Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL

Abstract

Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of ftsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of beta-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.

FIG. 1

Steady-state levels of FtsI and GFP-FtsI fusion proteins as determined by Western blotting with an anti-FtsI antibody. Molecular mass standards are indicated to the left of the blot, and the positions of FtsI and GFP-FtsI are shown to the right. The GFP fusion protein produced is indicated above each lane. The faint band at the position of GFP-FtsI in the first lane is due to cross-reaction of the primary antibody with a protein of unknown identity. The intensity of this band does not respond to IPTG. In contrast, the proteins identified as GFP-FtsI respond to IPTG and are also detected with anti-GFP antibodies (not shown). The strains used were MC4100, EC436, EC505, EC507, EC509, and EC511.

FIG. 2

Localization of GFP-FtsI, GFP-III, and GFP-FFI in cells fixed during exponential growth. Arrows indicate examples of FtsI rings at division sites. These are very faint in the case of GFP-III and not detected in GFP-FFI. In the case of GFP-FtsI, 10 of the 13 cells shown were judged to have an FtsI ring at the division site. The strains used were EC436, EC505, and EC507.

FIG. 3

Localization of FtsI in filaments formed by treating cells with FtsI-specific β-lactam antibiotics. Upper panels show strain EC522, expressing gfp-ftsI, after 75 min in the presence of cephalexin. (A) GFP. (B) DAPI. Lower panels show FtsI detected by IFM of strain MC4100 after 45 min in the presence of furazlocillin. Arrows indicate FtsI localized to potential division sites. (C) Fluorescein. (D) Propidium iodide.

FIG. 4

Dependence of localization of GFP-FtsI on FtsZ, FtsA, FtsQ, and FtsL. In each case, one or more filaments is shown together with nonfilamenting cells that do exhibit GFP-FtsI bands and serve as internal controls for microscopy and subsequent image processing. These latter are the short cells in the micrographs. Filaments are as follows: EC458 [ftsZ(Ts)] after 60 min at 42°C; EC436/pDSW259 (sfiA induced) 60 min after addition of arabinose; EC455 [ftsA(Ts)] after 45 min at 42°C; EC538 (FtsQ depletion) in glucose; EC607 (FtsL depletion) in glucose. Controls were EC436 after 60 min at 42°C, EC436/pDSW259 60 min after addition of glucose, EC436 after 45 min at 42°C, EC538 in arabinose, and EC607 in arabinose.

FIG. 5

Localization of ZipA-GFP in an ftsI23(Ts) mutant background (EC491). Cells were grown at 30°C prior to fixation (A) and were shifted to 42°C for 60 min prior to fixation (B).

FIG. 6

Dependency relationships for septal localization of several E. coli division proteins. The first event is polymerization of FtsZ into the Z ring at the future division sites. The other proteins then localize to that site in the order indicated. The positions of ZipA, FtsK, and FtsW are only partially established. Unpublished work from our lab indicates that ZipA localization requires a Z ring but not FtsA. FtsK requires FtsA but not FtsI. FtsW is known to localize, but what happens in fts mutant backgrounds is not known. The model is based on our own results in this paper, references , , , and , and reports from several other labs (2, 3, 5, 38, 53, 59).