Z-ring-independent interaction between a subdomain of FtsA and late septation proteins as revealed by a polar recruitment assay

Abstract

FtsA, a member of the ATPase superfamily that includes actin and bacterial actin homologs, is essential for cell division of Escherichia coli and is recruited to the Z ring. In turn, recruitment of later essential division proteins to the Z ring is dependent on FtsA. In a polar recruitment assay, we found that FtsA can recruit at least two late proteins, FtsI and FtsN, to the cell poles independently of Z rings. Moreover, a unique structural domain of FtsA, subdomain 1c, which is divergent in the other ATPase superfamily members, is sufficient for this recruitment but not required for the ability of FtsA to localize to Z rings. Surprisingly, targeting the 1c subdomain to the Z ring by fusing it to FtsZ could partially suppress a thermosensitive ftsA mutation. These results suggest that subdomain 1c of FtsA is a completely independent functional domain with an important role in interacting with a septation protein subassembly.

FIG. 1.

Effects of a deletion of subdomain 1c on FtsA function. (A) At the top are two linear schematics of FtsA-GFP and FtsAΔ1c-GFP fusions, showing restriction sites used in their construction. Black = deleted part of the 1c subdomain; gray = all of subdomain 1c; white = rest of FtsA and GFP. Restriction sites are shown below, in addition to the amino acid residues at the boundary of the deleted region of subdomain 1c in FtsAΔ1c. Below the schematics, the Thermotoga crystal structure of FtsA is shown, with subdomain 1c in yellow and the portion of this domain deleted to make FtsAΔ1c shown in blue. (B to E) Cells expressing FtsA-GFP from pWM633 (B and D) or FtsAΔ1c-GFP from pWM1909 (C and E) were grown in 40 μM IPTG for 4 h, and live cells were examined by fluorescence (B and C) or Nomarski (D and E) microscopy. (F) Complementation of an ftsA null strain (CH2/pDB280) by FtsA but not by FtsAΔ1c. CH2/pDB280 cells containing no extra plasmid, pET28-FtsA, or pET28-FtsAΔ1c were grown at 30°C, diluted 1:10, spotted onto LB plates, and incubated at the temperatures shown. At 42°C, the Cm^r pDB280 plasmid containing ftsA was lost, depleting cellular FtsA. Bar, 5 μm.

FIG. 2.

Localization of FtsA to the cell poles targets other cell division proteins. Cells were induced with arabinose (E), IPTG (C and D), or arabinose plus IPTG (A, B, and F to J) and examined by fluorescence microscopy. (A and C) DivIVA-FtsA(pWM1806) plus GFP-FtsI (in WM1488). (B and D) DivIVA-FtsA(pWM1806) plus GFP-FtsN (pWM1152) in TX3772. (E) DivIVA-GFP(pWM1461) in Top10. (F and G) IFM staining for FtsZ on cells used for panel B (Nomarski [F] or fluorescence [G]). (H and I) DivIVA-FtsA(pWM1806) plus FtsZ-GFP in TX3772 (Nomarski [HN] or fluorescence [I]). (J) DivIVA-FtsA(pWM1806) plus GFP-FtsQ(pWM1800) in TX3772. (K and L) Fluorescence intensity profiles along the length of a representative cell from panel E (K, light line), panel G (K, heavy line), panel B (L, light line), and panel J (L, heavy line). Arrows in panel H highlight visible inclusion bodies. Bar, 5 μm.

FIG. 3.

