Dimerization or oligomerization of the actin-like FtsA protein enhances the integrity of the cytokinetic Z ring

Abstract

In bacteria, the actin-like FtsA protein interacts with the tubulin-like FtsZ protein, helping to assemble the cytokinetic Z ring, anchor it to the cytoplasmic membrane and recruit other essential divisome proteins. FtsA also interacts with itself, but it is not clear whether this self-interaction is required for its full functionality. Here we describe new dominant negative missense mutations in Escherichia coli ftsA that specifically inhibit FtsA homodimerization and simultaneously cause disruption of Z rings. The negative effects of one mutation, M71A, were suppressed by altering levels of certain division proteins or by additional mutations in ftsA that promote increased integrity of the Z ring. Remarkably, when FtsA, FtsA-M71A, and other mutants of FtsA that compromise self-interaction were connected in a tandem repeat, they were at least partially functional and suppressed defects of an ftsZ84(ts) mutation. This gain of function by FtsA tandems further suggested that FtsA monomers cause deleterious interactions with FtsZ and that increased dimerization or oligomerization of FtsA enhances its ability to promote Z-ring integrity. Therefore, we propose that FtsZ assembly is regulated by the extent of FtsA oligomerization.

Fig. 1. FtsA-M71A has a dominant negative phenotype

(A) Mapping of Met71 of E. coli FtsA on the crystal structure of T. maritima FtsA. (B) Complementation of WM1115 (ftsA12ts) by FtsA (pWM2669) or FtsA-M71A (pWM2671) at 42°C. Each strain was grown at 30°C for 4 h in the absence of IPTG prior to spotting on plates, which contained indicated amounts of IPTG and were incubated at 42°C overnight. (C) DIC micrographs of WM1074 cells carrying pWM2780 (vector), pWM2669 (FtsA), or pWM2671 (FtsA-M71A) in the presence of the indicated concentrations of IPTG (mM). Scale bar is 10 μm. (D) Immunoblot of WM1074 cells pWM2780 (vector), pWM2669 (FtsA), or pWM2671 (FtsA-M71A) grown with the indicated concentrations of IPTG (mM) was probed with anti-FLAG (M2) (top) and anti-FtsZ (bottom).

Fig. 2. Inhibition of Z-ring assembly by overproduction of FtsA or FtsA-M71A

FtsZ (green) and nucleoids (red) were visualized by immunofluorescence and DAPI staining of WM1074 cells carrying pWM2669 (FtsA) (A-C) or pWM2771 (FtsA-M71A) (D, E) in the presence of the indicated concentrations of IPTG. Localization of the Z rings in WM1074 cells treated without (F) or with (G) cephalexin. Representative cells and widths of Z rings are shown at the right. Scale bar = 10 μm.

Fig. 3. Suppression of toxicity caused by overproduction of FtsA-M71A

(A) Colony viabilities of WM1074 (ftsA⁺), WM1659 (ftsA*) or WM2004 (ftsA⁺ ΔzapA) carrying either pWM2780 (vector), pWM2669 (FtsA), or pWM2671 (FtsA-M71A) in the presence of the indicated concentrations of IPTG. Plates were incubated at 30°C overnight. (B) Colony viabilities of WM1074 carrying pWM2671 (FtsA-M71A) plus either the compatible plasmids pKG110, pWM2765 (FtsZ), pWM3073 (ZipA), pWM3074 (ZapA) or pWM3075 (GFP-FtsN) in the presence of 0.2 mM IPTG (to induce production of FtsA-M71A) and the indicated concentrations of sodium salicylate (NaSal) to induce production of the other cell division proteins. (C) DIC micrographs of the same strains described in (B) in the presence of 0.1 mM IPTG and the indicated concentrations of sodium salicylate (NaSal). Scale bar = 10 μm. (D) Colony viabilities of WM1115 (ftsA12ts) carrying either pWM2780 (vector), pWM2669 (FtsA), pWM2671 (FtsA-M71A), pWM2781 (FtsA*), pWM2782 (FtsA-M71A+A*), pWM3077 (FtsA-I143L), pWM3078 (FtsA-M71A+I143L), pWM2702 (FtsA-E124A), or pWM3076 (FtsA-M71A+E124A) as described for Fig. 1B.

