A gain-of-function mutation in ftsA bypasses the requirement for the essential cell division gene zipA in Escherichia coli

Abstract

ZipA and FtsA are recruited independently to the FtsZ cytokinetic ring (Z ring) and are essential for cell division of Escherichia coli. The molecular role of FtsA in cell division is unknown; however, ZipA is thought to stabilize the Z ring, anchor it to the membrane, and recruit downstream cell division proteins. Here we demonstrate that the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA (FtsA*). Cells with ftsA* in single copy in place of WT ftsA or with ftsA* alone on a multicopy plasmid divide mostly normally, whether they are zipA+ or zipA-. Experiments with ftsQAZ and ftsQA*Z on multicopy plasmids indicate that ftsQAZzipA+ and ftsQA*ZzipA- cells divide fairly normally, whereas ftsQAZzipA- cells divide poorly and ftsQA*ZzipA+ cells display a phenotype that suggests their septa are unusually stable. In support of the idea that ftsA* stabilizes Z rings, single-copy ftsA* confers resistance to excess MinC, which destabilizes Z rings. The inhibitory effect of excess ZipA on division is also suppressed by ftsA*. These results suggest that the molecular mechanism of the FtsA* bypass is to stabilize FtsZ assembly via a parallel pathway and that FtsA* can replace the multiple functions of ZipA. This is an example of a complete functional replacement of an essential prokaryotic cell division protein by another and may explain why most bacteria can divide without an obvious ZipA homolog.

Figure 1

Immunoblot showing cellular ZipA in strains used in this study. Lane 1, TX3772 (WT); lane 2, pWM796 (ZipA-GFP) in WM1659 (ftsA*) + 1 mM IPTG; lane 3, WM1657 (ftsA* ΔzipA); lane 4, pWM1703 (ZipA) in WM1657 (ftsA* ΔzipA) + 1 mM IPTG; lane 5, pWM796 (ZipA-GFP) in WM1657 (ftsA* ΔzipA) + 1 mM IPTG; lane 6, WM1282 grown at 42°C for 4 h to deplete ZipA; lane 7, WM1282 grown at 30°C. ftsA* is the suppressor allele. Positions of 50-and 81-kDa protein markers are shown on the left.

Figure 2

Mostly normal cell division with ftsA* replacing WT ftsA. Shown are WT strain TX3772 (A and D), ftsA* zipA+ strain WM1659 (B and E), and ftsA* ΔzipA strain WM1657 (C and F). (A–C) Phase-contrast images. (D–F) Immunofluorescence microscopy for FtsZ (black staining) of the cells in A–C, respectively. (G–J) Phase-contrast images of nondividing WM1657 cells after growth in cephalexin for 2 h to accentuate the twisting at the potential septum. (Scale bar = 5 μm.)

Figure 3

FtsA* is sufficient to bypass the need for ZipA. Colony growth is shown at 32°C or 42°C on LB chloramphenicol plates without arabinose for the ZipA depletion (WM1282) strains containing the plasmids indicated.

Figure 4

Effects of overproduction of FtsA or FtsA* on cell morphology. (A–D) Representative phase-contrast images of pZA*Q in TX3772 (A), pZAQ in TX3772 (B), pZA*Q in TX3772 transduced with ΔzipA∷aph (C), and pZAQ in TX3772 transduced with ΔzipA∷aph (D). (E–J) Nomarski (E–G) and FtsZ immunofluorescence microscopy (H–J) of pZA*Q in TX3772 (E, F, H, and I) and pZAQ in TX3772 (G and J). Arrows in H and I highlight helical FtsZ patterns. (Scale bar = 5 μm.)

Figure 5

Molecular modeling of FtsA* (R286W) on the FtsA crystal structure and conservation of R286 across species. (A) A ribbon diagram of FtsA from T. maritima, with K287 highlighted as a ball-and-stick representation within the S13 domain. (B) An amino acid alignment of the region surrounding the FtsA S13 β-sheet (IKTTT in T. maritima) based on the presence or absence of the conserved arginine. From the top down, the groupings are γ, α, β, and δ Gram-negative proteobacteria, other Gram-negative proteobacteria, Gram-positive bacteria, and the most divergent species. Species in all caps have ZipA homologs; species underlined have the conserved arginine.