Polar recruitment of FtsN and FtsI by subdomain 1c of FtsA. Individual or representative fields of cells examined for GFP fluorescence are shown. (A and B) IFM, using anti-DivIVA, of cells induced with IPTG plus arabinose, expressing DivIVA-1c (pWM1814) plus GFP-FtsI (in WM1488) (A) or DivIVA-1c (pWM1814) plus GFP-FtsN (pWM1152) (B). (C to O) GFP fluorescence from live cells. Regular typeface indicates that expression of the fusion protein was induced by arabinose (all DivIVA derivatives) or IPTG (all GFP fusions), while italics indicate that the plasmid encoding the fusion protein was present but the expression of the fusion was not induced. (C and H) DivIVA-1c (pWM1814) plus GFP-FtsI (in WM1488); (D and E) DivIVA-LZ (pZD1396) plus GFP-FtsI (in WM1488); (F and G) DivIVA (pWM2045) plus GFP-FtsI (in WM1488); (I and J) DivIVA-1c (pWM1814) plus GFP-FtsN (pWM1152) in TX3772; (K and L) DivIVA-LZ (pZD1396) plus GFP-FtsN (pWM1152) in TX3772; (M) DivIVA-1c (pWM1814) plus GFP-FtsQ (pWM1800) in TX3772; (N) DivIVA-1c (pWM1814) plus GFP-FtsK (pWM1801) in TX3772; (O) DivIVA-1c (pWM1814) plus GFP-FtsW (pWM1818) in TX3772. Bar, 5 μm.

FIG. 4.

Immunoblotting of GFP and DivIVA fusion proteins in cells under assay conditions. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of cellular extracts or purified protein standards are shown, probed with rabbit polyclonal anti-GFP (A and B) or anti-DivIVA (C). (A) Immunoblotting of GFP-FtsN. Lane 1, protein molecular mass markers in kilodaltons; lanes 2 to 5, purified GFP protein (74, 45, 30, and 15 ng, respectively); lane 6, pWM1152 (GFP-FtsN) plus pWM1814 (DivIVA-1c) in TX3772 cells induced with IPTG; lane 7, same as lane 6 but induced with IPTG and arabinose; lane 8, cell lysate from TX3772. (B) Immunoblotting of GFP-FtsI. Lane 1, protein molecular mass markers in kilodaltons; lanes 2 to 4, purified GFP protein (120, 60, and 30 ng, respectively); lane 5, pWM1814 (DivIVA-1c) in WM1488 (GFP-FtsI) induced with IPTG; lane 6, same as lane 5 but induced with IPTG and arabinose; lane 7, cell lysate from TX3772 cells. (C) Immunoblotting of DivIVA-1c. Lane 1, protein molecular mass markers in kilodaltons; lanes 2 to 5, purified DivIVA protein (3,000, 2,000, 1,000, and 500 ng, respectively); lane 6, pWM1814 (DivIVA-1c) in WM1488 (GFP-FtsI) induced with arabinose; lane 7, DivIVA-GFP expressed from pWM1461 in Top10; lane 8, cell lysate from TX3772 cells.

FIG. 5.

Polar recruitment of FtsN and FtsI by subdomain 1c is independent of FtsZ and FtsA. (A and B) TX3772 cells coexpressing DivIVA-1c (pWM1814) and GFP-FtsN (pWM1152) with arabinose plus IPTG were fixed and stained for FtsZ by IFM. Panels A and B show the same field of cells visualized by Nomarski and fluorescence. (C to E) Cells were grown at 30°C, induced with IPTG (C) or arabinose plus IPTG (D and E), then shifted to 42°C for 20 min to inactivate FtsZ, and examined for GFP fluorescence. (D) GFP-FtsN (pWM1152) plus DivIVA-1c (pWM1814) plus ftsZ84(ts) in TX3772; (E) GFP-FtsI in WM1488 plus DivIVA-1c (pWM1814) plus ftsZ84(ts) in TX3772. (F) Cells induced with arabinose plus IPTG, coexpressing DivIVA-1c (pWM1814) and GFP-FtsA (pWM1333). Bar, 5 μm.

FIG. 6.

Tethering of subdomain 1c to FtsZ can partially suppress filamentation in an ftsA(ts) mutant, as shown in Nomarski images of WM1793 (ftsA12) cells containing or lacking the pBAD-FtsZ-1c plasmid and grown under the stated conditions. pBAD-FtsZ-1c (A) or no plasmid (B) at 30°C; no plasmid (C) or pBAD-FtsZ-1c (D) shifted from 30°C to 37°C for 90 min; no plasmid (E) or pBAD-FtsZ-1c (F) shifted from 30°C to 37°C for 3 h. Panel F is a composite of several fields of cells, emphasizing cells having difficulty dividing; arrows highlight elongated septa. Average cell size statistics from multiple fields of cells under each condition for panels C to F are listed in the appropriate panel. Bar, 5 μm.