The early divisome protein FtsA interacts directly through its 1c subdomain with the cytoplasmic domain of the late divisome protein FtsN

Abstract

In Escherichia coli, FtsN localizes late to the cell division machinery, only after a number of additional essential proteins are recruited to the early FtsZ-FtsA-ZipA complex. FtsN has a short, positively charged cytoplasmic domain (FtsN(Cyto)), a single transmembrane domain (FtsN(TM)), and a periplasmic domain that is essential for FtsN function. Here we show that FtsA and FtsN interact directly in vitro. FtsN(Cyto) is sufficient to bind to FtsA, but only when it is tethered to FtsN(TM) or to a leucine zipper. Mutation of a conserved patch of positive charges in FtsN(Cyto) to negative charges abolishes the interaction with FtsA. We also show that subdomain 1c of FtsA is sufficient to mediate this interaction with FtsN. Finally, although FtsN(Cyto-TM) is not essential for FtsN function, its overproduction causes a modest dominant-negative effect on cell division. These results suggest that basic residues within a dimerized FtsN(Cyto) protein interact directly with residues in subdomain 1c of FtsA. Since FtsA binds directly to FtsZ and FtsN interacts with enzymes involved in septum synthesis and splitting, this interaction between early and late divisome proteins may be one of several feedback controls for Z ring constriction.

Fig 1

Coaffinity purification of FtsA and FtsN from cobalt resin suggests an in vitro interaction. (A) Cells expressing His₆-FtsN (WM3428) or FLAG-FtsA (WM2700) from plasmids were induced, pelleted, and lysed. Resulting crude extracts were mixed together and incubated with Talon cobalt resin. The resin was washed, and His₆-FtsN was eluted with 150 mM imidazole. Crude extracts and eluates were immunoblotted with anti-His and anti-FLAG antibodies. (B) The protocol was repeated using crude extracts of His₆-FtsA (WM1260) and untagged FtsN (WM2022) expressed from plasmids. Anti-FtsN antibody was used to detect untagged FtsN.

Fig 2

The periplasmic region of FtsN is not required for interaction with FtsA in BACTH assays. (A) FtsN variants were constructed in the BACTH plasmid pKT25F and cotransformed into the cya strain DHM1 with pUT18c-ftsA (pWM3021). Chimeras constructed with the secreted form of alkaline phosphatase (′PhoA) and the cytoplasmic and transmembrane regions of the Agrobacterium tumefaciens protein VirB10 are also shown. Positive interaction between the FtsA and FtsN variants is indicated by a plus symbol. Complementation of an FtsN depletion strain (WM2355) by the FtsN variants and chimeras is also shown. ND, not determined. (B) Interaction between the chimeras and FtsA was determined using medium containing X-Gal (50 μg ml⁻¹) and IPTG (0.5 mM IPTG). All strains shown are in a DHM1 background, transformed with pUT18c-ftsA (pWM3021) and pKT25F-ftsN (pWM3772) (1), pKT25F-ftsN_1-128 (pWM3861) (2), pKT25F-ftsN_1-242 (pWM3862) (3), pKT25F-ftsN_Δ63-130 (pWM3863) (4), pKT25F-ftsN_{Δ63-130/Δ243-319} (pWM3864) (5), pKT25F-ftsN_129-242 (pWM3865) (6), pKT25F-ftsN_1-54′phoA (pWM3451) (7), pKT25F-virB10_1-60′phoA (pWM3866) (8), or pKT25F empty vector (EV). (C) Successful translocation of N_1-54-′PhoA and VirB10_1-60-′PhoA was confirmed in the phoA mutant strain DH5α, using BCIP-containing medium (50 μg ml⁻¹).

Fig 3

Coaffinity purification of FLAG-FtsA and His₆-FtsN_Cyto-TM. Crude extracts containing overexpressed FLAG-FtsA (WM2700) were combined with those containing His₆-FtsN_Cyto-TM (WM3616) or His₆-FtsN (WM3428) and treated as described in the legend to Fig. 1.

