ATP-binding site lesions in FtsE impair cell division

Abstract

FtsE and FtsX of Escherichia coli constitute an apparent ABC transporter that localizes to the septal ring. In the absence of FtsEX, cells divide poorly and several membrane proteins essential for cell division are largely absent from the septal ring, including FtsK, FtsQ, FtsI, and FtsN. These observations, together with the fact that ftsE and ftsX are cotranscribed with ftsY, which helps to target some proteins for insertion into the cytoplasmic membrane, suggested that FtsEX might contribute to insertion of division proteins into the membrane. Here we show that this hypothesis is probably wrong, because cells depleted of FtsEX had normal amounts of FtsK, FtsQ, FtsI, and FtsN in the membrane fraction. We also show that FtsX localizes to septal rings in cells that lack FtsE, arguing that FtsX targets the FtsEX complex to the ring. Nevertheless, both proteins had to be present to recruit further Fts proteins to the ring. Mutant FtsE proteins with lesions in the ATP-binding site supported septal ring assembly (when produced together with FtsX), but these rings constricted poorly. This finding implies that FtsEX uses ATP to facilitate constriction rather than assembly of the septal ring. Finally, topology analysis revealed that FtsX has only four transmembrane segments, none of which contains a charged amino acid. This structure is not what one would expect of a substrate-specific transmembrane channel, leading us to suggest that FtsEX is not really a transporter even though it probably has to hydrolyze ATP to support cell division.

FIG. 1.

Steady-state levels of Fts membrane proteins in cells depleted of FtsEX. The FtsEX depletion strain EC1335 was grown in LB0N Cam and either Ara or Glu for about 5 h, at which time the Glu culture was filamentous. Cells were fractionated, and FtsK, FtsQ, FtsN, or FtsI was detected by Western blotting. Only the relevant portion of each blot is shown. The position of a molecular mass marker and the protein detected are indicated to the left and right, respectively, of each blot. crude, crude extract; sol., soluble fraction; mem., membrane fraction.

FIG. 2.

ATP-binding site mutants of ftsE. (A) Cartoon of FtsE. Vertical black bars indicate invariant residues in an alignment of FtsE proteins from E. coli, Salmonella enterica serovar Typhimurium, Yersinia pestis, Vibrio cholerae, Haemophilus influenzae, Pseudomonas aeruginosa, and Pasteurella multocida. Sequences of the Walker A, Walker B, and ABC signature motifs are shown below, with residues targeted for mutagenesis indicated by arrows. (B) Steady-state level of mutant FtsE proteins. Strain EC251 (wild type) transformed with plasmids that direct expression of ftsE-3×HA alleles was induced with 0.1 mM IPTG, and whole cells were analyzed by Western blotting with anti-HA antibody. The location of molecular mass markers is shown at the left. FtsE-3×HA is predicted to be 28.5 kDa. The plasmids used were pDSW609, pDSW890, pDSW891, pDSW892, and pTH18-kr. (C) Complementation test. The ΔftsEX null mutant EC1215 was transformed with low-copy-number P_lac::ftsEX plasmids (or controls) that direct production of the indicated proteins. Transformants were grown overnight in LB Kan, adjusted to 10⁹ CFU/ml in LB0N, and 10-fold serial dilutions were spotted onto LB0N Kan with 0.1 mM IPTG. Plates were incubated overnight at 37°C and then photographed. The plating efficiency was ∼100% for all strains when plates contained LB rather than LB0N (not shown). The plasmids used were pTH18-kr, pDSW904, pDSW905, pDSW906, pDSW907, pDSW988, and pDSW989.

FIG. 3.

