Premature targeting of a cell division protein to midcell allows dissection of divisome assembly in Escherichia coli

Abstract

Cell division in Escherichia coli requires the recruitment of at least 10 essential proteins to the bacterial midcell. Recruitment of these proteins follows a largely linear dependency pathway in which depletion of one cell division protein leads to the absence from the division site of "downstream" proteins in the pathway. Analysis of events that underlie this pathway is complicated by the fact that a protein's ability to recruit "downstream" proteins is dependent on its own recruitment by "upstream" proteins. Hence, one cannot separate the individual contributions of various upstream proteins to any specific recruitment step. Here we present a method--premature targeting--for bypassing the normal localization requirements of a cell division protein and apply it to FtsQ, a protein recruited midway through the pathway. We fused FtsQ to the FtsZ-binding protein ZapA such that FtsQ was targeted to FtsZ rings independently of proteins FtsA and FtsK, which are normally required for FtsQ localization. Analysis of the resulting ZapA-FtsQ fusion suggests that FtsQ associates with a large complex of cell division proteins and that premature targeting of FtsQ can restore localization of this complex under conditions in which neither FtsQ nor the associated proteins would normally be localized.

Figure 1.

(A) The septal recruitment pathway for cell division proteins in E. coli. (B) Bacterial two-hybrid results summarized from several groups (Di Lallo et al. 2003; G. Karimova and D. Ladant, in prep.; M. Gonzalez and J. Beckwith, unpubl.). Cell division proteins are listed clockwise corresponding to their respective order in A. Lines indicate proposed interactions; circular arrows indicate proposed homodimerization. (C–E) Potential models for divisome assembly. (C) Linear assembly. (D) Cooperative binding by an upstream complex. (E) Sequential activities plus substrate recognition. (F) ZapA–FtsQ constructs used in this work. (Cyto) Cytoplasmic domain of FtsQ; (TM) transmembrane segment of FtsQ; (Peri) periplasmic domain of FtsQ. YFP and non-YFP constructs are identical except for the addition of YFP fused in frame to the N terminus of ZapA. (G) Proposed premature targeting of ZapA–FtsQ construct.

Figure 2.

Localization of YFP–ZapA requires FtsZ but not FtsA. YFP–ZapA and GFP–FtsQ localize in ftsZ84(Ts) or ftsA12(Ts) cells grown at 30°C (permissive temperature). When grown at 42°C (restrictive temperature), YFP–ZapA localizes in cells carrying the ftsA12(Ts) allele but not in those containing ftsZ84(Ts). GFP–FtsQ does not localize in either background at 42°C. The strains used are JOE95, JOE97, NWG128, and NWG129.

Figure 3.

YFP–ZapA–FtsQ colocalizes with FtsZ rings in cells depleted for FtsA or FtsK. (Top panel) Localization of CFP–FtsZ (FtsZ) with either YFP–ZapA–FtsQ (ZapA–FtsQ) or YFP–FtsQ (FtsQ) in ftsA12(Ts) cells at permissive (30°C) or restrictive (42°C) temperatures. (Bottom panel) Localization of the same in ΔftsK cells when FtsK expression from a complementing plasmid is induced with arabinose (Ara) or repressed with glucose (Glc). Examples of colocalization of YFP–ZapA–FtsQ with FtsZ–CFP in cells depleted for FtsK or FtsA are indicated by white arrowheads. Bar, 10 μm. The strains used are NWG327, NWG328, NWG373, and NWG374.

Figure 4.

ZapA–FtsQ restores localization of FtsL in cells depleted for FtsK, FtsA, or both. Localization of GFP–FtsL is shown in cells depleted for the indicated cell division protein(s) and expressing, as indicated, ZapA–FtsQ (A,D,G), wild-type FtsQ (B,E,H), or ZapA (C,F) from the λatt site. Representative images are shown. A quantitative summary of results for all cells analyzed is presented in Table 1, rows 1–8. Bar, 10 μm.

Figure 5.

ZapA–FtsQ restores localization of FtsI, but not FtsN. Cells express either GFP–FtsI (A–C) or GFP–FtsN (D–F) from chromosomal constructs and are depleted for the cell division proteins FtsA and FtsK as indicated. All cells express ZapA–FtsQ from the λatt site. Representative images from paired samples are shown. Note the short cells in D, indicating suppression of the FtsK depletion by expression of GFP–FtsN. (C) Examples of rings in a doubly depleted cell are indicated by white arrowheads. A quantitative summary of results including control cells expressing wild-type FtsQ from the λatt site is presented in Table 1, rows 9–20. Bar, 10 μm.

Figure 8.

Summary of observed recruitment by prematurely targeted FtsQ in cells depleted for FtsA and FtsK (A) or FtsA alone (B). (A) ZapA–FtsQ is able to recruit FtsL and likely FtsB, which restores the pathway through FtsI. (B) ZapA–FtsQ is also able to interact with FtsK, and this interaction with FtsK in turn supports association of multiple FtsQ molecules. FtsB and FtsW are shown in outline as their presence is not explicitly tested in this work, but implied from previous data, which indicate FtsI requires both FtsB and FtsW to localize to the septum.

Figure 6.

ZapA–FtsQ promotes localization of FtsK in cells depleted for FtsA. Cells express an FtsK–YFP fusion from the native ftsK locus and ZapA–FtsQ (A), FtsQ (B), or ZapA (C) as indicated from IPTG-inducible constructs integrated at the λatt site. Cells also carry the ftsA12(Ts) allele and were grown at 42°C to deplete FtsA. Representative images are shown. A quantitation of results is presented in Table 1, rows 21–23. Bar, 10 μm.

Figure 7.

Overexpression of FtsK promotes association of multiple FtsQ molecules. (B) When FtsK was overexpressed from multicopy plasmid pBAD44 (pNG137) in cells expressing both GFP–FtsQ and ZapA–FtsQ under conditions of FtsA depletion, localization of GFP–FtsQ was seen. If either wild-type FtsQ was expressed instead of ZapA–FtsQ (C) or empty vector was substituted for the FtsK plasmid (A), localization was significantly reduced. Representative images from paired samples are shown. A quantitation of results is presented in Table 1, rows 24–29.