LOCALIZATION OF CELL-DIVISION PROTEIN FTSK TO THE ESCHERICHIA-COLI SEPTUM AND IDENTIFICATION OF A POTENTIAL N-TERMINAL TARGETING DOMAIN

Escherichia coli cell division protein FtsK is a homolog of Bacillus s ubtilis SpoIIIE and appears to act late in the septation process, Td d etermine whether FtsK localizes to the septum, we fused three N-termin al segments of FtsK to green fluorescent protein (GFP) and expressed t hem 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 prot ein is a septum-targeting domain. Localized fluorescence was detectabl e 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 comp lement the ftsK44 mutation in trans, suggesting that the N- and C-term inal domains are functionally separable However, overproduction of Fts K-GFP resulted in a late-septation phenotype similar to that of ftsK44 , with fluorescent dots localized at the blocked septa, suggesting tha t high levels of the N-terminal domain mag; still localize but also in hibit FtsK activity., interestingly, under these conditions fluorescen ce was also sometimes localized as hands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In additi on, FtsK-GFP localized to potential division sites in cephalexim-induc ed and ftsI mutant filaments, further supporting the idea that FtsK-GF P fan target early, perhaps by recognizing FtsZ directly. This hypothe sis was supported by the failure of FtsK-GFP to localize in ftsZ mutan t filaments, In ftsK44 mutant filaments, FtsA and FtsZ were usually lo calized to potential division sites between the blocked septa. When th e ftsK44 mutation was incorporated into the Ftsk-GFP fusions, localiza tion to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.