Fig. 4. Mapping of E. coli FtsA-M71, E69 and intragenic missense suppressors of M71A on the three-dimensional structure of T. maritima FtsA

Front view (A) and rotated view (B). Each domain is shown in a different color: 1A (blue), 1C (yellow), 2A (red), and 2B (green). Alpha helix H1 is shown in orange. Strands S6, S7, S12, and S13 are highlighted by arrows.

Fig. 5. FtsA tandems are functional in vivo

(A) Schematic illustration of an FtsA tandem repeat (AA), which has an FLAG tag at the N-terminus of AA and a Ser-Arg dipeptide between FtsA subunits. (B) Complementation of WM1115 (ftsA12ts) by FtsA (pWM2669), AA (pWM3079), WT-71 (pWM3080), 71-WT (pWM3081), 71-71 (pWM3082), Δ1C-71 (pWM3083), Δ1C-Δ1C (pWM3084), or 69-69 (pWM3085), as described for Fig. 1B. (C) Immunoblots of representative cultures used for the spot assay (B) were probed with anti-FLAG (M2). Both blots show cultures producing AA (~88 kDa) as controls. (D) Immunoblots of WM1115 carrying pWM2785 (FtsA), pWM2777 (AA), pWM3036 (Δ1C-Δ1C), or pWM3086 (69-69) in the presence and absence of 1 mM IPTG were probed with anti-FLAG (M2).

Fig. 6. FtsA tandems support growth of WM1125 (ftsZ84) at 42°C

(A) Colony viabilities of WM1125 (ftsZ84ts) carrying either pWM2780 (vector), pWM2669 (FtsA), pWM2781 (FtsA*), pWM3079 (AA), pWM3082 (71-71), or pWM3084 (Δ1C-Δ1C) as described for Fig. 1B. (B) Distribution of lengths of WM1125 cells producing AA without IPTG (open bars) or with 0.1 mM IPTG (filled bars). (C-G) DIC micrographs of representative cells of WM1125 producing AA in the presence of 0.1 mM IPTG: normal (C), filaments and minicells (D), bent (E), branched (F), and twisted (G). (H-I) Localization of Z rings in WM1125 producing AA in the absence (H) and presence of 0.1 mM IPTG (I). FtsZ (green) was visualized by immunofluorescence microscopy, and nucleoids (pseudocolored red) by DAPI staining. Arrowheads highlight FtsZ polar localization. Scale bar = 10 μm.

Fig. 7. Functionality of FtsA mutant tandems with defects in binding to FtsZ and/or recruitment of later divisome proteins

(A) Complementation of WM1115 (ftsA12ts) by FtsA (pWM2785), Δ1C (pWM2789), R300E (pWM3121), AA (pWM2777), Δ1C-WT (pWM3034), Δ1C-Δ1C (pWM3036), 300-300 (pWM3122), 300-WT (pWM3123), 300-Δ1C (pWM3124), or (300+Δ1C)-WT (pWM3125, as described for Fig. 1B. (B) An immunoblot of representative cultures used for the spot assay (A) probed with anti-FLAG (M2). (C) Suppression of WM1125 (ftsZ84ts) carrying either pWM2784 (vector), pWM2785 (FtsA), pWM2777 (AA), pWM3036 (Δ1C-Δ1C), pWM3122 (300-300), pWM3123 (300-WT), pWM3124 (300-Δ1C), or pWM3125 [(300+Δ1C)-WT] as described for Fig. 1B.

Fig. 8. FtsA dimerization/oligomerization and cell division

(A) Proposed interaction of FtsA dimers/oligomers with FtsZ and the Z ring. FtsZ protofilaments (speckled ovals) initially form a scaffold for the binding of FtsA monomers (white ovals). Subsequently, FtsA dimerizes and/or oligomerizes, crosslinking FtsZ protofilaments and increasing the integrity of the Z ring. (B) Model of FtsA dimers (white ovals) and their potential interactions with FtsZ and later divisome proteins. Residue R300 is required for binding to FtsZ (speckled ovals), and domain 1C is required for recruitment of late proteins (hatched ovals). The ability of each potential FtsA dimer to promote cell division of an ftsA(ts) or ftsZ(ts) mutant is shown below.