Promiscuous targeting of Bacillus subtilis cell division protein DivIVA to division sites in Escherichia coli and fission yeast

Abstract

The Bacillus subtilis divIVA gene encodes a coiled-coil protein that shows weak similarity to eukaryotic tropomyosins. The protein is targeted to the sites of cell division and mature cell poles where, in B.subtilis, it controls the site specificity of cell division. Although clear homologues of DivIVA are present only in Gram-positive bacteria, and its role in division site selection is not conserved in the Gram-negative bacterium, Escherichia coli, a DivIVA-green fluorescent protein (GFP) fusion was targeted accurately to division sites and retained at the cell pole in this organism. Remarkably, the same fusion protein was also targeted to nascent division sites and growth zones in the fission yeast Schizosaccharomyces pombe, mimicking the localization of the endogenous tropomyosin-like cell division protein Cdc8p, and F-actin. The results show that a targeting signal for division sites is conserved across the eukaryote-prokaryote divide.

Fig. 1. Alignments and structural predictions for DivIVA-like proteins. Proteins similar to DivIVA were identified by BLAST searches and a multiple alignment was generated by CLUSTAL W. Each protein was then tested for the presence of a likely coiled-coil region and the letters a and d were placed above the alignment at positions directed by the COILS output (with minor adjustments to accord with the alignment). The arrow marks the position of a skip residue needed to maintain the phase of the heptad repeat (Lupas, 1996).

Fig. 2. DivIVA–GFP is targeted to division sites and cell poles in E.coli. Wild-type and mutant strains of E.coli expressing a DivIVA–GFP fusion were visualized by immunofluorescence microscopy. (A) DivIVA–GFP localization in a static field of E.coli cells (strain D4A). (B) DivIVA–GFP localization in a field of B.subtilis for comparison (strain 1757; see Edwards and Errington, 1997). (C–E) Time lapse images of a field of cells of E.coli cells (strain D4A), with (D) and (E) being taken, respectively, 40 and 80 min after (C). (F and G) DivIVA–GFP localization in filamentous cells of an ftsZts mutant of E.coli (strain Z4A) at the non-permissive temperature. (H) DivIVA–GFP localization in a minB mutant of E.coli (strain M4A). Scale bar represents 2 µm.

Fig. 3. Targeting of DivIVA–GFP to growth zones and cytokinetic rings in wild-type and mutant strains of S.pombe. All cultures were grown in the absence of leucine and thiamine. (A) Schizosaccharomyces pombe expressing full-length DivIVA fused to GFP (strain ESP1). (B) Schizosaccharomyces pombe expressing a truncated form of DivIVA fused to GFP (strain ESP2). (C) Confocal view of S.pombe strain ESP1 expressing full-length DivIVA fused to GFP. (D) As (C). Close up (1.5× magnified) of a typical cell with a DivIVA–GFP band. (E) Rotation of (D), showing the ring-like configuration of the DivIVA–GFP band. (F–I) Fluorescence (F and H) and phase-contrast (G and I) images of typical cdc8 mutant cells (strain 1815) expressing divIVA–gfp at the permissive (30°C; F and G) and non-permissive temperature (37°C; H and I). Arrows point to the positions of septa. (J and K) Effect of Lat-A on localization of DivIVA–GFP. Live cells of strain ESP1 were visualized either untreated (J) or 30 min after treatment with Lat-A (K). Scale bars represents 4 µm.