Effect of ATP-binding site lesions in FtsE on growth in LB0N. Cultures of the ΔftsEX null mutant EC1215 carrying various ftsEX plasmids (or controls) were grown to mid-log phase in LB Kan with 0.1 mM IPTG. At time zero, these cultures were diluted into LB0N Kan with 0.1 mM IPTG. The plasmids used were pTH18-kr (black circles), pDSW904 (medium-gray circles), pDSW905 (open circles), pDSW906 (black squares), pDSW907 (dark-gray squares), pDSW988 (light-gray squares), and pDSW989 (open squares).

FIG. 4.

Effect of FtsE(D162A) on cell division and septal ring assembly. Wild-type cells expressing both the indicated gfp construct and ftsE(D162A) (together with ftsX) were fixed, and GFP was visualized by fluorescence microscopy. Arrows indicate sites of Fts protein localization. The strains shown are EC437 (gfp-ftsI), EC439 (gfp-ftsL), EC441 (gfp-ftsN), EC449 (ftsZ-gfp), EC450 (zipA-gfp), and EC452 (gfp). All carry plasmid pDSW988 (P_lac::ftsE(D162A)X).

FIG. 5.

ATP-binding site mutants of FtsE localize to potential division sites. EC1215 (ΔftsEX) cotransformed with plasmids that express ftsX (pDSW621) and the indicated ftsE-3×HA allele (pDSW609, pTH18-kr [(−)], pDSW890, pDSW891, or pDSW892) were grown in LB0N containing antibiotics, Ara (to induce ftsX), and 0.1 mM IPTG (to induce ftsE). Cells were fixed, and FtsE was visualized by immunofluorescence microscopy, with a secondary antibody conjugated to Alexa 488 used to detect FtsE-3×HA. Membranes were stained with FM4-64. Arrows indicate sites of FtsE localization.

FIG. 6.

ATP-binding site mutants of FtsE support recruitment of FtsI and FtsN to septal rings. Left panel set: localization of GFP-FtsI. Cells were fixed and photographed under phase-contrast and fluorescence microscopy. Arrows indicate sites of GFP-FtsI localization. The cells shown are derivatives of EC1882 carrying plasmids pTH18-kr (empty vector), pDSW906 (ftsEX), pDSW904 (ftsE alone), pDSW905 (ftsX alone), pDSW907 [ftsE(K41Q) and ftsX], pDSW988 [ftsE(D162A) and ftsX], and pDSW989 [ftsE(E163A) and ftsX] in rows A to G, respectively. Right panel set: localization of FtsN. Cells were fixed, processed for immunofluorescence microscopy to visualize FtsN, and stained with FM4-64 to visualize membranes. Arrows indicate sites of FtsN localization. The cells shown are derivatives of EC1215 carrying the same plasmids used to study GFP-FtsI recruitment in the left panel set.

FIG. 7.

FtsX-GFP localizes in cells that lack FtsE. Fluorescence microscopy was used to image FtsX-GFP in live and fixed cells of EC251 (wild type) or EC1215 (ΔftsEX) harboring pDSW513 (P₂₀₄::ftsX-gfp). Arrows indicate sites of FtsX-GFP localization.

FIG. 8.

Topology of FtsX. (A) Model of FtsX. Arrowheads and black residues indicate junction points of FtsX′-′PhoA fusions. Arrowheads are colored based on their activity as either high (>125 units; black) or low (<12 units; white). Arrows with circled “+” or “−” symbols indicate charged residues predicted by some computer topology programs to be within the membrane. Shaded residues are highly conserved (≥80% identity) in an alignment of FtsX proteins from E. coli, Photorhabdus luminescens, Xylella fastidiosia, V. cholerae, H. influenzae, P. aeruginosa, Shewanella oneidensis, Photobacterium profundum, P. multocida, Xanthomonas axonopodis, and Neisseria meningitidis. (B) Western blot of FtsX′-′PhoA fusions. Lane numbers correspond to the fusions in panel A. “(−)” is a strain with the ′phoA fusion vector, which lacks a translational start site. “(+)” is an ftsI′-′phoA fusion. Molecular mass markers are indicated to the left of the blot.