Identification of chromosomal SlmA-binding sites. (A) Circular diagram of the E. coli chromosome with approximate locations of SlmA-binding sites shown as blue lines. Green, red, dark- and light-orange colored regions correspond to the Ori, Ter, Left, and Right macrodomains (MDs), respectively (26). (B) Sequence logo of consensus SlmA-binding sequence generated using weblogo. (C) The indicated concentrations of SlmA were incubated with unlabeled poly-dIdC (100 μg/mL) and 0.2 nM [³²P]-labeled SBS17-containing DNA (100 bp). The SBS17 site was centered within the probe fragment and flanked by adjacent chromosomal sequence. Protein–DNA complexes were separated from free probe by gel electrophoresis and detected using a phosphorimager. We do not currently know why an intermediate shifted complex is observed.

FtsZ GTPase activity is required for SlmA–SBS complexes to disrupt polymerization. (A) FtsZ GTPase activity measured at 18 °C by hydrolysis of α-[³²P]-GTP. Reactions contained 5 mM GTP, 5 μM FtsZ, 10 μM SlmA, or SlmA(R73D) and 30 bp SBS17 fragments (2 μM) as indicated. Results are the average of triplicate measurements and error bars show the SD. Reaction rates (molecules GTP hydrolyzed/min/FtsZ molecule) are listed. Due to overlap, not all lines are readily visible in the graph. (B) Sedimentation assays using FtsZ(D212N) were performed as in Fig. 1A. SlmA(WT) or its variants were included in the reactions along with 30 bp of SBS17 dsDNA as indicated.

SlmA is an FtsZ polymerization antagonist. (A) FtsZ polymerization assays. Purified FtsZ (5 μM) was incubated in polymerization buffer (50 mM PIPES, pH 6.7, 10 mM MgCl₂, 200 mM KCl) at room temperature for 20 min with or without SlmA(WT) or SlmA variants (5 μM) as indicated. GTP (5 mM) was then added and FtsZ polymers were sedimented by ultracentrifugation. Proteins found in the resulting pellet (P) and supernatant (S) fractions were separated on an SDS-polyacrylamide gel and visualized by Coomassie brilliant blue staining. (B) NO defect of slmA missense alleles. TB57 [P_ara::minCDE] cells and its derivatives with the indicated slmA allele were grown overnight in M9-arabinose medium. The resulting cultures were serially diluted in LB (1% NaCl), 5 μL of each dilution was spotted on LB (1% NaCl) agar lacking arabinose, and the plate was incubated at 30 °C overnight. (C–E) Localization of SlmA variants. Cells of HC246 [ΔslmA] with the integrated expression constructs (attHKTB99) [P_lac::gfp-slmA] (C), (attHKHC482) [P_lac::gfp-slmA(R73D)] (D), or (attHKHC505) [P_lac::gfp-slmA(T33A)] (E) were grown to an OD₆₀₀ of 0.5 in LB with 250 μM IPTG and visualized using GFP (C1, D1, and E1) and DIC (C2, D2, and E2) optics.

SBS binding activates SlmA. (A) Sedimentation assays were performed as in Fig. 1 A–B, except lower levels (250 nM) of SlmA were used and the reactions also contained 30 bp of WT or mutant SBS17 dsDNA fragments (50 nM) as indicated. (B and C) FtsZ polymers were formed in reactions containing FtsZ (2 μM) and GTP (2 mM) without (B) or with (C) the addition of SlmA (2 μM) and 30 bp of SBS17 dsDNA (400 nM). Reactions were spotted onto carbon-coated grids 5 min after GTP addition, negatively stained with uranyl formate, and visualized by electron microscopy. (Scale bar, 100 nm.)

Model for FtsZ regulation by SlmA. (A) In newborn cells, MinCD and SlmA prevent Z-ring assembly at the cell poles and over the nucleoid, respectively. For simplicity, MinCD oscillation is not shown. As the chromosome is replicated, SlmA bound to chromosomal SBSs segregates with the origin-proximal regions of the daughter chromosomes. This leaves the midcell zone free of inhibitors so that the Z ring can form as replication nears completion. (B) SlmA free in the cytoplasm is monomeric and inactive. Binding to SBSs stimulates the ability of SlmA to interact with FtsZ polymers. Upon associating with polymers, it promotes their breakdown. We propose it does so by disrupting FtsZ–FtsZ interactions at interfaces where GTP has been hydrolyzed to GDP. In our model, DNA binding activates SlmA in part by promoting dimerization. It is also possible that higher order oligomers are formed and that they further enhance SlmA activity.