Yu X C, Tran A H, Sun Q, Margolin W
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston 77030, USA.
J Bacteriol. 1998 Mar;180(5):1296-304. doi: 10.1128/JB.180.5.1296-1304.1998.
Escherichia coli cell division protein FtsK is a homolog of Bacillus subtilis SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in E. coli cells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the ftsK44 mutation in trans, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of ftsK44, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced and ftsI mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize in ftsZ mutant filaments. In ftsK44 mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the ftsK44 mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.
大肠杆菌细胞分裂蛋白FtsK是枯草芽孢杆菌SpoIIIE的同源物，似乎在隔膜形成过程的后期起作用。为了确定FtsK是否定位于隔膜，我们将FtsK的三个N端片段与绿色荧光蛋白（GFP）融合，并在大肠杆菌细胞中表达。所有这三个片段都足以将GFP靶向隔膜，这表明该蛋白仅前15%就足以作为隔膜靶向结构域。仅在含有可见细胞中部缢缩的细胞中可检测到定位荧光，这表明FtsK的靶向通常仅发生在隔膜形成的后期。最大的两个FtsK-GFP融合蛋白能够至少部分地反式互补ftsK44突变，这表明N端和C端结构域在功能上是可分离的。然而，FtsK-GFP的过量表达导致了类似于ftsK44的后期隔膜形成表型，荧光点位于受阻的隔膜处，这表明高水平的N端结构域可能仍然能够定位，但也会抑制FtsK的活性。有趣的是，在这些条件下，荧光有时也会定位为潜在分裂位点处的条带，这表明FtsK-GFP能够非常早期地靶向。此外，FtsK-GFP定位于头孢氨苄诱导的和ftsI突变体细丝中的潜在分裂位点，进一步支持了FtsK-GFP可能通过直接识别FtsZ而能够早期靶向的观点。ftsK-GFP在ftsZ突变体细丝中无法定位支持了这一假设。在ftsK44突变体细丝中，FtsA和FtsZ通常定位于受阻隔膜之间的潜在分裂位点。当ftsK44突变整合到FtsK-GFP融合蛋白中时，细胞中部的定位介于非常弱和无法检测之间，这表明FtsK44突变蛋白在靶向隔膜方面存在缺陷。
