bound SlmA, presumed to be a dimer, competes with membrane bound septal ring components, such as ZipA and FtsA, for binding FtsZ polymers. In subsequent steps, SlmA may play a rather passive role in that SlmA bound polymers may simply turn-over due to their intrinsic GTP hydrolytic activity. A more attractive possibility is that SlmA, perhaps in combination with some other factor (X), actively promotes the disassembly of the FtsZ polymers.

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