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 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.
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Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston 77030, USA.
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Margolin
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D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002352:Carrier Proteins; D002455:Cell Division; D003598:Cytoskeletal Proteins; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006602:Hexosyltransferases / Q000235:genetics / Q000502:physiology; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009097:Multienzyme Complexes / Q000235:genetics / Q000502:physiology; D002267:Muramoylpentapeptide Carboxypeptidase; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C065103:FtsA protein, Bacteria; C489310:FtsA protein, E coli; C096452:FtsK protein, E coli; C071525:FtsZ protein, Bacteria; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase