An altered FtsA can compensate for the loss of essential cell division protein FtsN in Escherichia coli

Abstract

FtsN is the last known essential protein component to be recruited to the Escherichia coli divisome, and has several special properties. Here we report the isolation of suppressor mutants of ftsA that allow viability in the absence of ftsN. Cells producing the FtsA suppressors exhibited a mild cell division deficiency in the absence of FtsN, and no obvious phenotype in its presence. Remarkably, these altered FtsA proteins also could partially suppress a deletion of ftsK or zipA, were less toxic than wild-type FtsA when in excess, and conferred resistance to excess MinC, indicating that they share some properties with the previously isolated FtsA* suppressor mutant, and bypass the need for ftsN by increasing the integrity of the Z ring. TolA, which normally requires FtsN for its recruitment to the divisome, localized proficiently in the suppressed ftsN null strain, strongly suggesting that FtsN does not recruit the Tol-Pal complex directly. Therefore, despite its classification as a core divisome component, FtsN has no unique essential function but instead promotes overall Z ring integrity. The results strongly suggest that FtsA is conformationally flexible, and this flexibility is a key modulator of divisome function at all stages.

Fig. 1. The cloned ftsA suppressor mutant (ftsA ^sup) permits cell division and colony formation in the absence of FtsN or FtsA

A–F. DIC micrographs of FtsN depletion (WM2355) derivatives containing pBAD18kan (A and B), pBAD18kan-FtsA (C and D) or pBAD18kan-FtsA^sup (E and F) grown at either 30°C (A, C and E) or 42°C to deplete FtsN (B, D and F) for 4 h after initial growth for 2 h at 30°C (no arabinose added). Inset in (A) shows cells of the parent W3110 strain. G–I. P1 transduction of ΔftsN::cat into W3110 containing pBAD18kan (G), pBAD18kan-FtsA (H) or pBAD18kan-FtsA^sup (I), selecting for Cm^R and Km^R at 37°C (no arabinose added). J. Complementation of the FtsA depletion strain WM1281 by FtsA* or FtsA^sup. WM1281 cells containing the indicated plasmids were either grown at 30°C or induced at 42°C for 2 h prior to spotting on plates, which were then incubated overnight at the indicated temperatures. K. Anti-FtsN immunoblot of lysates from strains WM2417, containing the chromosomal E124A suppressor and ΔftsN::cat, or parent strain W3110. Markers to the left are in kDa. The position of the FtsN protein band is shown to the right.

Fig. 2. Z rings and relative viability of ftsN ⁺ cells with the suppressor and suppressed cells lacking ftsN

A and B. Representative fluorescence/DIC overlays of logarithmically growing cells of EC448 (FtsZ-GFP) (A) or of WM2600, which is EC448 plus pBAD18kan-FtsA^sup and ΔftsN::cat (B). C and D. Colony viabilities of EC448 carrying derivatives of pBAD18kan (C) or W3110 derivatives (also containing leu::Tn5) with or without the chromosomal E124A allele (D), in the presence or absence of ftsN.

Fig. 3. Conservation of ftsA suppressor mutations in FtsA

Top. An alignment of segments comprising the K48, K117 and E124 residues involved in suppression. Conserved residues at these three positions, denoted by numbers at the top, are shown in bold. Species containing a likely homologue of FtsN, defined as an E-value < 0.001 after a BLAST search with E. coli FtsN, are underlined. Bottom. Positions of the three FtsA^sup mutations and R286W (FtsA*) in the corresponding crystal structure of Thermotoga maritima FtsA. shows the positions of the three mutations mapped onto the crystal structure of FtsA from Thermotoga maritima.

Fig. 4. Effects of various ftsA mutants on bypassing the requirement for FtsN and inhibiting colony formation

A. FtsN depletion strains (WM2355) containing pCSB1 plasmid derivatives expressing IPTG-inducible alleles of ftsA shown at left were tested for colony viability on plates at 30°C (permissive) or 42°C (to deplete FtsN). IPTG was present in the plates at 0, 0.1, 0.5 or 1 mM, as shown at the top. B. Immunoblots of representative cultures used for the 30°C spots were probed with polyclonal anti-FtsA antibodies. Both blots show cultures expressing WT FtsA as controls.

Fig. 5. Suppressor mutants of ftsA can suppress the loss of ftsK or zipA in addition to the loss of ftsN, with varying efficiencies

A. The ΔzipA::kan, ΔftsK::kan or ΔftsN::cat alleles were introduced by P1 transduction into W3110 carrying the plasmids indicated, and plated on selective plates containing 0.1 mM IPTG to show the frequency and size of the initial transductants. B–E. Colony viabilities of various combinations of ftsA suppressor mutants and cell division gene mutants are shown in each row. The ftsA suppressor mutants were expressed either from the native chromosomal locus in the absence of WT ftsA (C and D), or from plasmid pRR48 derivatives (supplemented with 0.1 mM IPTG) in addition to the WT ftsA in the chromosome (B and E).

Fig. 6. The E124A allele of FtsA is resistant to inhibition caused by overproduction of MinC. WM2886 (ftsA E124A) or WM2887 (WT ftsA) containing plasmid pWM2801, which expresses flag–minC under control of IPTG, were grown at 37°C with either 0 mM or 1 mM IPTG for 5 h. Cells were then harvested for microscopy and for immunoblotting

A–D. WM2886/flag–minC without (A) or with IPTG (B); WM2887/flag–minC without (C) or with IPTG (D). E. Immunoblot of cell extracts from (A)–(D), probed with anti-FLAG antibody, showing the overproduction of FLAG–MinC after IPTG addition. Equal cell density equivalents were loaded in each lane.

Fig. 7

GFP-FtsN localizes normally in cells with the E124A suppressor. Shown are representative fluorescence micrographs of (A) WM2886 (ftsA E124A), (B) WM2887 (WT ftsA) or (C) WM2417 (ftsA E124A, ΔftsN::cat) producing GFP-FtsN from pWM1152. Cells were grown in LB without IPTG for 4 h at 37°C to mid-exponential phase.

Fig. 8

GFP–TolA localizes to division septa of cells lacking FtsN when the E124A suppressor is present. Shown are representative DIC (left) and fluorescence (right) micrographs of GFP–TolA producing cells of (A) WM2886 (ftsA E124A), (B) WM2887 (WT ftsA) or (C) WM2417 (ftsA E124A, ΔftsN::cat). Cells were grown in LB without IPTG for 5 h at 37°C to mid-exponential phase.