Fig 4

Binding of purified FtsA to purified derivatives of FtsN and FtsN_Cyto. (A) Cartoon diagrams of the constructs used in this assay. The cylinders representing the different portions are color coordinated and refer to (i) the His₆ tag (red), (ii) full-length FtsN or FtsN_Cyto (blue), (iii) the FLAG tag (yellow), (iv) GCN4 or MtaN leucine zippers (black), and (v) FtsN_Cyto with DDEE replacing RRKK_16-19 (FtsN_Cyto-DDEE) (orange). Parallel and antiparallel leucine zippers are denoted by convergent and divergent arrows, respectively. The basic nature of the RRKK region is indicated by plus signs. Combined Western (C and D) and far-Western (E) results show binding of the purified His₆-GluGlu-FtsA probe to His₆-FtsN-FLAG and His₆-FtsN_Cyto-ParLeu-FLAG (lanes 1 and 2) but not to His₆-FtsN_Cyto-FLAG, His₆-FtsN_Cyto-DDEE-ParLeu-FLAG, or His₆-FtsN_Cyto-AntiLeu-FLAG (lanes 3, 4, and 5, respectively). The purified proteins were subjected to SDS-PAGE, and both loading and protein purity controls are shown in panel B by staining with Coomassie blue. His₆-GluGlu-FtsA, loaded to the right of lane 5, was used as a negative control for anti-FLAG and a positive control for anti-GluGlu. The membrane in panel C was incubated with anti-FLAG antibody, and those in panels D and E were incubated with anti-GluGlu. The four gels were loaded and run identically. The reaction conditions are described in Materials and Methods. (F) Alignment of FtsN cytoplasmic and transmembrane domains from the indicated species. The conservation of the basic patch of residues corresponding to residues 16 to 19 in E. coli is highlighted in red.

Fig 5

Coaffinity purification of His₆-FtsA-1c and FtsN. Crude extracts containing untagged FtsN (WM2022) were combined with those containing His₆-FtsA-1c-83-176 (WM1835) induced at various IPTG concentrations (0, 0.01, 0.10, and 1.00 mM). Untagged FtsN was detected with anti-FtsN antibody.

Fig 6

FtsA-1c-83-176 binding to FtsN and to FtsN_Cyto. Combined Western (B and C) and far-Western (D) results show binding of purified His₆-FtsA-1c-83-176, used to probe membranes containing His₆-FtsN-FLAG (lane 1, upper arrow, in panel B), His₆-FtsN_Cyto-ParLeu-FLAG (lane 2, lower arrow, in panel B), His₆-FtsN_Cyto-AntiLeu-FLAG (lane 3), or His₆-FtsA-1c-83-176 (lane 4), transferred from SDS-PAGE gels. The lower band in panel B, lane 1, is a degradation product that is also visible in panel A, lane 1. Panel A shows loading and protein purity controls after SDS-PAGE and Coomassie blue staining. Molecular mass markers are shown to the left of each panel. The membrane in panel B was incubated with anti-His antibody, and those in panels C and D were incubated with anti-FtsA. The upper band in panels C and D, lanes 4, may be a trace contaminant in the FtsA-1c-83-176 preparation recognized only by the anti-FtsA antibody. The four gels were loaded and run identically, and the reaction conditions are described in Materials and Methods. (E) Cartoon diagram of the FtsA 1c subdomain (residues 83 to 176), containing alpha helix H2 and beta strands S5, S6, and S7, from the T. maritima structure (53).

Fig 7

Overproduction phenotypes of FtsN and FtsN_Cyto-TM. (A) Histogram of length distributions of cells with pBAD33 (WM4049), pBAD33-FtsN_Cyto-TM (WM4050), or pBAD33-FtsN (WM4051) after induction of expression with 0.2% arabinose for 2 h. Data were obtained from two independent experiments. (B to D) Representative micrographs obtained for the cells quantified in panel A. (B) pBAD33 vector (WM4049); (C) FtsN_Cyto-TM (WM4050); (D) FtsN (WM4051). Bar = 5 μm. Colony morphotypes shown in panels E, F, and G depict the typical phenotypes observed for the strains represented in panels B, C, and D, respectively, when cells were spot diluted onto LB agar plates containing 0.2% arabinose.