SepF Increases the Assembly and Bundling of FtsZ Polymers and Stabilizes FtsZ Protofilaments by Binding along Its Length

Abstract

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90°light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZ⌬16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366 -382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division.

Identification ofEscherichia coliZapC (YcbW) as a Component of the Division Apparatus That Binds and Bundles FtsZ Polymers

“…This further highlights the variety of Z-ring components in different bacterial phyla that are known (or suspected) to modulate the assembly dynamics of FtsZ but appear to be phylogenetically unrelated. Other examples are SepF in Gram positives and cyanobacteria (40,41,54,56,77), EzrA (14,35,76) and UgtP (87) (Ftn6) (54) in cyanobacteria, FzlA (32) and KidO (67) in Caulobacter crescentus, and FipA in mycobacteria (79). Evidently, bacteria evolved different Z-ring-associated modulators of FtsZ assembly that are finely tuned to their specific needs.…”

ZipA Is Required for FtsZ-Dependent Preseptal Peptidoglycan Synthesis prior to Invagination during Cell Division

“…However, orthologues or functional homologues may be expressed in Mycobacterium tuberculosis (59), Bacillus subtilis (58), and Neisseria gonorrhoeae (28). The B. subtilis protein SepF may bundle FtsZ polymers and tether them to the membrane in a manner similar to that of ZipA (36).…”

The Conserved DNA-Binding Protein WhiA Is Involved in Cell Division in Bacillus subtilis

“…The protein ZapA cross-links FtsZ-polymers and promotes polymer bundling (3,4). In Gram-positive bacteria and cyanobacteria the protein SepF supports the bundling of FtsZ polymers, as well (5)(6)(7)(8). The absence of SepF results in irregular division septa (9).…”