The FtsH protein accumulates at the septum of Bacillus subtilis during cell division and sporulation - PubMed (original) (raw)

The FtsH protein accumulates at the septum of Bacillus subtilis during cell division and sporulation

W Wehrl et al. J Bacteriol. 2000 Jul.

Abstract

The ftsH gene encodes an ATP- and Zn(2+)-dependent metalloprotease which is anchored to the cytoplasmic membrane via two transmembrane segments in such a way that the very short amino- and the long carboxy termini are exposed to the cytoplasm. Deletion of the ftsH gene in Bacillus subtilis results in a pleiotropic phenotype such as filamentous growth. This observation prompted us to ask whether ftsH is involved in cell division. A translational fusion was constructed between the complete coding region of ftsH and gfp(+) the latter carrying five point mutations to obtain enhanced fluorescence. We detected that the FtsH protein accumulates in the midcell septum of dividing cells, and during sporulation first in the asymmetrically located septa of sporulating cells and later in the membrane which engulfs the forespore. These observations revealed a new function of FtsH.

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Figures

FIG. 1

FIG. 1

The ftsH-gfp+ gene fusion inserted at the chromosomal amyE locus of the B. subtilis chromosome. (A) Schematic drawing of the ftsH-gfp+ translational fusion fused to the xylose-regulatable promoter P_xylA_ and sandwiched between _amyE_-front and _amyE_-back (not drawn to scale). (B) Partial DNA sequence of the ftsH-gfp+ fusion. Indicated is the DNA sequence at the immediate beginning of ftsH with the first two codons, the hybrid _Bam_HI-_Bgl_II site, the last two codons of ftsH (K and E), two foreign codons introduced by the _Nhe_I restriction site (A and S), the first and the last codon of gfp, and the downstream _Pst_I and the hybrid _Bgl_II-_Bam_HI sites.

FIG. 2

FIG. 2

FtsH-GFP+ localization in live cells. Cells were grown either in Luria broth (A to C) or in DS medium (22) (D to G) in the presence of 0.1% xylose to induce ftsH-gfp+. FtsH-GFP was visualized in live cells as follows. Cells from 1.5 ml of growth medium were sedimented, resuspended in 200 μl of Tris-HCl buffer (pH 7.4), and mixed with 500 μl of 4% agarose; 10 to 20 μl was applied to a glass slide, covered with a coverglass, and allowed to cool for ∼1 min. A Leica (Heidelberg, Germany) TCS/SP confocal scanning laser microscope equipped with a argon-ion laser for excitation at 488 nm was used. Detection occurred at 510 nm, and the data from the channel were collected with fourfold averaging at a resolution of 512 by 512 pixels and processed using Corel Photo Point 7. Original magnification, ×20,000.

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